Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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TITILE ~F THE I1~TVE1'~T'I"IOI~T
~EI~''I"I~I'~T A~I'~Tllf~ AL~CIJI'~IJ1~LAT"I~I~T ~F ~IJECCELaTLdTIJILAI~
'~"~I~IJIh~I~TEI'~TT~y 1~I'~TI~ hI~~TEII~~T~ I'DE 'I~~EI~ THEI~EFI~~IylZ
REL~,TED APPLI~ATIONS/PATENTS ~ IN~ORPOI~ATION DY
I~FEREI~~T~E
A claim of priority is made to U. S. Provisional Application No. 60/455,767,
filed March 19, 2003 and to U.S. Application Serial No. 10/741,313, filed
December
19, 2003. Reference is made to U. S. Application Serial No. 09/995,054, filed
November 27, 2001.
Each of the applications and patents cited in this text, as well as each
document or reference cited in each of the applications and patents (including
during
the prosecution of each issued patent; "application cited documents"), and
each of
the PCT and foreign applications or patents corresponding to and/or claiming
priority from any of these applications and patents, and each of the documents
cited
or referenced in each of the application cited documents, are hereby expressly
incorporated herein by reference. More generally, documents or references
cited in
this text; and, each of these documents or references ("herein-cited
references"), as
well as each document or reference cited in each of the herein-cited
references
(including any manufacturer's specifications, instructions, etc.), are hereby
expressly
incorporated herein by reference.
FIELD OF THE INVENTION
The present invention relates generally to the field of proteomics and to
fields which can utilize subcellular proteomes. More in particular, the
instant
invention relates to methods for the fractionation of a proteome of a
biological
sample to achieve improved detection and analysis of proteins comprising said
proteome, in particular, the detection and analysis of low-abundance proteins.
In a
further aspect, the instant invention relates to the parallel separation and
isolation of
different types of subcellular organelles from any biological sample by
continuous-
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flow ultracentrifugation. Further, the method of the instant invention
provides for
purity, enrichment, accumulation, and integrity of isolated subcellular
organelles and
for proteins contained therein, thereby offering an enhanced strategy to study
and
analyze subcellular proteoms, especially low-abundance proteins.
~A~L;~~1~~1CT1~~T~D
Proteomics attempts to mderstand biological phenomena-e.g., disease,
cellular differentiation, growth cycles, and evolution-via a detailed
knowledge and
appreciation of the functions, subcellular or extracellular locations,
interactions,
activities, and quantities for each and every protein of a cell and/or tissue.
Such an
understanding will greatly advance, for example, the diagnosis, treatment,and
prevention of disease. Proteomics finds applicability in, for example, drug
discovery, preclinical and clinical research, clinical diagnostics, veterinary
medicine,
forensics, agrochemistry and biotherapeutics.
Compared to the field of genomics, however, proteomics is regarded as
having a significantly higher level of complexity. This complexity results
from the
dynamic changes in protein content, localization, post-translational
modifications,
and protein-protein interactions, typically as a function of time. These
changes vary
among individuals, tissues, cells and organelles, and occur in response to,
for
example, growth, differentiation, senescence, enviromnental changes and
disease.
At present, there is no single strategy that can sufficiently address all
levels
of the proteome organization. Furthermore, monitoring dynamic proteome changes
such as, for example, protein localization, requires special techniques for
proteome
analysis at the organelle level.
Subcellular fractionation techniques traditionally have been among the key
methods in cell biology and biochemistry for isolating and characterizing
organelles
(~onifacino et al., (2000), Supplement 3-6, John Wiley ;Sons, Inc.,1V~').
These
procedures exploit various separation techniques, such as, density gradient
centrifugation, free-flow electrophoresis and ligand affinity chromatography.
In
most cases, preparations of subcellular organelles are optimized for a single,
targeted
organelle prepared from distinct sources. Apart from isolating the targeted
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organelle, the remainder of the preparation is generally regarded as debris
and
discarded.
One example of isolating a targeted organelle is described in Price et al.
((1973), Analytical Biochemistry 54:239-246), wherein the authors describe
isolation and separation of intact chloroplasts from spinach brie by
continuous-flow
tonal centrifugation in a CF-6 rotor in gradients of colloidal silica. The
authors
report the recovery of chloroplasts which is supported by phase contrast
microscopy
and concentration of chloroplast specific proteins per unit of chlorophyll
material.
In another example, Cline and Dagg ((1978) Methodological Developments in
Biochemistry, Longman, p.61-70) report separation of chloroplasts from other
plant
cell components using continuous sample-flow with isopycnic banding tonal
rotors
such as J-I and RK-II.
Other reports of monitoring dynamic changes in the proteome at the
subcellular level are described in the articles mentioned below.
Dreger et al., ((2003), Mass. Spec., 22:27-56) reports that monitoring
dynamic proteome changes at the organelle level, such as, for example, protein
translocation events, is an especially difficult task because most
fractionation
techniques are designed to enrich for a single type of organelle. The authors
report
that there is a need in the art to develop new cellular fractionation
techniques for
monitoring at least two types of organelles in parallel in order to provide
one skilled
in the art with the elucidation of organelle-specific protein translocations.
Dreger et al., ((2003) Eur. J. Biochem., 270:589-599) reports the need for
improving techniques for monitoring protein translocation events as several
proteins
may be associated with certain subcellular structures only in certain
physiological
states. V6~hile the authors state that it is possible to separate major
cellular fractions,
such as, for examples, cytosolic and nucleoplasmic fractions, the authors
report that
these studies provide limited information on the dynamic proteome changes to
one
skilled in the art as they do not enrich for organelles and, as a result, do
not elucidate
organelle-specific protein translocation.
Additionally, Gerner et al., ((2000) J. Biol. Chem. 275:39018-39026)
analyzes the effect of Fas-induced apoptosis on the cellular localization of
the TCP
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lA protein. This study does not provide one skilled in the art, however, with
infonmation as to which specific organelle in the cytosol has acquired the TCP-
lA
protein. Obtaining this information would be useful for designing specific
therapeutics aimed at blocking or enhancing specific protein tTanslocation
events.
similar studies were reviewed by I~uber et al. ((2003) Circulation Research,
92:962-968). W this recent 2003 review, the authors note that the present
state of the
art allows for fractionation of cells by differential centrifugation into
three major
components such as cytosol, nuclei and membranes. Similar to the Gerner et al.
article, these studies do not provide information on the dynamic changes in
the
organelle-specific protein localization.
At present, there still exists a need in the art to develop subcellular
fractionation techniques wherein at least two types of organelles can be
simultaneously enriched, accumulated and separated while maintaining high
purity
and intactness, thereby increasing the detection threshold for proteins, such
as, for
example, low-abundant proteins. A need also exists in the art to develop
fractionation techniques whereby subtypes of subcellular organelles can be
accumulated and separated in sufficient quantity and qualitatively resolved
whereby
the proteomic profiles of the subtypes of subcellular organelles can be
determined.
SiIMMARY OF THE INVENTION
One aspect of the invention relates to separation and accumulation of
organelles, such as subcellular organelles, from a sample, preferably a
biological
sample. The separation and accumulation of the organelles are performed by,
for
example, fractionation by a continuous-flow process. The continuous-flow
process,
in turn, utilizes centrifugal force, such as that generated by a centrifuge.
In an
embodiment, a continuous-flow ultracentrifuge is used to separate and
accumulate
organelles. It is understood, however, that other continuous-flow processes
can be
used and that the instant invention is not limited to the use of an
ultracentrifuge. The
contents of the organelles are fractionated. For example, the organelles can
be lysed
and the proteome released therefrom. The proteins and peptides from the
proteome
can be separated by, for example, chromatography, electrophoresis, continuous-
flow
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centrifugation or other art-recognized techniques. The separated proteins and
peptides can be characterized and quantitatively analyzed by a number of
techniques
such as, for example, mass spectrometry. Afterwards, the proteins can be
identified,
if possible, characterized and used for dovmstream applications.
Ieilore specifically, both the separated and accmnulated subcellular
organelles, and the separated and accumulated low-abundance proteins, can be
used
in downstream applications. Such applications include, for example, selling
the
subcellular organelles and/or low-abundance proteins, leasing the subcellular
organelles and/or low-abundance proteins, licensing the subcellular organelles
and/or low-abundance proteins, protecting an intellectual property interest in
the
subcellular organelles and/or low-abundance proteins and placing information
about
the subcellular organelles and/or low-abundance proteins into a database which
can
optionally be provided to third parties.
Against this background, and in accordance with one embodiment of the
present invention, a method is provided for enriching and accumulating
organelles
from a sample comprising the organelles, having the steps of a) releasing the
organelles from the sample; b) introducing the organelles to a density
gradient
within a continuous-flow centrifuge; c) applying a centrifugal force
sufficient for at
least two types of organelles to migrate within the density gradient; and d)
collecting
the at least two types of subcellular organelles from the density gradient so
as to
utilize the at least two types of subcellular organelles.
In another embodiment of the invention, a method is provided for
accumulating low abundance proteins from organelles, having the steps of a)
releasing the organelles from a sample comprising the organelles; b)
introducing the
organelles to a density gradient within a continuous-flow centrifuge; c)
applying a
centrifugal force such that organelles enrich and accumulate within the
density
gradient; d) collecting the organelles from the density gradient; e) lysing
the
organelles to form a proteome; and f) collecting the low-abundance proteins
from
the proteome.
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In a further embodiment of the invention, a method is provided for separating
at least t~,~o types of organelles from a biological sample comprising the at
least two
types of organelles, having the steps of: a) releasing the at least two types
of
subcellular organelles from the sample in a homogenate; b) continuously
flowing the
homogenate over a density gradient and applying a centrifugal force in an
amount
sufficient for each of the at least tv~o types of organelles to enter and
migrate in the
density gradient to a position in the density gradient such that the density
of the
gradient and the buoyant density of each respective organelle are
substantially equal;
and c) isolating the at least two types of organelles from the density
gradient.
In a still another embodiment of the invention, a method for enriching and
accumulating at least two types of organelles from a biological sample, having
the
steps of a) obtaining the biological sample from tissue or cell material; b)
homogenizing the tissue material or lysing the cell material to form an
organelle
homogenate; c) feeding said organelle homogenate into a continuous-flow
ultracentrifuge having a density gradient; d) applying a centrifugal force
such that at
least two types of organelles migrate and accumulate within the density
gradient;
and e) collecting the at least two types of organelles from the density
gradient so as
to utilize the at least two types of organelles.
lii yet another embodiment of the invention, a method is provided for
accumulating low abundance proteins from a subcellular organelle, having the
steps
of a) releasing the subcellular organelles from a sample comprising the
subcellular
organelles; b) introducing the subcellular organelles to a density gradient
within a
continuous-flow centrifuge; c) applying a centrifugal force such that
subcellular
organelles migrate and accumulate within the density gradient; d) collecting
the
subcellular organelles from the density gradient; e) lysing the subcellular
organelles
to form a proteome suspension; f) collecting the low-abundance proteins from
the
proteome suspension; and g) utilizing the low-abundance protein in a process
selected from the group consisting of selling the low-abundance proteins,
leasing the
low-abundance proteins, licensing the low-abundance proteins, protecting an
intellectual property interest in the low-abundance proteins, placing
information
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about said low-abundance proteins into a database and viewing information
about
the low abundance proteins in a database.
In a still another embodiment of the invention, a method is provided for
purifying and accumulating subcellular organelles from a biological sample
comprising said subcellular organelles, having the steps of: a) introducing
said
biological sample into a centrifuge, said centrifuge comprising a density
gradient
solution adapted to separate into discrete layers, each of said layers having
a holding
capacity; and b) centrifuging said biological sample in a continuous mode to
produce said accumulated and purified subcellular organelles in said discrete
layers
within said density gradient solution, wherein each of the at least two types
of
subcellular organelles migrate within separate discrete layers within said
density
gradient solution, wherein said at least two types of subcellular organelles
are
accumulated at a concentration at or immediately below the holding capacity of
said
at least two discrete layers, and wherein said at least two accumulated
subcellular
organelles are substantially intact.
In a yet further embodiment of the invention, a method is provided for
accumulating subcellular organelles, having the step of using a continuous-
flow
ultracentrifuge to obtain said. subcellular organelles from a biological
sample in
sufficient yield and purity so as to isolate and detect a low-abundance
protein
therefrom.
In another embodiment of the invention, a method is provided for
accumulating at least two different types of subcellular organelles, having
the step of
using a continuous-flow ultracentrifuge to obtain said at least two different
types of
subcellular organelles from a biological sample in sufficient yield and purity
so as to
isolate and detect a low abundance protein therefrom.
In a still further embodiment of the present invention, a method is provided
for analysing proteomic profiles of at least two types of subcellular
organelles as a
function of time, having the steps of: a) releasing the at least two types of
subcellular
organelles from a biological sample at a first time; b) introducing the at
least two
types of subcellular organelles to a density gradient within a continuous-flow
ultracentrifuge; c) applying a centrifugal force such that the at least two
types of
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subcellular organelles migrate within the density gradient; d) collecting the
at least
two types of subcellular organelles from the density gradient; e) isolating
and
purifying proteins from said at least two types of subcellular organelles to
determine
a proteomic profile of said at least two types of subcellular organelles at
said first
time; fJ releasing the at least two types of subcellular organelles from a
second
biological sample at a second time; g) repeating steps b) through d); h)
isolating and
purifying proteins from said at least two types of subcellular organelles to
determine
a proteomic profile of said at least two types of subcellular organelles at a
second
time; and i) analyzing the proteomic profiles at said first and second times
to detect
changes in said proteomic profiles as a function of time.
In a still further embodiment of the invention, a method is provided for
analyzing the translocation process of a translocation protein of a biological
sample,
said translocation process relating to the intracellular movement of the
translocation
protein as a function of time from a first organelle to a second organelle of
said
biological sample, said function of time having at least two time points,
having the
steps of: (a) determining the relative amounts of said translocation protein
in said
first and second organelle of a first biological sample, said first biological
sample
being isolated at a first time point, comprising the steps of: homogenizing
the first
biological sample under conditions sufficient to release said first and second
organelles into a homogenate, said first and second organelles each comprising
a
subcellular proteome, introducing said homogenate to a density gradient within
a
continuous-flow ultracentrifuge, applying a centrifugal force to said
homogenate
such that the first and second organelles migrate within the density gradient,
removing said first and second organelles from said density gradient,
solubilizing
the subcellular proteomes of the first and second organelles, detecting said
translocation protein in the first and second organelles of the first
biological sample,
measuring the level of detected translocation protein in the first and second
organelles of the first biological sample, determining the relative amounts of
said
translocation protein in said first and second organelle of a second
biological
sample, said second biological sample being isolated at a second time point
and
repeating the above steps; and analyzing said translocation process of said
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translocation protein as said function of time by comparing the measured
levels of
said detected translocation protein in the first and second organelles for
each of said
biological samples isolated at each of said time points.
In yet another embodiment of the invention, a method is provided for
obtaining proteins from subcellular organelles and sub-types thereof, having
the
steps of-. a) releasing the subcellular organelles and sub-types thereof from
a
biological sample; b) introducing the subcellular organelles and sub-types
thereof to
a density gradient within a continuous-flow ultracentrifuge; c) applying a
centrifugal
force such that the subcellular organelles and sub-types thereof migrate and
accumulate within the density gradient in a single run; and d) collecting the
subcellular organelles and sub-types thereof from the density gradient and
obtaining
the proteins therefrom.
These and other embodiments of the invention are provided in or are obvious
from the following detailed description of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
The following detailed description given by way of example, but not
intended to limit the invention solely to the specific embodiments described,
may
best be understood in conjunction with the accompanying drawings in which:
FIG. 1 is a flow chart depicting the method of separation and accumulation
of organelles. an embodiment of the invention.
FIG. 2 is a flow chart depicting the method of protein characterization arid
quantitation.
FIG. 3 depicts the percentage of mitochondria, endoplasmic reticulum,
Golgi, and plasma membrane in collected fractions for rat liver.
FIG. 4 depicts the enrichment of mitochondria, endoplasmic reticulum,
Golgi, and plasma membrane in collected fractions for rat liver.
FIG. 5 depicts the percent (%) integrity for preparations of (1) endoplasmic
reticulum (76.3%), (2) mitochondria (72.6%), (3) Golgi bodies
(~9.3°/~), and (4)
plasma membrane (72.7%).
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FIG. 6 depicts transmission electron micrographs comparing the organelle
content and ultrastructure of a crude extract sample of rat liver cells and an
endoplasmic reticulum fraction as prepared by the method of the present
invention.
FIG. 7 depicts the percentage of mitochondria, endoplasmic reticulum,
Golgi, and plasma membrane in collected fractions for HeLa cells.
FIG. 8 depicts the enrichment of mitochondria, endoplasmic reticulum,
Golgi, and plasma membrane in collected fractions for HeLa cells.
FIG. 9 depicts the level of enrichment of a specific organelle by the method
of the present invention.
FIG. 10 depicts the quantitated signals for each fraction shown in FIG. 7.
FIG. l ldepicts the percentage sucrose content for collected post-
centrifugation fractions of homogenized and centrifuged HeLa cells.
FIG. 12 depicts a comparison of 2D gel electrophoresis analysis on the crude
extract (CE) sample and a fraction of endoplasmic reticulum (ER).
FIG. 13 depicts the results of 2D gel electrophoresis analysis of HeLa cell
crude extract, a Golgi fraction, and a plasma membrane fraction.
FIG. 14 depicts the mass spectrometry data for spots 12, 13, and 14 of FIG.
13.
FIG. 15 shows the results of 2D gel electrophoresis analysis of rat liver cell
crude extracts and an endoplasmic reticulum fraction.
FIG. 16 shows the results of 2D gel electrophoresis analysis of rat liver cell
crude extracts and a mitochondria fraction.
FIG. 17 shows the results of 2D gel electrophoresis analysis of rat liver cell
crude extracts and a Golgi fraction.
FIG. 18 shows the results of 2D gel electrophoresis analysis of rat liver cell
chide extracts and an plasma membrane fraction.
FIG. 19 shows a flow chart to provide information pertaining to the method
of the invention to third parties.
FIG. 20 shows a flow chart to protect intellectual property flowing from the
method of the invention.
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FIG. 21A and 21D show results of homology searching using peptide
sequences obtained from the method of the invention.
FIG. 22A and 22D show the detection limit of proteins using 2D-gel analysis
and the estimated amounts of biological material required to reach the protein
detection limit, relative to the protein copy number. FIG 22A and 22D relate
to cells
and tissues, respectively.
These and other embodiments are disclosed, or are obvious from and
encompassed, by the following Detailed Description.
DETAILED DESCRIPTION
As seen in Figures 1 and 2, an embodiment of the invention involves
obtaining a biological sample in the form of a tissue or a cell; homogenizing
the
tissue and/or lysing the cell to provide for a homogenate; optionally
clarifying to
remove certain material, such as, for example, nuclei; feeding the homogenate
into a
continuous-flow ultracentrifuge having a density gradient therein; applying a
centrifugal force to the homogenate to separate and accumulate intact
organelles;
collecting the organelles; and using the organelles in further downstream
processes.
One such downstream process involves obtaining low-abundance proteins from the
organelles by lysing the organelles to release the proteome; separating and
accumulating the low-abundance proteins therefrom; characterizing,
quantitizing
and, if possible, identifying the low-abundance proteins; and using the low-
abundance proteins in further downstream processes.
Obtaining the sample. As seen in Figure 1, the method of the invention can
be applied to any biological sample known to one of ordinary skill in the art,
or any
sample comprising a biological sample, isolated or obtained from any source
using
any method known to the spilled artisan.
For the purposes of the invention, a "biological material," which can have
the same meaning as a "biological sample," "biological specimen," or
"biological
substance," or any other similar variation known to a spilled artisan, refers
to any
type of biological material known to one of ordinary skill in the art,
including, for
example, whole cells, cellular extracts, tissues, homogenized cells or
tissues, protein
solutions, subcellulax structures, such as, for example, organelles and
organelle
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subtypes, or any other material that one of ordinary skill in the art would
consider to
be a biological material. A biological material can also be any solution,
mi~~ture,
suspension, substance, buffer, or any of the like, such as, for example, a non-
biological solution, such as, for example, a phosphate buffer, that comprises
a
biological material added thereto, such as, for example, an organelle or
organelle
subtype. hilore specifically, a biological material of the invention can be
obtained
from any known source, living or dead, such as, for example, an organ, bodily
fluid,
blood, serum, plasma, saliva, tears, feces, urine, semen, mucous, tissue,
tissue
homogenate, cellular extract, or spinal fluid, derived from any knowxn
organism or
part thereof or virus, including, but not limited to, for example, any
prokaryote or
eukaryote; vertebrate or invertebrate; or any organism, such as, for example
an
animal, a mammal, a human, a bird, a horse, a fish, a rodent, an insect, or
plants, etc.
or any combinations thereof.
In one embodiment, the biological sample is a cell. A "cell," in accordance
with the present invention, is meant in the ordinary biological sense as the
smallest,
membrane-bound body capable of independent reproduction. In a broader sense,
cells can be either eukaryotic or prokaryotic. In addition, it will be
appreciated that
a cell can be obtained from a multicellular organism, a tissue, a cell or
tissue culture,
a virus-infected cell in a cell culture, or from any biological sample. It
will be
further appreciated that a cell, especially a eukaxyotic cell, contains
subcellular
structures, including, for example, organelles and other subcellular
structures.
"Organelle" and "subcellular organelle," which have the same meaning in
the invention, are understood by one of ordinary skill in the art in the
ordinary
biological sense. An organelle includes any type of complex structure that
forms a
component of a cell and typically performs a characteristic function. The
invention
contemplates any organelle from my biological sample known to one of ordinary
skill in the art, such as, for exaanple mitochondria, chloroplasts,
peroxisomes, Colgi
apparatus, endoplasmic reticulum, nuclei, proteosomes, ribosomes, and others,
including, any known or unknown sub-types of organelles, such as, for example,
smooth and rough mitochondria, early and late endoplasmic reticulum, or any
sub-
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type or sub-population of a particular organelle that would be understood or
discoverable by one of ordinauy skill in the art.
In accordance with the present invention, an organelle "sub-type" or 'gsub-
population99 can refer to a sub-portion of a particular organelle population
in a cell
that is distinct in some manner from the remainder of the same type of
organelles of
that same population. For example, organelle sub-types include differences
based
on, for example, the overall size and shape of the organelle, the density of
the
organelle, the characteristic protein population that is expressed, the
composition of
the organelle membrane, or any other physiological or morphological
distinction that
would be known to the skilled artisan. Some organelles contain membranes,
which
are called "organelle membranes."
It is also understood that organelles have, for example, characteristic sets
of
biomolecules, in particular, characteristic sets of proteins that make up
subsets of the
whole protein complement of a cell as subsets of the whole proteome of a cell.
The
subset of proteins associated with a subcellular stnicture, such as, for
example, an
organelle, or those proteins forming a subset of the entire protein complement
of a
cell, tissue, or genome can be referred to as a "subcellular proteome." In the
particular case of an organelle, the subcellular proteome associated with the
organelle-specific proteins-those proteins that are contained within and/or
directly
or indirectly bound, integrated, or attached to the organelle membrane-can be
referred to as the "organelle proteome." An organelle subtype can have a
proteome
that is unique in its composition such that it can be distinguished from the
proteome
that is formed from the combination of some or all of each of the remaining
sub-
types of organelles comprising the organelle. '
One skilled in the art will appreciate that the coining of the term "proteome"
is generally given credit to Marc Wilkins of Macquarie University (Australia),
who
defined the proteome as "all proteins expressed by a genome, cell or tissue."
For
example, and for the purposes of the present invention, the term proteome
refers to
the entire protein complement and includes all of the e~~pressed proteins, of
a
genome, cell, tissue, or organelle. As such, the proteome can be thought of as
a
dynamic collection of proteins expressed by a genome, cell or tissue that can
change
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in accordance with a variety of different factors, such as, for example, the
growth
and/or differentiation stage of a cell, internal and external environmental
factors,
disease factors, and any other factors known to the skilled artisan.
hl some cases, it will be appreciated that certain organelles, such as, for
example, mitochondria and chloroplasts, contain their own chromosomes which
can
express some of the proteins associated with the chromosome-containing
organelle.
However, the skilled artisan will understand that the majority of proteins
that
constitute an organelle proteome are expressed by the cell's chromosomes and
are
transported into the organelle of interest vis-a-vis a variety of mechanisms,
such as,
for example, translocation and vesicular delivery.
For the purposes of the invention, the term "proteomics" refers to the effort
to establish the properties including, for example, identities, quantities,
structures
and biochemical and cellular functions, of all the proteins in an organism,
organ,
tissue, extracellular space, cell, or organelle, or any combination thereof,
and how
these properties vary in space, time and physiological state. It will be
further
appreciated that proteomics investigates the nature of cellular processes
through the
characterization of the many defining properties and behaviors of proteins,
such as,
for example, protein expression profiles, post-translational modifications,
intracellular localizations, and protein-protein interactions, with a view to
space,
time, and physiological state. Froteomics includes not only the identification
and
quantification of proteins, but also the determination of their localization,
modifications, interactions, activities, and, ultimately, their function.
Homogenizing/lysing the biological sample. Refernng again to Figure 1,
once the biological sample is obtained, the biological sample is homogenized
and/or
lysed. The product of the homogenization step is typically referred to as a
homogenate. A homogenate is meant to have the same meaning as recognized in
the
art. Tlaus9 a homogenate is the form of the biological sample following
homogenization and/or lysing of the biological sample. The process of
homogenization and/or lysis is further explained below.
The methods and materials used for homogenization and/or lysis are
generally known in the art. In accordance with the present invention, the term
14
CA 02518555 2005-09-14
WO 2004/083405 PCT/US2004/008655
"homogenization" and related terms, such as, for example, homogenize or
homogenizing, can refer to any of a variety of techniques used by one ~f
ordinary
skill in the art to achieve the disruption of tissues into smaller and more
uniform
components, such as cells and extracellular material comprising the tissue.
For
example, homogenization of a tissue can refer to the breaking up of the tissue
into
individual cells such that the cells become separated and/or detached from
each
other and from any extracellular material. The terms homogenization and/or
lysis
can also refer to the step of disrupting cells, for example, cells of a
tissue, into their
subcellular components. Thus, in accordance with the present invention, the
homogenized tissues or homogenized and/or lysed cells can result in the
release of
the subcellular components, including, for example, the organelles. By
"release" of
intracellular components from the cell, such as, for example, organelles, it
is meant
that the intracellular components no longer remain confined by a cellular or
plasma
membrane.
One of ordinary skill in the art will appreciate the variety of approaches
available to carry out the disruption of a tissue and/or cell. It will be
appreciated that
lysis and/or disruption can result in the disruption of the cellular membrane
such that
the intracellular components, such as, for example, organelles, are released.
The
homogenization and/or lysis conditions can be adjusted so that the cellular
membrane is disrupted while minimizing the disruption of the organelle
membranes.
Methods for adjusting these conditions to achieve the lysis of the cellular
membrane
while minimizing the lysis of the organelle membranes are known and can be
found,
for example, in Current Protocols in Cell Biolo~y (1999), Ed. J.S. Bonifacino
et al.
and Subcellular Fractionation: A Practical Approach, (1997), Ed. J.M. Graham
et al.
The present invention contemplates any technique for homogenizing and/or
lysing a biological sample known or that will become available to one of
ordinary
skill in the art, such as, for example, any chemical-based, mechanical-based,
pressure-based, or temperature-based technique. For example, such methods can
include applying a liquid shear force to the cells and/or tissue by passing
the cells
and/or tissue through the narrow annulus of a ball-bearing and a metal block
in a
syringe ("ball-bearing homogenizer"); forcing the cells and/or tissue under
high-
CA 02518555 2005-09-14
WO 2004/083405 PCT/US2004/008655
pressure through a small orifice; exposing cells and/or tissue to nitrogen gas
under
high pressure and then forcing through a needle valve, such as, for example, a
syringe valve; sonicating the cells to disrupt the cell membrane; contacting
the cells
and/or tissues with detergents, such as, for example, Tween-20 or sodium
dodecylsulfate ("SIBS"); contacting the tissue and/or cells with a solution
that
provides osmotic stress, such as, for example, an isoosmotic medium,
hypoos111ot1c
medium, such as, for example, a sucrose solution of 0.1 Molar; and applying
shear
forces, such as, for example, introducing the tissues and/or cells into a
tissue blender
(such as a WaringTM blender, blaring Laboratory, CT); or any combination of
the
above methods or any other additional methods known to the skilled artisan.
One of ordinary skill in the art will appreciate that specific sources and/or
types of tissues from any biological material (such as, for example, heart,
pancreas,
glands, muscle, bone, kidney, skin, liver, lung, brain, or blood, or other
organ, and
specific sources of cells, including, for example, tissue culture cells, cell
culture
cells, or any type of cell in suspension) can be homogenized in accordance
with a
technique or procedure that is designed for a particular tissue and/or cell.
For
example, liver cells may have a homogenization method that is designed for the
homogenization or lysis of that particular type of cell. Information on the
many
techniques of cell and tissue homogenization and/or cell lysis can be found in
commercially-available handbooks, such as, for example, Sambrook J. et al.,
Molecular Cloning: a Laboratory Manual, 2"a edition, 1989, Cold Spring Harbor
Laboratory Press.
The term "substantially intact" refers to the relative degree of integrity of
the
subcellular components, especially the organelles, at any point during the
method of
the invention, including the point at which the organelles are released from
the cells
and/or tissues following homogenization and/or lysing or during or after the
continuous-flow process, such as, for example, the continuous-flow
centrifugation
process, or at any other point during the method of the invention. Whether the
organelles are substantially intact can be determined by any known method to
one of
ordinary skill in the art, such as, for example, by quantitative enzymatic
assays of
organelle-specific markers, Western blots to organelle-specific markers, or by
visual
16
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WO 2004/083405 PCT/US2004/008655
inspection using microscopy, such as, for example transmission electron
microscopy
(TEI~). In the use of microscopy, the skilled artisan will appreciate the
morphological characteristics and features of any and all types of organelles
from
any biological source and/or cell or tissue type and will understand how to
judge
whether a given organelle is intact based on the particular morphological
characteristics.
In one embodiment, organelle integrity can be enzymatically measured. For
example, an organelle preparation of interest, such as, for example, a
preparation of
mitochondria according to the inventive method, can be centrifuged to pellet
the
insoluble portion, which can comprise intact organelles and portions thereof,
such
as, for example, organelle fragments. The supernatant contains any soluble
components, including any soluble proteins and/or enzymes, released from a
fragmented organelle of interest. Next, the relative levels or quantities of
an
organelle-specific marker, such as, for example, an enzyme that is particular
to a
given organelle of interest, can be measured with respect to both the
organelle pellet
and the remaining supernatant fraction. It will be appreciated that the
pelleted
organelles may have to be lysed prior to measuring or detecting the organelle-
specific marker.
One of ordinary skill in the art will appreciate that different subcellular
organelles will have different and distinct "organelle-specific markers" that
can be
detected, assayed or probed with an antibody in order to determine the
enrichment
factor of a particular organelle. For example, cytochrome-c oxidase and/or
Tom20
(l8kDa) can be used to detect mitochondria; beta-hexosaminidase and/or beta-
galactosidase can be used to detect lysosomes; peroxidase can be used to
detect
endosomes; alkaline phosphodiesterase I and/or NaKATPase (150 kDa) can be used
to detect plasma membrane; alpha-mannosidase II and/or G1VI130 (130 kl~a)
and/or
Pl 15 (115 kDa) can be used to detect the Golgi apparatus; catalase can be
used to
detect peroxisomes; lactate dehydrogenase can be used to detect the cytosolic
fraction; and l~TA and/or BiP/GI~P78 (78 kDa) can be used to detect rough
endoplasmic reticulum. Preferably, antibodies against mitochondrial-specific
Tom20 (18 kDa), endoplasmic reticulum-specific BiP/GRP78 (78 kDa), plasma
17
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WO 2004/083405 PCT/US2004/008655
membrane-specific IVaKATPase (150 kDa), Golgi-specific GIeiI130 (130 kl~a),
and
Golgi-specific P115 (115 kI~a) can be used to detect and quantify the presence
of the
specific organelles in the fractions of the centrifuged biological samples of
the
present invention using any suitable means known to the skilled artisan, such
as, for
example, western blotting and immunoblotting. These antibodies can be obtained
from commercial sources, such as from EI~ >3I~SCIENCES (C~), STI~ESSGEI~T
(Victoria, ~C Canada), and from academia.
Thus, integrity is assessed by separating an organelle preparation into
soluble
(supernatant) and insoluble (solid pellet) fractions, assaying or detecting am
organelle-specific marker in both fractions, and then comparing the relative
levels or
quantities from both fractions. Generally, it will be appreciated that the
higher
relative level of quantity of the organelle-specific marker contained in the
insoluble
fraction (as compared to the soluble fraction) corresponds to a higher degree
of
organelle integrity. Preferably, the invention contemplates equal to or
greater than
about 60%, 70%, 80% or over 90% intactness of the organelles at any stage of
the
inventive method prior to the stage of lysing the organelles.
In one embodiment, organelle integrity can be calculated by dividing the
relative quantity of the organelle-specific marker measured for the insoluble
fraction
by the sum of the relative quantities of organelle-specific marker in both
fractions
multiplied by 100 to yield a percent (%) intactness (or integrity). For
example, a
preparation of mitochondria can be centrifuged to form a pellet of
mitochondria (and
fragments of mitochondria such as those mitochondria that have been disrupted
and/or lysed thereby releasing the intra-organelle soluble materials, such as,
for
example, soluble mitochondria) proteins, including a mitochondria)-specific
marker)
and a supernatant comprising soluble components of disrupted and/or lysed
organelles. The relative level of mitochondria)-specific protein and/or enzyme
(mitochondria) marker, such as Tom20) can then be determined for both the
soluble
and the insoluble fractions. Percent integrity can then be calculated by
dividing the
quantity of mitochondria) marker in the insoluble fraction by the sum of the
mitochondria) marker quantities of both the insoluble and soluble fractions
l~
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WO 2004/083405 PCT/US2004/008655
multiplied by 100 to obtain a percentage that reflects the relative portion of
the
mitochondria) preparation containing intact mitochondria.
It will be appreciated by the skilled artisan that a buffer is generally used
during the homogenization process. The invention contemplates any suitable
buffer
known to one of ordinary skill in the art including, for example, detergents,
such as~
for example, Triton-X, sodium dodecylsulfate (SDS), and the like, salts, such
as, for
example, sodiwn chloride, proteinases, such as, for example, proteinase K,
inhibitors
of DNA and RNA degrading enzymes, and any other additional components suitable
for use in a homogenization buffer. The skilled artisan will appreciate that
the
composition of the buffer can depend on the type and/or source of biological
sample.
Optional clarification step. Referring again to Figure 1, a cell andlor tissue
homogenate of the biological sample, wherein~the homogenate comprises intact
organelles,is typically "clarified" to remove certain intracellular
components, such
as nuclei. Nuclei, typically blockother components in a sample, such as, for
example, other organelles, from entering the gradient. Thus, the nuclei can be
removed from the sample prior to the continuous-flow centrifugation process of
the
invention.
Any method suitable for the removal of the nuclei is contemplated by the
instant invention, including, but not limited to, centrifugation. For example,
to
clarify a homogenate using a centrifuge, any centrifuge known to one of
ordinary
skill in the art, such as a batch or analytical centrifuge at an appropriate
relative
centrifugal force (RCF) (x g), , such as, for example from about 500 x g to
about
40,000 x g can be used. The centrifuge separates, for example, the nuclei by
applying a centrifugal force to the homogenate to cause the nuclei, but not
the
remaining organelles, to migrate towards one end of the centrifuge tube, for
example, towards the bottom of a centrifuge tube. In one embodiment, a low-
speed
clarification centrifuge known in the art can be used to clarify the
homogenate. The
low-speed clarification centrifuge can be a continuous-flow centrifuge.
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WO 2004/083405 PCT/US2004/008655
ECen~~ffuge. As seen in Figure 1, once the biological sample is homogenised
and/or the cell is lysed, the homogenate and/or product derived therefrom is
introduced to a density gradient within a continuous-flow centrifuge.
For the purposes of the present invention, a "continuous-flow centrifuge" is a
type of centrifuge or ultracentrifuge that can have a rotor with an inlet and
generally
an outlet wherein a sample material can be introduced into the rotor through
the
inlet, allowed to contact a gradient while in the rotor, and allowed to exit
through the
outlet. A continuous-flow centrifuge can encompass a semi-continuous-flow
centrifuge.
Ayly known configurations of the continuous-flow centrifuge and the
continuous-flow centrifuge rotor are contemplated by the present invention.
For
example, the continuous-flow rotor can have an inlet or an inlet and an outlet
such
that a sample can be continuously or intermittently introduced through the
inlet and
continuously or intermittently released through the outlet. The rotor can also
have
an inlet without an outlet, allowing the sample to be continuously introduced
into the
rotor, but not continuously released. Where the rotor is spinning, a gradient
can be
pre-formed or pre-established. The sample that is released from the outlet can
also
be continuously or intermittently recirculated or reintroduced into the rotor
through
the inlet to provide multiple "passes" of the sample material over the
gradient. The
invention contemplates any number of passes over the gradient sufficient to
enrich
and accumulate the organelles.
The gradient can be removed from the rotor of the continuous-flow
centrifuge at the end of a run while the rotor continues to spin. In its
place, a fresh
gradient material can be added into the moving rotor. Once the new gradient is
established in the rotor, another biological sample, such as the homogenate of
another biological sample, can be introduced into the rotor and allowed to
contact
the gradient. In this sense-where the operation of the continuous-flow
centrifuge is
such that a first gradient, having a first biological sample separated
therein, is
removed while the rotor is spinning or rotating and replaced with a fresh
volume of
gradient material while the rotor continues to spin for the separation of a
second
biological sample-is termed "continuous-flow mode." Continuous-flow mode is
CA 02518555 2005-09-14
WO 2004/083405 PCT/US2004/008655
not limited to adding and removing only a first and second gradient, but
rather, any
number of gradients can be successively added a~ld removed from the centrifuge
rotor to separate any number of biological samples in succession all while the
rotor
continues to spin, e.~., without having to shut down or stop the rotor of the
centrifuge.
The invention further contemplates that a biological sample of the invention,
such as a homogenate of a~biological sample, can be loaded into the continuous-
flow
centrifuge in a manual, automatic, or semi-automatic manner. For example, a
robotics system, including any appropriate sensors or electronics, can be
employed
in a suitable manner to achieve the automatic or semi-automatic loading of the
biological sample into the rotor of the continuous-flow centrifuge. In
addition to the
loading of the biological sample, the gradient material can also be loaded
into the
rotor of the continuous-flow centrifuge in a manual, automatic or semi-
automatic
manner and can employ any suitable robotics, sensors, electronics, or
computers
systems and/or software for the controlling and/or programming of the
automated or
semi-automated systems.
Examples of suitable continuous-flow centrifuges are those manufactured by
Alfa Wassermann, Inc. (West Caldwell, NJ) including, but not limited to,
models
KII, PKII and RK Some representative rotor models include, but are not limited
to,
AW K3-3200, AW PK3-1600, AW PK3-800, AW PK3-400 , AW PK3-200, and
AW PK3-100. Rotors of higher and lower volume are contemplated to fall within
the scope of the invention.
Other continuous-flow centrifuges can be utilized by the invention. These
include, for example, Beckman CF32Ti, Beckman JCF-Z-standard core, Beckman
JCF-Z small pellet core, Beckman JCF-Z large pellet core, Beckman Z60, Sorvall
SS34/KSB, Sorvall TZ-28/GK, Sorvall TCF-32 (P32CT with 940 ml core), Sorvall
TCF-32, and those manufactured by Hitachi, such as, for example, centrifuges
CC40, CP40Y, C40CT2-H, C40CT and CP60Y. The Hitachi centrifuges are
distributed by Kendro.
In another embodiment, the continuous-flow ultracentrifuge is a rate tonal
ultracentrifuge. Zonal rotor assemblies have been used for many years and
21
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WO 2004/083405 PCT/US2004/008655
considerable literature is available on the subj ect. Information about zonal
rotors is
included in most purification handbooks and biochemistry texts. Specific
information can be found in Anderson, An IfatYOduetiora to Pazftiele ~'ePaf-
atioaas in
tonal Centnifu~es (National Cancer Institute Teilonograph No. ~ 1, 1966);
Anderson,
Separation of bS"ub-Cellulaf~ Cofnponents and T~ir-uses by Cotnbinecl leate
and
Isop~enie tonal Cent~-ifu~atiora (National Cancer Institute Monograph No. 21,
1966); and, Anderson, Preparative Zonal Centv~ifu~ation, in lVlethods of
biochemical Analysis (1967), all of which are incorporated herein by
reference.
For the purposes of the invention, a centrifuge "run" refers to the moment
when a sample is added to a rotor, either with the rotor already in motion and
having
a preformed gradient or with the rotor stopped, until the sample is processed
by the
centrifuge, including any number of passes for example, one pass (no
recirculation
of sample), two passes (sample is recirculated once), three passes (sample is
recirculated twice), etc. The passes can be carried out such that the rotor is
not
stopped or slowed. Further, the sample can also be continually recirculated
for any
period of time. It is also contemplated that a centrifuge run can occur at a
constant
or variable speed.
In an embodiment, the centrifuge run utilized by the invention can be a
single run. For example, the migration, separation and accumulation of the
subcellular organelles and subtypes thereof are performed in one centrifuge
run.
Typically, preparation for and conducting a continuous-flow ultracentrifuge
run is either manually performed, automated, for example, by a computer, or a
combination of both manually performed and automated. Preferably, computers
and
software are utilized for controlling the centrifuge and calculating a
centrifugation
protocol. Such computers and software provide the operator with operating
parameters displayed in "real-time" on a control screen. ~iutomated programs
can
also be run from pre-stored files, or manually through a control scr een.
In an embodiment, during each centrifuge run, on-line data monitoring and
recording of set parameters, run parameters, and alarm status are made and are
down-loaded to the system memory. Such downloading may also be directed to an
extenial data storage location.
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WO 2004/083405 PCT/US2004/008655
A separation protocol, computer-automated, manually-performed, or a
combination of both, typically involves manipulation of a number of variables.
Such variables include, for example, the physical characteristics of the
target
organelle; formation of the gradient; and the calculation of run parameters.
The physical characteristics of the target organelle useful for defining a
separation protocol include, for example, the sedimentation coefficient (Sao,)
and
buoyant density of the target organelle. Such values are useful, for example,
for
reducing the number of trial and error experiments. (See, Rickwood et al.,
Centrifugation Essential Data, BIOS Scientific Publishers Limited 1994,
Publisher J
Wiley & Sons; Preparative Centrifugation: A Practical Approach, Edited by D
Rickwood, Oxford University Press 1921; and Methods in Enzymology, Vol. 182:
Guide to Protein Purification, Edited by Murray P. Deutscher, Academic Press
1990).
The separation protocol also typically involves knowledge of the gradient. A
gradient can include, but is not limited to, a density gradient. The density
gradient,
in turn, can be, for example, a continuous gradient, a discontinuous gradient
or a
step gradient. The choice of gradient material depends on, for example, the
product,
impurity stabilities and product densities. Commonly used gradient materials
include any suitable gradient material known to one of ordinary skill in the
art and
that can be obtained commercially or prepared by the skilled artisan. Gradient
materials include, but are not limited to: an alkali metal solution, such as,
for
example, cesium chloride (CsCI), cesium sulfate (Cs2S04), potassium tartrate,
or
potassium bromide; nonelectrolyte solutes, such as, for example, sucrose,
mannitol,
or glycerol; polysaccharides, such as, for example, Ficoll~ 400 (Pfizer, CT);
iodinated nonelectrolytes, such as, for example, metrizamide, IVycodenz~
(Nycomed, Inc., NJ), Iodixanol~, or Optiprep~; Percoll~ (colloidal silica
coated
with polyvinylpyrrolidone) (Pfizer, CT), or any other suitable material known
to one
of ordinary skill in the art.
It will be appreciated that the gradients comprised of alkali metals, although
corrosive, can create high densities with low viscosity. For example, cesium
chloride, which is frequently used as a gradient material, can achieve high
density
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CA 02518555 2005-09-14
WO 2004/083405 PCT/US2004/008655
that is typically up to approx. 1.9 g/cm3. In another example, potassium
bromide
can also form 111gh de11S1t1eS, but only at elevated temperatures, e.g.
25° C. Such
elevated temperatures may be incompatible with the stability of the proteins
of
interest.
Examples of gradients mentioned above include Nycodenz~, ~ptiprep'~,
Iodixanol'~ and sucrose. Sucrose is a cost-effective gradient material and
utilizes a
sufficient density range for most operations (up to approx. 1.3 g/cm3). The
viscosity
of a sucrose gradient allows for the formation of a steep gradient used for
banding
product, or, alternatively, to create a wide product capacity in the same
rotor. The
steep gradient is typically efficient for a continuous flow operation if, for
example,
entry of the non-target protein is to be minimized. The viscosity of sucrose
is also a
desirable attribute to forming steep gradients for long periods of time in a
continuous
flow rotor. By contrast, a low-viscosity solution, such as CsCl, may need the
addition of a higher-viscosity material, such as glycerol, to increase
viscosity and
minimize gradient erosion during a continuous-flow run.
The invention contemplates using any type of gradient having any
concentration profile. The "concentration profile" will be known by the
skilled
artisan as the variation in the concentration of the gradient medium or
material along
a path perpendicular to the gradient in the horizontal, vertical, diagonal, or
any
direction there-between. As such, the gradient can be a "linear gradient," a
"convex
gradient," a "concave gradient," or a "discontinuous gradient," or any other
suitable
form known to the slcilled artisan.
Sucrose is a preferred density gradient material. Table 1 describes the
theoretical separation requirements for the separation of mitochondria,
endoplasmic
reticulum, plasma membrane, and Golgi apparatus contained in a homogenized
biological sample using sucrose density gradients.
Tr~bd~ 1. TITeo~~etical sepczt-cztion t-eqasi~eynerats f~r Tiona~~enized
bi~Z~~i~czl
sample.
Component ~ Amount I Banding in I Density range Separation
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~~cr ~se~ ~~a2~iti~n
Mitochondria16% of cell 42.5% 1.19 g/cm3 5,000 xg, l
Omin
protein 1.17-1.21 g/cm3 10,000xg 25
min
16% of cell
protein
Endoplasmic5% of cell 37%*"~ 1.16 g/cm3*~* 100,000xg,
protein 120 min
Reticulum 24% of cell 1.06-1.23 smooth 150,000 xg
50 min
protein 1.18-1.23 rough
Plasma 2% of cell 37% 1.16 g/cm3 80,000 xg,
protein 60 min**
Membrane 0.4_2.5%of 1.12-1.14 100,000 xg
60 min
homogenate
Golgi 1% of cell 33 to 36%**1.14 to 1.15 g/cm3100,000 xg,
protein 55 min
1.12-1.16 150,000 xg
20 min
* Derived from the density data using sucrose tables
** based on a step gradient
*** based on banding similarity to plasma membrane
As described above and defined herein, a continuous-flow centrifuge run can
include a number of passes. For example, a homogenized biological sample can
be
passed twice through the continuous-flow centrifuge of the invention. The
first pass
can be carried out at 20,000 RPM in a PK-3-800 rotor using a flow rate of 20
ml/min
(1.2 L/hr). As such, the materials over 487 Svedberg's (S) are expected to
enter the
gradient. The following parameters can be used for the first run:
G force core 24.,379 xg
bowl 29,562 xg
I~ factor 121.94.
Time to pellet 15.00 min
Transient time 20.00 min
CA 02518555 2005-09-14
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Svedberg value 4.875
The second pass in turn can be can-ied out 4.0,000 RPM. As such, the
materials over 1225 were expected to enter the gradient. The following
parameters
can be used for the second run:
G force core 97,515 xg
bowl 118,250 xg
I~ factor 30.49
Time to pellet 15.00 min
Transient time 20.00 min
Svedberg value 122 S
Alternatively, the second pass can be carried out at 35,000 RPM in a PK-3-
800 rotor using a flow rate of 20 ml/min (1.2 L/hr). As such, the materials
over
1595 are expected to enter the gradient. The following parameters can be used
for
such an alternative pass:
G force core 74,660 xg
bowl 90,535 xg
K factor 39.82
Time to pellet 15.00 min
Transient time 20.00 min
Svedberg value 159 S
The length of time used to carry out the centrifugation at a particular RPM
value determines whether a particular material will pellet out, which in turn,
typically depends on the Svedberg value of the material. For example, using
the
PIE-3-800 rotor at 35,000 RPM, the material over 53S typically pellets out in
45
minutes. In the case of 120 minutes, the material over 19.95 typically pellets
out. In
both instances, the I~CF values at the core and bowl would be 74,660 xg and
90,535
xg, respectively.
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WO 2004/083405 PCT/US2004/008655
used on the known theoretical sedimentation ranges of the organelles, for
example, mitochondria, plasma membrane, endoplasmic reticuhtm, and Colgi
apparatus, as shown below, the time required for pelleting can be estimated.
For
example, the known sedimentation ranges of mitochondria, plasma membrane,
endoplasmic reticulum, and (aolgi apparatus are as follows: 10,000 to 50,000
S; 50
t~ 1,0 an 0,00~ to 500,00~ S, 1 t~ 5,~00 S; and 1,00~ t~ 1~,000 S,
respectively.
The time needed to pellet out an organelle at different speeds can be
determined. For example, based on centrifugation at 20,000 RPM in the PIE-3-
800
rotor at a 20 ml/min sample flow rate, the times to pellet the following
components
are shown in the following table:
Component Svedberg constantTime to pelletCapture rate
(min)
Mitochondria 10 000 S 0.73 100%
Mitochondria 50 000 S 0.15 100%
P.M. 50 S 146.33 0%
P.M. 1 000 S 7.32 100%
P.M. 100 000 S 0.07 100%
P.M. 500 000 S 0.01 100%
E.R. 1 S 7316 0%
E.R. 5 000 S 1.46 100%
Golgi 1000 S 7.32 100%
Golgi 10 OOOS 0.73 100%
At 35,000 RPM in turn, the times to pellet the following components in a
PIE-3-800 rotor at a 20 ml/min sample flow rate are as follows:
~'Lo~nponent ~vcdberg constantTimc to pelletCapture rate
(min)
P.M. 50 S 224.29 0%
E.R. 1 S 11214 0%
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WO 2004/083405 PCT/US2004/008655
Alternatively, at 40,000 RPM, the times to pellet the following components
in a PIE-3-800 rotor at a 20 ml/min sample flow rate are as follows:
(Component ~veclber g c~n~tantTime t~ pelletCapttare rate
(min)
P.I~L. 50 S 36.58 0/~
E.lf~. 1 S 1829 0%
The time to band a particular component having a particular Svedberg
constant can be determined. For example, predictions can be made based on
centrifugation at 35,000 RPM using a first pass of 45 min and a second pass of
120
min in a PK-3-800 rotor as seen in the table below. The table also shows
whether
the banding is completed after the 45 min and 120 min passes.
Component Svedberg constantTime to band Banding complete
(min) 45min / 120
min
Mitochondria 10 000 S 0.24 Yes / Yes
Mitochondria 50 000 S 0.05 Yes / Yes
P.M. 50 S 47.78 No / Yes
P.M. 1 000 S 2.39 Yes / Yes
P.M. 100 000 S 0.02 Yes / Yes
P.M. 500 000 S 0.005 Yes / Yes
E.R. 1 S 2389 No / No
E.R. 5 000 S 0.48 Yes / Yes
Golgi 1000 S 2.39 Yes / Yes
Golgi 10 OOOS 0.24 Yes / Yes
In one embodiment, the continuous-flow e.~ltracentrifuges contemplated
herein can be used with different size rotors with differing geometries so as
to
provide for a scalable separation. For e~cample, the continuous-flow
ultracentrifuge
of the invention can be configured with different size rotors, such as, for
example, a
15-inch or 30-inch rotor. It will be appreciated that the geometry of the
rotor used in
28
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WO 2004/083405 PCT/US2004/008655
the instant invention can affect the volume of the sample that can be
processed, the
narrowness of the sedimentation path, and the total resistance time required
f~r
separation. Fuz-ther, the continuous-flow ultracentrifuge rotors contemplated
by the
invention can operate in a "reorienting gradient pattern" wherein the gradient
moves
from loading position (horizontal position) to operational position (vertical
position)
and back to the loading position to allow for product collection. During use
of the
rotors contemplated by the invention, the flow path of the sample material can
enter
the rotor at either end (top or bottom end) through a center port of the core,
which
then can flow through long thin tubular shafts to exit at attached product
lines or
tubes.
In another embodiment, a scale separation is performed using the same rotor
length but changing the configuration of the rotor core to either reduce or
increase
volume. For example, as described in co-pending U.S. Application Serial No.
09/995,054, incorporated herein by reference, the method typically involves
using
cores of different designs, such as those having radially projecting "fins."
In an
embodiment, varying the dimensions of the fins modulates the volume displaced
by
a rotor core. For example, scale down is usually achieved by maximizing the
fin
size, thereby reducing the volume available for a centrifuge run. Scale up, in
turn, is
typically obtained by minimizing the fin size, thereby allowing for more
volume in
the centrifuge run.
In order to carry out a scale separation utilizing different sized rotors,
such as
those manufactured by, for example, Alfa Wassermann, Inc., a number of .
parameters are typically considered. These parameters include, but are not
limited
to, the Rm~ of the bowl, Rm;" of the core, xg-force at the bowl, xg-force at
the core,
time to pellet, transient time, I~ factor and sample flow rates. Such
parameters can
depend upon the Svedberg value of a particle being separated.
For example, the separation parameters for a particle of 1,000 S are
described below for a rotor, such as those manufactured by Alfa Wassermann,
Inc.
The rotor Rmax (maximum radius) in centimeters, rotor Rm;n (minimum radius) in
centimeters, and the ultracentrifuge (UCF) rotor maximum speed (rpm) are
typically
known and are specified by the manufacturer of the rotor and are incorporated
herein
29
CA 02518555 2005-09-14
WO 2004/083405 PCT/US2004/008655
by reference. Its also known to a skilled artisan that the rotor volume (rnl)
and the
maximum flow rate (L/hr or ml/min) of the rotor are readily available from the
manufacturer and axe incorporated herein by reference.
A parameter that is calculated is the rotor relative centrifugal force (RCF)
(xg). (see Rickwood, 1994.). RCF can be calculated using the following
eduation:
RCF = 11.18 x R x (Q/1,000) 2, where RCF = relative centrifugal force (xg), R
=
radius (cm), and Q = speed (revolutions per minute). For example, a particle
of
1,000 S can be separated in a PK3-800 rotor based on the following parameters:
RmaX 6.6 cm, Rm;n 5.45 cm, Rotor maximum speed 40,500 rpm. The calculation is
as
follows:
RCF = 11.8 x 6.6 x (40,500/1,000)2
RCF = 73.788 1,640.25
RCF = 12,1030.76 xg
RCF = 121,000 xg
Likewise, for a rotor having a Rm;" value of 5.45 cm, the RCF can be
calculated as 99,900 xg.
Another parameter that is calculated is the duration of the nm, which is a
function of the K factor. The duration of the run is typically referred to as
"run
time" or "time to sediment." In order to determine the duration of the run for
a
1,000 S particle, the K factor of the rotor can be determined from the
literature or
calculated as follows:
K = 2.53 ~ 1011 LN (R~~/RM~)
~2
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WO 2004/083405 PCT/US2004/008655
For example, the K factor of a PK3-800 rotor (Rma~ 6.6 cm, I~m;n 5.45 cm) for
a 1,000 S particle and a rotor maximnn speed of 4.0,500 I~I~I can be
calculated as
follows:
K = 2.53 ~ 1011 LN (6.6/5.45)
4~0 500
K = 2.53 ~ 1011 LN (6.6/5.45)
40 5002
154.244 0.19145
K = 29.53.
K can be also calculated for altenlate speeds. For example, at speeds of
35,000 rpm or 20,000 rpm, the following formula is typically used:
Knew = K(Qmax/Qnew)Z
~max - rotor maximum speed (revolutions per minute)
Qnew - new set speed (revolutions per minute).
Thus, to calculate the K factor at a speed of 20,000 rpm:
Knew = K(Qmax/Qnew)2
Knew = 29.53(40500/20000)2
Knew = 29.53 x 4.100
Knew = 121.
Similarly, the K factor for the set speed of 35,000 rpm is calculated as 39.
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Upon deternlination of the K factor, the run time can then be calculated. For
e~ample9 sedimentation time (T) can be calculated as follows:
T = ISIS
T - time to sediment (hours)
S - sedimentation coefficient (S).
Thus, for a 1,0005 particle centrifuged in the PK3-800 rotor at a speed of
20,000 rpm, the nm time can be calculated as follows:
T = K/S
T = 121/1000
T = 0.121 hours
T= 7m16s.
W another embodiment, the run time can be calculated in an alternative
manner. More specifically, the following formula can be used to determine the
run
time for a second rotor in a scalable centrifuge run:
Trotor2 = Trotorl x (K rotorl / K rotor2)~
wherein T rotorl 1S the sedimentation for a first rotor, T rotor2 1S the
sedimenation time
for a second rotor, K rotors is the K factor for the first rotor, and K rotorz
is the K factor
for the second rotor.
Yet another parameter that can be calculated is the sample flow rate. The
sample flow rate is a function of the sedimentation time (T) and is calculated
as
follows:
F= VF/T
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WO 2004/083405 PCT/US2004/008655
F - flow rate (L/hr)
VF - flow through volmne (L)
T - time to sediment
The PI~3-800 rotor typically has a 50°!° flow through
volume. Thus, for a
1,0005 particle running at 20,000 I~Fl~I, the flow rate can be calculated as:
F= VF/T
F= 0.4 / 0.121
F = 3.3 L/hr
F = 5 5 ml/min.
During a centrifuge run in an embodiment of the invention, the organelles
become enriched and are accumulated (wherein accumulated can also mean
amplified) within the density gradient. In one embodiment, at least two or
more
types of organelles and/or subtypes thereof are enriched and accumulated in a
density gradient by a continuous-flow ultracentrifuge. In another embodiment,
the
at least two or more types of organelles axe accumulated until the gradient
becomes
saturated with the at least two or more types of organelles. The continuous-
flow
method of the invention advantageously accumulates the at least two or more
types
of organelles and/or subtypes thereof in a quantity sufficient to isolate and
identify,
for example, low-abundance proteins, known and/or unidentified, that are
present in
the at least two or more types of organelles and/or subtypes thereof. The
continuous-flow method of the invention also advantageously allows for the
accumulation and enrichment of large amounts of specific subtypes of
organelles
having a less complex proteome in relation to the entire proteome ofthe
population
of an organelle in the biological sample.
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For the purposes of the present invention, "enrichanent" is defined as an
increase in fold (e.~., 1.1 ~, 2~~, 5~~, 10~, 50~, etc.) of an organelle or
protein
thereof at a location in a gradient, as measured under normalized conditions,
relative
to the same organelle or protein in a biological sample. In more general
terms,
enrichment relates to the increase in the relative quantity of an organelle or
a
plurality of organelles in a pauticular gradient fraction as compared to the
relative
amount of the same organelle or plurality of organelles in the original
biological
sample. Enrichment is also a form of purification of two organelle populations
in
the homogenate in that it separates organelle types into discrete sections of
the
density gradient that correspond to the density of the organelle type.
A common approach for determining the enrichment of an organelle or
protein thereof at a specific location in a gradient, in particular, at a
specific gradient
fraction, is to perform Western analysis on an organelle-specific marker, such
as any
of those previously mentioned. In particular, Western analysis is typically
carned
using normalized quantities (e.g.,standardized and/or comparable amounts of
materials) of both the gradient fraction of interest resulting from the
separation and
accumulation method of the invention and of the corresponding original
biological
sample. Enrichment is then calculated by dividing the relative amount of the
measured organelle-specific marker in the gradient fraction of interest to the
amount
in the corresponding original biological sample.
For example, and as a first step, the total protein concentrations of the
gradient fraction of interest and the original corresponding biological sample
are
normalized using art-recognized techniques, such as, for example, a Bradford
or
Lowry protein assay. Reagents and materials for such assays can be prepared by
the
skilled artisan in accordance with known procedures (e.g., Current Protocols
in
Biochemistry, John Wiley ~ Sons, Inc., 1999, Edited by Juan S. Bonifacino) or
plarchased from commercial sources (e.g., QIACEN, INC., CA). The determination
of the total protein concentrations of both the gradient fraction and the
original
biological sample includes the step of solubilizing the proteins, especially
the
insoluble proteins therein, such as, for example, membrane proteins. The
solubilizing step typically includes, for example, a suitable detergent, such
as SDS
34
CA 02518555 2005-09-14
WO 2004/083405 PCT/US2004/008655
or Triton-X. ~nce the proteins are solubilized in each of the samples, the
insoluble
material, such as residual membrane material and/or debris, is pelleted by
Celltrlfugatloll and the remaining supernatant, which contains the solubilized
proteins, is removed. The protein concentration of the supernatant is then
measured
using standard methods, such as the Bradford and Lowry assays mentioned above.
As a second step, comparable amounts-which can be equivalent-of the
supernatant of the gradient fraction and the corresponding original biological
sample
are separately electrophoresed in the same or in different apparatuses using a
suitable protein-separation material, such as, for example, polyacrylamide.
Typically, one-dimensional polyacrylamide gel electrophoresis is used.
The separated proteins axe transferred by the art-recognized technique of
blotting to a suitable support medium (.e.g., "blot paper"), such as, for
example,
nitrocellulose. Next, the relative quantities of the organelle-specific marker
can be
determined by the art-recoguzed technique of Western analysis. Typically in
Western analysis, a primary antibody specific to the organelle-specific marker
is
allowed to react with the separated proteins on the blot paper over a suitable
period
of time wherein the primary antibody will bind to the organelle-specific
marlcer in an
amount that is directly proportional to the amount of organelle-specific
marker
present on the blot.
The relative amount of primary antibody is then measured by any suitable
means, such as, for example, introducing and detecting a secondary antibody
specific for the first antibody. The primary and/or secondary antibodies can
be
covalently linked to a detectable moiety, such as, for example, a fluorescent
molecule, an enzyme, or a chromophore. In the case of an enzyme, a detectable
enzyme substrate, such as, a chromatogenic or fluorescent substrate, can be
used to
detect the primary and/or secondary antibody. The amount of primary and/or
secondary antibody present on the blot can then be measured and represented in
a
digital format,such as pixels.
For example, the enrichment of mitochrondria in a mitochondria-containing
fraction can be determined by Western blot analysis by measuring the relative
quantities of a mitochondrial-specific marker in normalized quantities of
protein
CA 02518555 2005-09-14
WO 2004/083405 PCT/US2004/008655
from the mitochondrial fraction of interest and the corresponding original
biological
sample. The detection of the mitochonduial-specific marker in the gradient
fraction
and the original biological sample can be detected vis-a-vis a fluorescently-
labeled
primary and/or secondary antibody and through the use of digital imaging
and/or
photography to detect and quantify the fluorescence signals of the antibodies
present
on the blot. Any art-recognised instrumentation and/or computer software
detecting
and measuring the strength of the fluorescent signals of the primary and/or
secondary antibodies can be used, such as those available from MOLECULAR
DYNAMICS, INC (CA).
The number and/or intensity of the digital signal corresponds to the relative
amount of primary and/or secondary antibody on the blot, which in turn
corresponds
to the relative quantity of organelle-specific marker on the blot, which in
turn
corresponds to the relative quantity of the organelle of interest in the
samples.
Enriclnnent is determined as the ratio of the relative amount of the organelle-
specific
marker measured from the gradient fraction of interest to that measured from
the
original biological sample.
The organelles become enriched and accumulated during the continuous-
flow centrifuge run according to the method of the invention. For example, and
as
explained above, the density gradient can be established in the rotor of the
continuous-flow centrifuge prior to introducing the biological sample. As
such, the
gradient material can be added to the continuous-flow rotor and then
centrifuged at a
speed sufficient to establish the gradient. Once the gradient is established,
the
biological sample can then be introduced into the rotor while the rotor
continues to
rotate. As described previously, the biological sample is typically a
homogenate of a
biological sample and contains organelles, cytosol components, and possible
membrane fragments. Optionally and prior to introducing the biological sample
to
the rotor of the continuous-flow centrifuge, the biological sample can be
clarified to
remove large particulate matter, such as cellular debris and nuclei, as
previously
explained.
As previously explained, the biological sample can be introduced into the
rotating rotor of the continuous-flow centrifuge in a continuous manner. For
36
CA 02518555 2005-09-14
WO 2004/083405 PCT/US2004/008655
example, the biological sample is fed into the rotor while the rotor continues
to spin.
The speed of the rotor can remain constant or it can be increased or decreased
vJhile
the biological sample is being added. The sample caaz be introduced into the
rotor
using any suitable means, including, but not limited to, a peristaltic pump.
Further
and as explained previously, the introduction of the sample into the rotor can
be
carried out in any suitable manual, automatic, or semi-automatic manner aazd
can
include the use of any suitable robotics and/or computer control systems.
Also, any
suitable volume of biological sample can be introduced into the rotor,
including, for
example, any volume that is less, equal to, or greater than the volume of the
gradient
material in the rotor.
As the biological sample enters and begins to flow through the rotor, it
comes into contact with the density gradient therein. The density gradient has
a
proximal end and a distal end whereby the proximal end is at a lower density
than
the distal end. Moving from the proximal end of the gradient to the distal
end, the
gradient increases in density in accordance with a particular density proFle.
As
explained previously, the density profile, which can also be referred to as
the
concentration profile, of the gradient can be, for example, linear, convex, or
concave. The density gradient can also be regarded as comprising different
"sections," where each section has a proximal end at a first density and a
distal end
at a second density where the second density is greater than the first
density.
Whether a particular component of the biological sample enters the gradient
is determined by both the physical characteristics of the component as well as
the
parameters used by the continuous-flow centrifuge. Such physical
characteristics,
including, for example, the component's sedimentation value and buoyant
density,
and centrifugation parameters, such as, for example, RCF (xg) at the rotor and
flow
rate of the biological sample, were previously described herein. The
centrifugation
parameters, including the RCF (xg) and the flow rate, can be increased or
decreased
during the operation of the centrifuge to affect the entrance of different
components
into the gradient. The parameters of the centrifuge, especially the RCF (xg)
can be
changed throughout the operation of the continuous-flow centrifuge, including
during the introduction of the biological sample.
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WO 2004/083405 PCT/US2004/008655
~nce a component of the biological sample enters the proximal end of the
gradient, the centrifugal force applied to the component by the centrifugation
process causes the component to migrate through the density gradient a rate
that is
dependent, in part, on the physical characteristics of the component,
including, the
buoyant density and the sedimentation coefficient of the component. The
component migrates through the gradient until reaching an isopynic point where
it
becomes enriched based on its buoyant density.
During the centrifuge run, further biological sample can be introduced into
the centrifuge, as described previously, so as to accumulate the components of
the
biological sample. For example, when mitochondria and subtypes thereof are
enriched in a section of the gradient equal to their buoyant densities,
addition of
more biological sample containing mitochondria and subtypes thereof into the
centrifuge results in the accumulation of the mitochondria and subtypes
thereof at
that section of the gradient. ,
Collecting organelles. As seen in Figure l, once the centrifuge run is
completed, the organelles that have migrated into the gradient are collected.
Any
art-recognized technique for collecting organelles falls within the scope of
the
present invention. For example, organelles can be collected by removing a
volumetric fraction of the gradient, either manually, automatically, or some
combination thereof, and stored and/or placed into a vessel, such as, for
example, a
sample tube. Any suitable fraction volume is contemplated, such as, for
example
1/10,000t~', 1/1,000th, 1/100th, or 1/l0th of the total volume of the
gradient, or any
other suitable volume thereof. The volumetric fractions can be the same or
different
volumes. Further, once collected, the different volumetric fractions can be
combined together.
The fractions can also be collected on the basis of a specified density range.
In one embodiment, a fraction can be regarded as the gradient material between
and
including a first density point and a second density point, where the first
and the
second density points are different. For example, one can collect as a
fraction, all
the gradient material between and including 10% to 15% sucrose. The density of
the
gradient at a particular fraction can be estimated or measured using a
commerciallv-
38
CA 02518555 2005-09-14
WO 2004/083405 PCT/US2004/008655
available refraction index analyzer, for example, I~1VIA 4500,1~XA 156, or
R~~A
170 (A1~T~~T PAAR, GI~IBH, Austria).
Any other method, automated, semi-automated, or manual, for the collection of
gradient fractions is contemplated and within the scope of the present
invention.
With automated or semi-automated systems for collection of fractions from the
gradient, the invention contemplates any suitable robotics system, including
any
suitable sensors, electronics, or other useful and/or necessary components. An
automated or semi-automated system for collecting gradient fractions, which
can be
referred to as automated or semi-automated fraction collectors, can also be
controlled and/or programmed using any suitable software or colnputer
system.The
automated and semi-automated fraction collector can be a stand-alone device
or, in
another embodiment, integrated with the continuous-flow centrifuge as an on-
board
device.
Analysis of organelles. According to Figure 1, once the organelles are
collected, the organelles are analyzed by art-recognized methods. For
example,. the
organelles in the collected fractions can be identified and/or characterized
using any
suitable methodology l~nown in the art, such as, for example, Western blot
analysis,
enzymatic assays, immunofluorescence microscopy with fluorescently-labeled
antibodies specific to organelle-specific markers, and microscopy, including,
for
example, electron microscopy, or any other known method. By these methods, for
example, the organelle composition of a fraction can be assessed and
characterized,
for example, with respect to the relative amounts of different types of
organelles
present in the fraction. For example, by performing a Western blot analysis on
a
fraction and testing for the presence of organelle-specific markers, such as,
for
example, mitochondria, endoplasmic reticulum, plasma membrane, and Golgi, one
can assess the relative amounts of each of these organelles comprising the
fraction.
Information on the preceding protocols can be found in commercially-available
literature, such as, for example, Current Protocols in Cell Biolo~y, John
Wiley ~
Sons, W c., 1999, Edited by Bonifacino et al. or Current Protocols in
NJ:olecular
Biolo~y, John Wiley & Sons, Inc., 1999, Edited by Juan S. Bonifacino.
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Also, the integrity of the organelles can be determined by any suitable
method in the ant, such as, for example, quantitative enzynatic assays,
Western blots
to organelle-specific marker proteins and electron microscopy experiments.
Transmission electron microscopy (TElVI) can be used to identify the
organelles and
to qualitatively characterize the integrity of the organelles vis-~.-vis their
morphologies (e.g., size, shape, structural organization, and density), which
can
generally correlate with the function of the organelle. In other words, an
organelle
that has a higher degree of integrity generally would have a more intact
function.
Organelle applications. The organelles obtained by the invention can be
used in the field of proteomics, as well as other fields. Such other fields
include, but
are not limited to, genomics, neurochemistry, irrununochemistry, biochemistry,
histology, botany, plant biochemistry, physical anthropology, forensics and
pathology, and combinations thereof. A skilled artisan would understand how
organelles can be utilized in these disciplines. Further, the organelles
obtained by
the method of the invention can be used for the development of diagnostics,
pharmaceuticals, chemicals and vaccines, useful in the fields of, for example,
human, animal, livestock and pet care.
Protein Characterization and Quantitation. Figure 2 relates to the
characterization and the quantitation of the proteins present in the
organelles. The
separation, enrichment, and accumulation of subcellular organelles and other
subcellular structures of interest according to the method of the invention
can be
thought of as a method for "pre-fractionating" a proteome of the biological
sample
since the proteome of the cell is divided up into the distinct types of
subcellular
organelles and structures. Thus, once the organelles are separated and
purified, the
proteome of the intact whole biological sample is effectively fractionated
into sub-
proteomic constituents. The process of the invention reduces the complexity of
the
proteome of the biological sample and facilitates the subsequent analysis of
the
protein constituents of the proteome.
~,yse organelles. As seen in Figure 2, the accumulated organelles are lysed
by any technique known in the art. Lysing is typically performed to disrupt
the
membrane of the organelle in a manner sufficient to release the contents of
the
CA 02518555 2005-09-14
WO 2004/083405 PCT/US2004/008655
organelle. The contents of the organelle include, for example, the proteome of
the
organelle.
~epar~te p~~tein~ ~~ad pep~idle~. ~nce enrichment, accumulation, and lysis
of the subcellular organelles are achieved, the protein constituents of each
of the
isolated organelles (e.g., the subcellular proteomes of each organelle) can be
analyzed to facilitate the detection of a protein of interest, such as, a low-
abmdance
protein. Different ways to analyze large populations of proteins and peptides,
such
as, the subcellular proteome of an organelle, are known in the art. One of
ordinary
skill in the art may select the most appropriate protein isolation and
purification
techniques without departing from the scope of this invention.
An example of a particular method is two-dimensional (2D) gel
electrophoresis. Two-dimensional gel electrophoresis of a complex protein
solution,
such as a subcellular proteome, results in a pattern of separated, typically
referred to
in the art as resolved, polypeptides which can then be further investigated as
to their
identity. For example, Western blotting can be used to identify a specific
type,
class, or specific protein or fragment thereof through the probing with a
specific
antibody. Additionally, mass spectroscopy can be used to determine the
identity of a
resolved protein in a gel by comparison of molecular weight profiles of the
resultant
polypeptide fragments generated and detected by the mass spectrometer with
information contained in a mass spectrometry database or whole-genome sequence
or polypeptide database.
Detection and identification processes can be automated or semi-automated.
Also, robotics or high-throughput instrumentation known to one of ordinary
skill in
the art can be used.
Other technologies useful for studying proteins include, for example, liquid
chromatography, such as normal or reversed phase, using HPLC, FPLC and the
like;
size exclusion chromatography; immobilized metal chalets chromatography;
affinity
chromatography; any other chromatographic method; protein binding analysis;
yeast
two-hybrid analysis; three-dimensional structure studies; gel electrophoresis,
such
as, 1D and 2D; and most recently, proteinlpolypeptide microarrays, and
bioinformatics.
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Another technique within the sc~pe of the invention to separate and proteins
and peptides is multidimensional liquid chromatography ("1~DLC'~), also
referred to
as "MudPIT." MudPIT is used as an alternative to two-dimensional gel
electrophoresis to identify a different, and partially overlapping, set of
proteins in a
proteome. W stead of using an initial protein separation step like two-
dimensional
gel electrophoresis, the complete proteome of the biological sample, such as,
a
gradient fraction enriched for an organelle, is first digested with trypsin.
The
resulting complex mixture of peptides is resolved by MDLC using a combination
of
strong anion exchange (SAX) and reverse-phase (RP) columns, and the separated
peptides are analyzed by tandem mass spectrometry (MS/MS). The information
gained from MS/MS of the peptides is then used to predict protein identity.
Proteome analysis is typically performed by combining the high-resolution
separation technique of 2D-GE with the highly sensitive identification
capabilities of
matrix-assisted laser desorption-ionization ("MALDI") mass spectrometry.
Several
strategies based on this combination have been developed. Most recently,
approaches based on ESI/MS/MS have emerged as complementary or alternative
techniques for proteome analysis. Such approaches include global proteolytic
digestion of a complex sample followed by partial separation of the
proteolytic
mixture using one or more iterative in-line chromatography steps, followed by
analysis of the peptides using MS/MS , usually via an electrospray ionization
interface. Independently from the strategy used to obtain the data, the
experimentally obtained masses of digested peptides are introduced into
database-
searching programs in order to match the obtained values with those
theoretically
calculated for the tryptic peptides derived from all proteins within a given
database.
Characterize and quantitate proteins and peptides. Refernng again to
Figure 2, techniques to characterize and quantitate the proteins, such as low-
abundance proteins, and peptides derived from the invention, include, for
example,
any known biochemical approach, enzyme assay, antibody innnunoreaction, ligand
analysis, protein/peptide mass spectrometry, substrate analysis, or
combinations
thereof. The type of experimentation used to validate the function of the
protein
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WO 2004/083405 PCT/US2004/008655
typically depends on, and is guided by, the knowledge as to the predicted
function of
the protein.
In one embodiment, relative quantitation of protein levels can be obtained
from 2D gels by comparing the intensity of protein/peptide spots in digitized
versions of the gel image using computer software such as, for example,
Phoretix
ZIP Evolution from l~Tonlinear Dynamics. ~ther methods that do not involve 2D
gels
can be used such as isotope-coded affinity tags (ICA.T) (APPLIED BI~SYSTEMS,
CA).
The ICAT method uses heavy and light versions of a reagent that react with
proteins. In addition to this 'isotope coding', the reagent has a chemical
group,
iodoacetamide, that reacts with cysteine suflhydryl groups, and an affinity
tag,
biotin, to facilitate purification. An ICAT experiment typically involves
reacting
one proteome with the light version of the reagent and another proteome with
the
heavy version. The labelled proteomes are then combined together and analyzed
using a suitable workflow instrument. For example, labelled peptides produced
by
trypsin are affinity purified from non-labelled peptides to reduce the
complexity of
the peptide mixture under analysis. The affinity-purified peptides are then
separated
and analyzed by MS.
Mass spectra of ICAT-labelled peptides typically contain pairs of ions that
differ in mass equal to the difference in the masses of the heavy and light
reagents.
Because the peptides are being measured in the same mass spectrum, it is
possible to
obtain a relative quantitation of the peptides and therefore of the proteins
in the two
proteomes. ICAT is useful for quantitating proteomes or sub-proteomes that are
not
amenable to two-dimensional gel electrophoresis.
identify pr~teins. Any identification or analytical technique available to a
skilled artisan may be used to identify the proteins and peptides obtained by
the
invention. Technologies useful for identifying and studying proteins include,
for
example, mass spectrometry, co-immunoprecipitation, affinity chromatography,
protein binding analysis, yeast two-hybrid analysis, three-dimensional
structure
studies, and most recently, protein/polypeptide microarrays and
bioinformatics.
Some of the more common identification techniques include 2D-GE combined with
43
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MALI)I; ESI/IVIS-MS; and tandem mass spectrometry (MS-MS), usually via an
electrospray ionization interface.
In one embodiment, the invention isolates and purifies proteins, in
substantially pure form, particularly one or more low-abundance proteins, from
the
organelles accumulated by the method herein. For example, the low-abundance
proteins can be removed from a polyacnylamide gel, such as the two-dimensional
polyacrylamide gels of the invention, and purified therefrom using standard
techniques. The low-abundance protein can also be purified using other art-
recognized techniques, such as, for example, immunoprecipitation or
immunoaffinity chromatography using antibodies specific to a particular low-
abundance protein of interest. Further and as explained in greater detail
herein, the
gene coding for a low-abundance protein of interest can be cloned and
expressed in
a host organism, and isolated and purified using art-recognized techniques.
The low-abundance proteins of the invention are not meant to be limited to
any particular class. Low-abundance proteins can be classified as such based
on
their relative quantity or copy numbers in the cell. For example, it is known
that a
typical cell has about 109 protein molecules, having at least 104 unique
protein
species and having a "dynamic range," with respect to copy number, of orders
of
magnitude (i.e., from less than 102 copies to greater than 10~). The "dynamic
range"
is the range that proteins in a cell show from the lowest number of copies to
the
highest number of copies. About 9,000 proteins in a cell are present in fewer
than
about 1,000 copies per cell and are lcnown as the "low-abundance proteins."
The
sum of the low abundance proteins in a cell generally constitutes less than
about 3%
of the cell's mass. For example, tyrosine kinases are present in the range of
30-40
copies per cell. Further, certain low abundance proteins may be present in the
about
picoMolar (pM) or 10-9 to the about femtoMolar (fM) or 10-12 concentrations,
for
example at about 10-9, at about 10-12, or at about below 10-9 concentrations.
Low-abundance proteins are generally difficult to detect using lmown protein
analytical instuumentation and/or methods. For example, low-abundance proteins
in
the context of 2D gel electrophoresis can be difficult to detect as "spots"
(an
electrophoretically-separated polypeptide on a gel) based on low copy numbers
44
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and/or their overlap with more prevalent proteins. The present invention
contemplates any low-abundance protein, even low-abundance proteins present in
less than about 750, 500, 250 or 100 copies per cell, or even in about one
copy per
cell, known or unlmown, intracellular or extracellular (such as proteins in
the
interstitial space, neurotransmitters and signaling proteins).
J~rOteara appigc~ta~a~~ 0f claa~-~t~t:e~-ized l~a~~~~n and ~anh~nOVYn
pr~ttegn~.
Referring again to Figure 2, there are many ways to utilize the proteins
obtained by the methods of the invention, such as the low-abundance proteins.
For
example, the proteins obtained by the method of the invention can be used for
the
development of diagnostics, pharmaceuticals, chemicals and vaccines, useful in
the
fields of, for example, human, animal, livestock and pet care.
One application of the invention provides for a method of analyzing
proteomic changes among two sets of biological samples or as a function of
time.
The time relates to the point when the biological sample is taken, such as a
biopsy.
In this embodiment, at least two types of subcellular organelles are released
from a
biological sample, typically by an art-recognized homogenization or lysing
procedure. The at least two types of subcellular organelles are then
introduced to a
density gradient within a continuous-flow ultracentrifuge. A centrifugal force
is
applied, preferably greater than or about 100,000 x g, such that the at least
two types
of subcellular organelles migrate within the density gradient. In one
embodiment,
centrifugation is performed in a single run. After centrifugation, the at
least two
types of subcellular organelles are collected from the density gradient. The
proteins
from the at least two types of subcellular organelles are then isolated and
purified to
determine a proteomic profile of the at least two types of subcellular
organelles at
the first time. This process can also be performed with a single type of
subcellular
organelle.
A second biological sample is provided and the at least two different types of
subcellular organelles axe released therefrom. The at least two types of
subcellular
organelles are then introduced to a density gradient within a continuous-flow
ultracentrifuge; and a centrifugal force is applied such that the at least two
types of
subcellular organelles migrate within the density gradient, preferably in a
single run.
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After centrifugation, the at least two types of subcellular organelles are
collected
from the density gradient. The proteins from the at least two types of
subcellular
organelles are isolated and purified to determine a proteomic profile of the
at least
two types of subcellular organelles at a second time. This part of the process
can
also be carried out with one type of organelle. Finally, the proteomic
profiles at the
first and second times are analyzed by art-recognized techniques to detect
changes in
the proteomic profiles as a function of time. Such ari invention finds
applicability,
for example, in analysis of disease states and when comparing proteomic
profiles of
individuals or different groups of individuals.
In another protein application embodiment, protein translocation events can
be analyzed using the method of the present invention. More specifically, the
translocation process relates to the intracellular and/or intercellular
movement of a
translocation protein and/or translocation proteins as a function of time. The
relative
amounts of the translocation protein in a first and second types of organelles
of a
first biological sample are first determined. The procedure includes, for
example,
homogenizing the first biological sample under conditions sufficient to
release the
first and second organelles into a homogenate, wherein the first and second
organelles each comprise a subcellular proteome. The homogenate is then
introduced into a density gradient within a continuous-flow ultracentrifuge. A
centrifugal force is applied to the homogenate so that the first and second
organelles
migrate within the density gradient. The first and second organelles are
removed
from the density gradient, and the subcellular proteomes of the first and
second
organelles are subsequently solubilized. After solubilization, the
translocation
protein in the first and second organelles of the first biological sample is
then
detected and the level of the detected translocation protein is measured.
A second biological sample is similarly processed along the lines of the first
biological sample. That is, the second biological sample is homogenized under
conditions sufficient to release the first and second organelles into a
homogenate,
wherein the first and second organelles each comprise a subcellular proteome.
The
homogenate from the second biological sample is then introduced into a density
gradient within a continuous-flow ultracentrifuge. A centrifixgal force is
applied to
46
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the homogenate so that the first and second organelles migrate within the
density
gradient. The first and second organelles are removed from the density
gradient, and
the subcellular proteomes of the first and second organelles are subsequently
solubilized. After solubilization, the trallSloCat1o11 protein in the first
and second
organelles of the second biological sample is then detected and the level of
the
detected translocation protein is measured. .
After the translocation protein and/or translocation proteins of the first and
second biological samples is detected and measured, the translocation process
is
analyzed. For example, the translocation process of the translocation protein
as a
function of time is determined by comparing the measured levels of the
detected
translocation protein in the first and second organelles for each of the
biological
samples at the first and second times.
The invention further contemplates, as indicated at Fig. 19(A)(3-4), that the
information pertaining to the analysis and separation of organelle proteins
and the
detection and/or identification of low-abundance proteins thereof can be
provided to,
transmitted to, or stored in a database to be accessed at a later point in
time by the
same or another user. The invention contemplates that any data generated or
collected during the method of separating said proteins of a proteome or
detecting a
low-abundance protein can be transmitted or transferred to a third party. For
example, image data relating to the pattern of resolved proteins on a two-
dimensional gel or information pertaining to the different levels of
expression of the
resolved proteins of a gel can be transmitted electronically, for example by
email, or
over the Internet or a network to a third party, to or from a database, to a
laboratory,
individual, or research group. The data can also be transferred (e.g" posting)
electronically to a network, such as the World Wide Web or other global
communications networks.
~ne of ordinary skill in the art will appreciate that the databases of the
present invention can have many different forms and/or structures and can use
any
known protocols for electronic storage and retrieval of information. The
invention
further contemplates providing access to the database for commercial purposes.
47
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Access can be electronic access over a global communications network, such as
the
World Wide W ~b.
~nce the low-abundance protein is identified by a detection method
contemplated by the invention, such as by mass spectrometry, the complete
amino
acid sequence of the protein or protein fragment can be obtained from a whole-
genome sequence database. The invention further contemplates the assessment of
the putative function of a low-abundance protein of interest by comparative
sequence analysis methods. such methods are widely known in the art and
pertain
to computer software available locally on a desktop computer or workstation or
available over a network, such as the World Wide Web, that employ algorithms
for
comparing an amino acid sequence of interest (e.g., the "query sequence") with
the
amino acid sequences contained in a database to identify a polypeptide having
a
similar sequence whose function is already known. This general approach can be
identified as "homology searching." Homology searching does not positively
identify a function for a query sequence but only establishes a likelihood
that a
particular sequence shares the same or similar function. Experimentation can
be
carried out to further confirm or validate the function of a protein of
interest, such
as, for example a low-abundance protein.
Thus, the low-abundance proteins of the invention can be assigned predicted
function based on comparative sequence analyses (e.g., homology searching) to
protein sequences in various databases, such as, for example GenBank, Swiss-
Prot,
and Protein Data Bank, etc. The term "percent identity" in the context of
amino acid
sequence refers to the residues in the two sequences which are the same when
aligned for maximum correspondence. There are a number of different algorithms
known in the art which can be used to measure sequence similarity or identity.
For
instance, polypeptide sequences can be compared using NCBI BLASTp and/or
FASTA, a program in GCG version 6.1. FASTA provides alignments and percent
sequence identity of the regions of the best overlap between the query and
search
sequences.
Alternatively, in the context of DNA or RNA, nucleotide sequence similarity
or homology or identity can be determined using the "Align" program of Myers
and
48
CA 02518555 2005-09-14
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Miller, ("Optimal Alignments in Linear Space", CA~IOS 4, 11-17, 1988) and
available at NCFI. The terms "similarity" or "identity" or "homology", f~r
instance,
with respect t~ a nucleotide sequence, is intended t~ indicate a quantitative
measure
of homology between two sequences. The percent sequence similarity can be
calculated as (N,.~f - N~If)~° 100/IV,.~f, wherein N~;f is the total
number of n~n-identical
residues in the two sequences when aligned and wherein N,.ef is the number of
residues in One of the sequences. Hence, the DNA sequence AGTCAGTC will have
a sequence similarity of 75% with the sequence AATCAATC (N,.ef = 8; N~1J=2).
Alternatively or additionally, "similarity" with respect to sequences refers
to the
number of positions with identical nucleotides divided by the number of
nucleotides
in the shorter of the two sequences wherein alignment of the two sequences can
be
determined in accordance with the Wilbur and Lipman algorithm (Wilbur and
Lipman, 1983 PNAS USA 80:726), for instance, using a window size of 20
nucleotides, a word length of 4 nucleotides, and a gap penalty of 4, and
computer-
assisted analysis and interpretation of the sequence data including alignment
can be
conveniently performed using commercially available programs (e.g.,
Intelligenetics
TM Suite, Intelligenetics Inc. CA). When RNA sequences are said to be similar,
or
have a degree of sequence identity with DNA sequences, thymidine (T) in the
DNA
sequence is considered equal to uracil (U) in the RNA sequence.
Once a putative or predicted function is ascertained for a given protein of
interest, especially a low-abundance protein, a patent application can be
drafted and
filed with the with the appropriate national and/or international patent
office. The
application can be directed to, for example, the protein of interest whose
function is
predicted from homology searching. The claims can be directed to, for example,
the
amino acid sequence of the protein of interest, its utility based on its
predicted
function, 0r any cloning vector or expression vector carrying the DNA encoding
said
protein of interest.
The present invention, as seen in Fig. 19(C)(8), further contemplates
validating the predicted function of a protein of interest, such as a low-
abundance
protein. Validation can be carried out using biochemical, immunological,
physiochemical, protein structural, and genetic techniques, any of which are
known
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to one of ordinary skill in the art. In one embodiment, as seen in Fig.
190)(7), the
invention contemplates cloning the nucleic acid sequence encoding the protein
of
interest. Different strategies can be used to clone the gene, gene fragment,
or
nucleotide sequence encoding a protein of interest. For example, a degenerate
nucleotide probe can be crafted based on the sequence of tlae protein of
interest aazd
used to screen a DNA or cDNA library for a plasmid or vector clone carrying
the
encoding piece of DNA. W another example, a nucleotide sequence encoding the
DNA of interest can be amplified by PCR using primers that are based on the
sequence of the protein of interest. Further, cloning steps can be
subsequently
carried out to obtain the transcriptional control regions of the encoding
nucleotide
sequence. The nucleotide sequences can be obtained not only from the original
1 source of biological material, but also from another source of biological
material
sharing similar sequences.
Once the encoding nucleotide sequence is cloned, it can be further
engineered into an expression vector, expressed in a host cell, isolated, asld
then
further analyzed to assess and ascertain by experimentation the function of
the
protein of interest. Thus, the polypeptides of the present invention, such as
the
detected low-abundance proteins, are produced recombinantly and may be
expressed
in unicellular hosts. In order to obtain high expression levels of foreign DNA
sequences in a host, the sequences can generally be operably linked to
transcriptional and translational expression control sequences that are
functional in
the chosen host. Preferably, the expression control sequences, and the gene of
interest, can be contained in an expression vector that further comprises a
selection
maxker.
The DNA sequences encoding the polypeptides of this invention may or may
not encode a signal sequence. If the expression host is eukaryotic, it
generally is
preferred that a signal sequence be encoded so that the mature glycoprotein is
secreted from the eukaryotic host.
An amino terminal methionine may or may not be present on the expressed
polypeptides in the compositions of this invention. If the terminal methionine
is not
CA 02518555 2005-09-14
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cleaved by the expression host, it may, if desired, be chemically removed by
standard technlqlles.
A wide variety of expression host/vector combinations may be employed in
expressing the DNA sequences encoding the WNV polypeptides used in the
pharmaceutical compositions and vaccines of this invention. Useful expression
vectors for eukaryotic hosts, include, for example, vectors comprising
expression
control sequences from SV40, bovine papilloma virus, adenovirus, adeno-
associated
virus, cytomegalovirus and retroviruses including lentiviruses. Useful
expression
vectors for bacterial hosts include bacterial plasmids, such as those from E.
coli,
including pBluescript~, pGEX-2T, pUC vectors, col E1, pCRl, pBR322, pMB9 and
their derivatives, pET-15, wider host range plasmids, such as RP4, phage DNAs,
e.g., the numerous derivatives of phage lambda, e.g. 7~GT10 and AGT11, and
other
phages. Useful expression vectors for yeast cells include the 2~, plasmid and
derivatives thereof. Useful vectors for insect cells include pVL 941.
In addition, any of a wide variety of expression control sequences, sequences
that control the expression of a DNA sequence when operably linked to it, may
be
used in these vectors to express the polypeptides used in the compositions of
this
invention. Such useful expression control sequences include the expression
control
sequences associated with structural genes of the foregoing expression
vectors.
Examples of useful expression control sequences include, for example, the
early and
late promoters of SV40 or adenovirus, the lac system, the trp system, the TAC
or
TRC system, the T3 and T7 promoters, the major operator and promoter regions
of
phage lambda, the control regions of fd coat protein, the promoter for 3-
phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid
phosphatase, e.g., PhoS, the promoters of the yeast-mating system and other
constitutive and inducible promoter sequences known to control the expression
of
genes of prokaryotic or eukaryotic cells or their viruses, and various
combinations
thereof.
The term "host cell" refers to one or more cells into which a recombinant
DNA molecule is introduced. Host cells of the invention include, but need not
be
limited to, bacterial, yeast, animal, insect and plant cells. Host cells can
be
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CA 02518555 2005-09-14
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unicellular, or can be grown in tissue culture as liquid cultures, monolayers
or the
like. Host cells may also be derived directly or indirectly from tissues.
A wide variety of unicellular host cells are useful in expressing the DIVA
sequences encoding the polypeptides used in the pharmaceutical compositions of
this invention. These hosts may include well known eukaryotic and prokaryotic
hosts, such as strains of ~'. c~li, hseascl~tn~iaezs, ~ezcillus,
Sta~ept~~zyces, fungi, yeast,
insect cells such as Sp~cl~ptef~a fi°u~ipea-ela (SF9), animal cells
such as CI~~ and
mouse cells, African green monkey cells such as C~S 1, C~S 7, BSC 1, BSC 40,
and BMT 10, and human cells, as well as plant cells.
A host cell is "transformed" by a nucleic acid when the nucleic acid is
translocated into the cell from the extracellular environment. Any method of
transferring a nucleic acid into the cell may be used; the term, unless
otherwise
indicated herein, does not imply any particular method of delivering a nucleic
acid
into a cell, nor that any particular cell type is the subject of transfer.
An "expression control sequence" is a nucleic acid sequence which regulates
gene expression (i.e., transcription, RNA formation and/or translation).
Expression
control sequences may vary depending, for example, on the chosen host cell or
organism (e.g., between prokaryotic and eukaryotic hosts), the type of
transcription
unit (e.g., which RNA polymerase must recognize the sequences), the cell type
in
which the gene is normally expressed (and, in turn, the biological factors
normally
present in that cell type).
A "promoter" is one such expression control sequence, and, as used herein,
refers to an array of nucleic acid sequences which control, regulate and/or
direct
transcription of downstream (3') nucleic acid sequences. As used herein, a
promoter
includes necessary nucleic acid sequences near the start site of
transcription, such as,
in the ease of a polymerase II type promoter, a TATA element.
A "constitutive" promoter is a promoter which is active under most
environmental and developmental conditions. An "inducible" promoter is a
promoter which is inactive under at least one environmental or developmental
condition and which can be switched "on" by altering that condition. A "tissue
specific" promoter is active in certain tissue types of an organism, but not
in other
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tissue types from the same organism. Similarly, a developmentally-regulated
promoter is active during some but not all developmental stages of a host
organism.
Expression control sequences also include distal enhancer or repressor
elements which can be located as much as several thousand base pairs from the
start
site of transcription. They also include sequences required for RNA formation
(e.g.,
capping, splicing, 3' end formation and poly-adenylation, where appropriate);
translation (e.g., ribosome binding site); and post-translational
modifications (e.g.,
glycosylation, phosphorylation, methylation, prenylation, and the like).
The term "operably linked" refers to functional linkage between a nucleic
acid expression control sequence (such as a promoter, or array of
transcription factor
binding sites) and a second nucleic acid sequence, wherein the expression
control
sequence directs transcription of the nucleic acid corresponding to the second
sequence.
It should of course be understood that not all vectors and expression control
sequences will function equally well to express the polypeptides mentioned
herein.
Neither will all hosts function equally well with the same expression system.
However, one of skill in the art may make a selection among these vectors,
expression control sequences and hosts without undue experimentation and
without
departing from the scope of this invention. For example, in selecting a
vector, the
host typically should be considered because the vector is replicated in it.
The
vector's copy number, the ability to control that copy number, the ability to
control
integration, if any, and the expression of any other proteins encoded by the
vector,
such as antibiotic or other selection marlcers, should also be considered.
In selecting an expression control sequence, a variety of factors should also
be considered. These include, for example, the relative strength of the
promoter
sequence, its controllability, and its compatibility with the DNA sequence of
the
peptides described in this invention, in particular with regard to potential
secondary
structures. Unicellular hosts should be selected by consideration of their
compatibility with the chosen vector, the toxicity of the product coded for by
the
DNA sequences encoding the glycoproteins used in a pharmaceutical composition,
their secretion characteristics, their ability to fold the polypeptide
correctly, their
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CA 02518555 2005-09-14
WO 2004/083405 PCT/US2004/008655
fermentation or culture requirements, and the ease of purification from them
of the
products coded for by the I~I~TI~ sequences.
within these parameters, one of skill in the art may select various
vector/expression control sequence/host combinations that will express the
I)NA
sequences encoding the products used in the pharmaceutical compositions on
fermentation or in other large scale cultures.
The polypeptides described in this invention may be isolated from the
fermentation or cell culture and purified using any of a varietyof
conventional
methods described elsewhere herein. One of ordinary skill in the art may
select the
most appropriate isolation and purification techniques without departing from
the
scope of this invention. If the polypeptide is membrane bound or suspected of
being
a lipoprotein, it may be.isolated using methods known in the art for such
proteins,
e.g., using any of a variety of suitable detergents.
Once the function of the protein of interest is known or validated by
experimentation, one may have in possession valuable. intellectual property
that can
be protected by applying for a national or international patent directed to
the protein
of interest, such as, for example, a low-abundance protein of interest, its
amino acid
sequence, its function and/or biological activity, its concomitant nucleotide
sequence, and the cloning vectors and expression vectors harboring the
concomitant
nucleotide sequence. In particular, the validated function of the protein of
interest
may indeed establish the utility requirement for obtaining a national or
international
patent. The information generated by the above steps, in particular the
validated
function of the protein of interest, such as a low-abundance protein, can also
be
distributed or transmitted to a third-party user, such as, for example, a
pharmaceutical company, a biotechnology company, a database service, a
bioinformatics company, or a private or public research institute. The
invention
contemplates, as indicated at Fig. 20(C)(11-12), that the information
pertaining to
the analysis and separation of organelle proteins and the detection and/or
identification of low-abundance proteins thereof can be provided to,
transmitted to,
or stored in a database to be accessed at a later point in time by the same or
another
user.
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WO 2004/083405 PCT/US2004/008655
The present invention further encompasses a method of transmitting data, for
example disclosing the amino acid sequence of the identified protein or the
nucleic
acid molecule encoding said identified protein, information on the disease-
related
proteome profile of a specific organelle or organelles, information on the
changes in
proteome profile of a specific organelle or organelle upon application of a
specific
stimulus, such as, for example a drug, each transmitted by digital means, such
as by
facsimile, electronic mail, telephone, or a global communications network,
such as
the World Wide Web. For example, data can be transmitted via website posting,
such as by subscription or select/secure access thereto and/or via electronic
mail
and/or via telephone, IR, radio, television or other frequency signal, and/or
via
electronic signals over cable and/or satellite transmission and/or via
transmission of
disks, compact discs (CDs), computers, hard drives, or other apparatus
containing
the information in electronic form, and/or transmission of written forms of
the
information, e.g., via facsimile transmission and the like. Thus, the
invention
comprehends a user performing according to the invention and transmitting
information therefrom; for instance, to one or more parties who then further
utilize
some or all of the data or information, e.g., in the manufacture of products,
such as
therapeutics, assays and diagnostic tests and etc. This invention comprehends
disks,
CDs, computers, or other apparatus or means for storing or receiving or
transmitting
data or information containing information from methods and/or use of methods
of
the invention. Thus, the invention comprehends a method for transmitting
information comprising performing a method as discussed herein and
transmitting a
result thereof.
Further still, the invention comprehends methods of doing business
comprising performing or using some or all of the herein methods or
organelles,
proteins, compounds, compositions, or products derived therefrom, and
conununicating or transmitting or divulging a result or results thereof,
advantageously in exchange for compensation, e.g., a fee. Advantageously, the
communicating, tr ansmitl:ing or divulging of information is via electronic
means,
e.g., via Internet or email, or by any other transmission means herein
discussed.
Thus, the invention comprehends methods of doing business involving the
CA 02518555 2005-09-14
WO 2004/083405 PCT/US2004/008655
organelles, proteins, compositions, compounds, and products derived therefrom,
and
methods of the invention.
Thus9 a first party, 6'cllent" can request information, e.g., via any of the
herein mentioned transmission means - either previously prepared information
or
information specially ordered as to a particular amino acid sequence of a
detected
low-abundance prote111S - of a second party, '6vendor'9, e.g., requesting
information
via electronic means such as via intemet (for instance request typed into
website) or
via email. The vendor can transmit that information, e.g., via any of the
transmission means herein mentioned, advantageously via electronic means, such
as
Internet (for instance secure or subscription or select access website) or
email. The
information can come from performing some or all of a herein method or use of
a
herein method in response to the request, or from performing some or all of a
herein
method, and generating a library of information from performing some or all of
a
herein method or use of a herein algorithm. Meeting the request can then be by
allowing the client access to the library or selecting data from the library
that is
responsive to the request.
Accordingly, the invention even further comprehends collections of
information, e,g., in electronic form (such as forms of transmission discussed
above), from performing or using a herein method or apparatus.
The present invention is additionally described by way of the following
illustrative, non-limiting Examples that provide a better understanding of the
present
invention and of its many advantages. .
EXAMPLES
The following examples are set forth to illustrate various embodiments in
accordance with the present invention. The following exarrlples, however, are
in no
way meant to limit the present invention.
E~'~AI'~l~Tl.dE 1. l~A~AaL.~I.~EL ~~~ILATI~I'~T~ PIT 11~I1FT1~ATTl~hI Al's
ENRICHMENT
56
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WO 2004/083405 PCT/US2004/008655
~~' M~T~CH~~1~1~1~, ~~L~~, ~1'~~T~~PIL,A~T~dI~
1I~~1L°~~~TTL~1I~9 A1~'J~D PLA~l~A P~~ly~II~~Al~'~T~ lL~I~~I
LTl~lE~ T~~~~JlE
Liver- lz~rnogenizczti~n. Approximately 100g of rat liver was harvested from
male Wistar rats (150-200g) that vJere fasted ovenught prior to tissue
isolation.
Livers were homogenized in five volumes of homogenization buffer (O.SM
sucrose,
20mM HEPES-I~OH, SmM MgCI2 supplemented with an EDTA-free Protease
Inhibitor Cocktail from Roche) utilizing a blaring blender (10 seconds low, 10
seconds high, and 10 seconds low). Following homogenization, a post-nuclear
supernatant was obtained by centrifugation at 4-5000 x g for 10 minutes.
Following
the first post-nuclear spin, the supernatant was decanted carefully. The post-
nuclear
supernatant was equilibrated to isotonic conditions by addition of an equal
volume
of dilution buffer (20mM HEPES-I~OH, pH 7.2, SmM MgClz).
Continuous flow ultf°acentrifugation. For continuous flow
centrifugation,
sucrose gradient was established in the PI~3-800 rotor after which the rat
liver
homogenate was fed into the machine. A flow rate of approximately 20 ml/min
was
used and the PI~II was operated initially at 20,000 rpm for the first pass and
then at
maximum speed, 40,000 rpm for the second pass. Samples from the effluent were
captured and later analyzed to determine the capture efficiency for the target
organelles. Organelles vere given additional time after all the homogenate had
been
fed to the system to reach their banding densities. The rotor was brought to a
controlled stop and the rotor contents were unloaded from the bottom in 25 ml
aliquots.
In another experiment, the PI~3-800 rotor was filled with buffer (250mM
sucrose, 20n~ HEPES-I~OH, pH 7.2, SmM MgCl2) and air was removed from the
system by spinning the rotor at 10,000 rpm. Flow through the lines was
increased to
300 ml/min and flow through the rotor was reversed several times until air had
been
cleared from the system. The rotor was brought to a stop and the gradient
material
(i.e. sucrose) was pumped to fill half the rotor volume (approximately 400
ml).
57
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The rotor was accelerated under automatic operation to the maximum speed
(35,000 rpm or 409000 rpm). Flow of buffer was allowed to continue at
approximately 40 ml/min during gradient formation. ~nce the homogenate pool
was ready for processing, the rotor speed was reduced to 20,000 rpm. The
homogenate was fed at 20m1/min and the effluent material was collected and a
sample was retained for later analysis.
The feed was switched back to buffer and the rotor speed was increased to
35,000 or 40,000. The effluent collected from the 20,000 rpm feed was then re-
fed
to the PKII at 20 ml/min. The effluent was collected and a sample was retained
for
later analysis.
The feed was switched back to buffer and the lines were cleared. The flow
was then shut off and the material in the rotor was allowed to band for 45
minutes or
2 hours. The rotor was brought to a controlled stop and fractions were
immediately
collected. Aliquots were prepared and stored at -80°C. Working aliquots
were
maintained at 4°C for immediate analysis.
Identification of organelles followifzg cehtrif~cgation. After centrifugation,
the integrity , separation, and enrichment of the isolated organelles were
determined
by Western blotting, enzymatic assays and electron microscopy. The results of
these
experiments are summarized in Figures 3-6.
FIG. 3 shows the relative distribution of mitochondria, Golgi, endoplasmic
reticulum, and plasma membrane and sub-types thereof in different fractions of
sucrose gradient following separation and accumulation of these organelles as
described above. The X axis of the figure corresponds to each of the fractions
measured for organelle content. The Y axis indicates percentage of these four
organelles and sub-types thereof, detected at the corresponding sucrose
gradient
fractions, relative to the population within the range of gradient examined
for each
of these organelles and sub-types thereof. The ~2 axis shows the percentage of
sucrose for each corresponding fraction of the gradient. FIG. 3 indicates the
distribution of each of these organelles and sub-types thereof in distinct and
well-
defined locations in the gradient.
58
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FIG. 4 shows the relative enrichment of mitochondria, Golgi, endoplasmic
reticulum, and plasma membrane and sub-types thereof in different fractions of
sucrose gradient following separation and accumulation of these organelles as
described above. The X axis of the figure corresponds to each of the fractions
measured for relative organelle marker response. The Y axis indicates relative
organelle marker response (pixels) of these four organelles and sub-types
thereof,
detected at the corresponding sucrose gradient fractions, relative to the
population
within the range of gradient examined for each of these organelles and sub-
types
thereof. The Y2 axis shows the percentage of sucrose for each corresponding
fraction of the gradient. FIG. 4 shows the relative enrichment of each of
these
organelles and sub-types thereof in distinct and well-defined locations in the
gradient using the method of the invention.
FIG. 5 shows the high integrity level of the isolated organelles-above
values typically seen in the art. The data shows that endoplasmic reticulum,
mitochondria, Golgi apparatus, and plasma membrane, and sub-types thereof,
attained integrity levels of 76.3% (endoplasmic reticulum), 72.6%
(mitochondria),
89.3% (Golgi), and 72.7% (plasma membrane), respectively. Integrity was
determined by comparing the level of an organelle-specific enz5nnatic activity
between the soluble and insoluble phases of the organelle preparations of the
invention. The enzymatic activity of the insoluble fraction (organelles) was
compared relative to the total enzymatic activity determined for both the
soluble
(supernatant) and the insoluble fractions.
Integrity for endoplasmic reticulum was determined collectively by
quantitative enzymatic assays, Western blots to organelle-specific marker
proteins
and electron microscopy experiments. In particular, pellets and supernatants
were
assayed in parallel for organelle-specific marker er~ymes and proteins.
Detection of
the marker in the pellet at a level> 60% is indicative of
intactness/integrity. In
contrast, detection of the marker protein in the supernatant is an indication
that the
outer periphery of the organelle is compromised. For Western blots, the same
antibodies were used to detect organelle-specific markers as used for the
method of
59
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determining purity. Namely, anti-BiP/GRP78 antibody (BIB BI~SCIENCES) was
used to detect endoplasmic reticulun ~.
Transmission electron microscopy (TEM) was also employed to qualitatively
characterize the integrity of the organelles vis-a-vis their morphologies
(size, shape,
structural organization), which correlates with function. To determine
organelle
intactness by electron microscopy, samples from the fractionation procedure
were
collected immediately following the centrifuge run to avoid potential damage
from
further manipulation. Samples were selected based on the expected density
range as
reported in the literature for the respective organelles. Selected fractions
were
pelleted and fixed in a solution of 4% formaldehyde, 1 % glutaraldehyde in 0.1
M
phosphate buffer, pH 7.4 and stored at 4°C until needed for
preparation. Samples
were embedded, sectioned, stained with uranyl acetate and lead citrate and
observed
using a Zeiss electron microscope.
FIG. 6 compares the TEM of a crude extract sample and an endoplasmic
reticulum fraction following the fractionation method described above. As
compared to the TEM of the crude extract, it can be seen that the subcellular
structures present in the ER fraction are almost exclusively endoplasmic
reticulum.
This observation qualitatively illustrates the high degree of purity and
enrichment
obtained by the fractionation method of the invention. Further upon
inspection, the
ultrastructure of the organelles in both the crude and the ER samples aa-e
seemingly
well-intact, consistent with the high level of integrity as determined
quantitatively
(FIG. 2).
EXAMPLE 2. PARALLEL ISOLATION, PURIFICATION AND
ENRICHMENT
OF MITOCHONDRIA, ENDOPLASMIC RETICULUM,
~OTL~I, Al'j~I~ P1LASI~A_I~EMBI~AI'~~TE F~R
PR~TlE~MlIC AI'~TAll~~~I~ FR~T'~ HELA ~'LETIaL~
HeLa cells were cultured in Joklik modified SMEM (Sigma, #61100-103)
that was supplemented with sodium bicarbonate (Amresco, #0865), 10% fetal
bovine serum (Paragon BioServices, #30101121) and 50 ug/ml gentamycin
(Amresco, #0304). Cells were scaled up from roller bottles into a 40 L fully-
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controlled bioreactor for inoculation into a 200 L bioreactor. The reactor was
seeded at a density of 1.0x105 cells/ml.
Three days later, cells were har~%ested from the reactor and concentrated by
tangential flow filtration to a volume of 8 liters, which were subsequently
centrifuged at 2000 rpm for 12 minutes. The cell pellet was washed and
resuspended in I~PHS (Invitrogen, #14190-136) and then centrifuged again at
2000
rpm for 12 minutes. The supernatant was removed and the cell pellet was stored
at -
80°C in 30 g aliquots.
HeLa cell pellets were removed from -80°C storage. The pellets
were
thawed, pooled and homogenized in five volumes of homogenzation buffer (0.25M
sucrose, 20mM HEPES-KOH, pH 7.2, SmM MgCl2, EDTA-free Protease Inhibitor
Cocktail from Roche) utilizing a Dounce homogenizer (25 strokes). Following
homogenization, a post-nuclear supernatant was obtained by centrifugation at
4000 x
g for 10 minutes. Following the first post-nuclear spin, the supernatant was
decanted. The nuclear pellet was then reprocessed to generate a second post-
nuclear
supernatant utilizing a blender (10 sec. Low, 10 sec. High, and 10 sec. low)
(in 5
volumes of buffer) and same centrifugation parameters used above. The second
post-nuclear supernatant was decanted and combined with the first post-nuclear
supernatant. The resultant pooled homogenate was used in the PKII for
fractionation of the organelles. Aliquots of the crude homogenate were stored
at -
80°C for later analysis.
To gauge the overall organelle content of a given fraction and to compare
between fractions, the refractive index for each sample was determined using
an
Abbe refractometer. Percent (%) sucrose may be calculated from refractive
index
measurements. Alternatively, it may be obtained through conversion tables of
refractive index to percent sucrose in reference texts such as the CRC
Handbook of
Chemistry and Physics (Ed. R. feast, CRC Press Inc., 58th Edition). FIG. 11
depicts the percentage sucrose content for collected post-centrifugation
fractions of
homogenized and centriguged HeLa cells. This figure relates directly to the
fractions illustrated in FIGS. 7 and 8 (described below) and this example.
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In order to test for intactness and enrichment of the isolated organelles, the
isolated fractions were subjected to a combination of electron microscopy
analysis,
Western blotting and succinate dehydrogenase enzymatic assay.
To test for intactness of organelle isolation, samples from the fractionation
were collected immediately following the run to avoid potential damage from
further
manipulation. Samples were selected based on the expected density range as
reported in the literature for the respective organelles. Selected fractions
were
pelleted and fixed in a solution of 4% formaldehyde, 1 % glutaraldehyde in 0.1
M
phosphate buffer, pH 7.4 and stored at 4°C until needed for
preparation. Samples
were embedded, sectioned, stained with uranyl acetate and lead citrate and
observed
using a Zeiss electron microscope.
In order to standardize Western blotting and enzymatic assays, the protein
concentrations of the organelle-containing fractions were determined by
Bradford
assay (BIO-R.AD, #500-0006). Samples were incubated with Coomassie reagent for
five minutes at room temperature, and the absorbance was measured (595nm). A
standard curve was generated using BSA (Pierce, #23210).
After determining the protein concentration of the organelle-containing
fractions, the fractions were ascertained as to their organelle composition by
screening each of the fractions by Western (immunoblot) blot using antibodies
to
known organelle-specific markers. Equal quantities of protein extracts from
the
organelle-containing fractions were resolved by polyacrylamide gel
electrophoresis
followed by the detection of the organelle-specific markers using appropriate
antibodies. For example, anti-Tom20 antibody (BD BIOSCIENCES) was used to
detect mitochondria, anti-GM130/P115 antibody (BD BIOSCIENCES) was used to
detect Golgi, anti-BiPlGRP7~ antibody (BD BIOSCIENCES) was used to detect
endoplasmic reticulum, and anti-NaI~ATPase antibody (LTI~TTI~. OF IOWA) was
used
to detect plasma membrane.
To carry out polyacrylamide gel electrophoresis, samples were mixed with 4
x NuPAGE SDS sample buffer (T1~T~ITROGEN, #NP0007) and SO mM DTT prior
to being loaded into either 1.0 mm x 10 well or 1.5 mm x 15 well, 4-12% Bis-
Tris
gradient minigels (INVITROGEN, #NP0335 or NP0323). Samples were
62
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electrophoresed for approximately 4~0 minutes at 150 ~ using MSS SDS running
buffer. For total protein analysis, gels were stained for 0.5 hours in
Coornassie blue
in 40% methanol, 10~/o acetic acid and subsequently destained in a 10%
methanol,
10% acetic acid solution. Immunoreactive bands were detected using ECL
detection
(#I~N2108, ECL Western Slotting Analysis System, AMEI~SH~I, TNC.) and
quantified using Kodak I2igital Science 1D Image Analysis software (I~~D).
In addition to Western blotting, enzymatic essays, for example, the succinate
dehydrogenase enzymatic assay, were carried out to further assess the
integrity of
the isolated organelles of the recovered fractions. For these experiments,
each 50 ul
sample of organelle fraction was incubated with 0.3 ml of a O.O1M solution of
sodium succinate (Sigma, #52378) in 0.05 M phosphate buffer, pH 7.5. Following
incubation at 37°C for 10 minutes, 0.1 ml of a 2.5 mglml solution of p-
Iodonitrotetrazolium violet (INT) (Sigma, #I8377) in 0.05 M phosphate buffer,
pH
7.5 was added. The tubes were incubated at 37°C for 10 minutes. The
reaction was
stopped with the addition of 1.0 ml of ethyl acetate: ethanol: trichloroacetic
acid in a
ratio of 5:5:1 (v,v,w). The tubes were centrifuged at 1.5,000 rpm for 1 minute
before
measuring the absorbance at 490nm. The results of these experiments are
summarized in Figures 7-11.
FIG. 7 shows the relative distribution of mitochondria, endoplasmic
reticulum, and plasma membrane and sub-types thereof in different fractions of
sucrose gradient following separation and accumulation of these organelles as
described above. The X axis of the figure corresponds to each of the fractions
measured for organelle content. The Y axis indicates percentage of these three
organelles and sub-types thereof, detected at the corresponding sucrose
gradient
fractions, relative to the population within the range of gradient examined
for each
of these organelles and sub-types thereof. The Y2 axis shows the percentage of
sucrose for each corresponding fraction ofthe gradient. FIG. 7 indicates the
distribution of each of these organelles and sub-types thereof in distinct and
well-
defined locations in the gradient.
FIG. 8 shows the relative enrichment of mitochondria, endoplasmic
reticulum, and plasma membrane and sub-types thereof in different fractions'of
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sucrose gradient following separation and accumulation of these organelles as
described above. The ~ axis of the figure corresponds to each of the fractions
measured for relative organelle marker response. The ~' axis indicates
relative
organelle marker response (pixels) of these three organelles and sub-types
thereof,
detected at the corresponding sucrose gradient fractions, relative to the
population
within the range of gradient examined for each of these organelles and sub-
types
thereof. The Y2 axis shows the percentage of sucrose for each corresponding
fraction of the gradient. FIG. ~ shows the relative enrichment of each of
these
organelles and sub-types thereof in distinct and well-defined locations in the
gradient using the method of the invention.
EXAMPLE 3: COMPARATIVE ENRICHMENT STUDIES USING
HELA CELLS
Referring to the experimental conditions presented in Example 2 above,
comparative enrichment was studied in accordance with the following data.
FIG. 9 and 10 illustrate the comparative levels of enrichment achieved by the
method of the invention. Emiclunent can be determined qualitatively either
using
Western blots or enzymatic assays of organelle-specific markers and/or enzymes
contrasting the signal/activity from the particular fraction of interest to
the
signal/activity present in another fraction or in the original crude extract
of the
biological sample prior to fractionation. Relative enrichment can be
determined
based upon the accumulation of the marker protein in the organelle fraction
relative
to another organelle fraction. Further, enrichment can be measured by the
activity
of an organelle-specific marker enzyme for an organelle of interest relative
to the
activity of the same marker enzyme in another fraction or in the crude
homogenate.
FIG. ~ shows a Western blot of NaKATPase as detected by antiNaI~AATPase
antibody from each of the fractions of the biological sample.
FIG. 10 shows the measured level of NaI~ATPase from each of the fractions
of the sample. A comparison of FIGs. 9 and 10 indicate that fractions 14 and
15
have the highest.relative level of Na.KATPase. Since NaKATPase is the
organelle-
64
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WO 2004/083405 PCT/US2004/008655
specific marker for plasma membrane, the data suggests that fractions 14. and
15
have the greatest concentration of plasma membrane.
To determine organelle integrity and enrichment by Western blotting and/or
enzymatic assays, firstly, protein content was determined by a Bradford based
assay
(Bio-Rad, #500-0006). Samples were incubated with Coomassie reagent for five
minutes at rooam temperature, and the absorbance was measured (S~Snm). A
standard curve was generated using BSA (Pierce, #23210)
Prior to western blotting, samples were mixed with 4 x NuPAGE SDS
sample buffer (INVITROGEN, #NP0007) and 50 mM DTT prior to being loaded
into either 1.0 mm x 10 well or 1.5 mm into x 15 well, 4-12% Bis-Tris gradient
minigels (INVITROGEN, #NP0335 or NP0323). Samples were electrophoresed for
approximately 40 minutes at 150 V using MES SDS running buffer. For total
protein
analysis gels were stained for 0.5 hours in Coomassie blue in 40% methanol,
10%
acetic acid and subsequently destained in a 10% methanol, 10% acetic acid
solution.
To carry out polyacrylamide gel electrophoresis, samples were mixed with 4
x NuPAGE SDS sample buffer (INVITROGEN, #NP0007) and 50 mM DTT prior
to being loaded into either 1.0 mm x 10 well or 1.5 rmn x 15 well, 4-12% Bis-
Tris
gradient minigels (INVITROGEN, #NP0335 or NP0323). Samples were
electrophoresed for approximately 40 minutes at 150 V using MES SDS running
buffer. For total protein analysis, gels were stained for 0.5 hours in
Coomassie blue
in 40% methanol, 10% acetic acid and subsequently destained in a 10% methanol,
10% acetic acid solution. Immunoreactive bands were detected using ECL
detection#RFN2108, ECL Western Blotting Analysis System,AMERSHAM, INC.)
and quantified using Kodak Digital Science 1D Image Analysis software (KODAK).
For the succinate dehydrogenase enzymatic assay, each 50 ul of the homogenate
was
incubated with 0.3 ml of a O.OlM solution of sodium succinate (Sigma. #52378)
in
0.05 M phosphate buffer, pH 7.5. Following incubation at 37°C for 10
minutes. 0.1
ml of a 2.5 mg/ml solution of p-Iodonitrotetrazolium violet (INT) (Sigma,
#I8377)
in 0.05 M phosphate buffer, pH 7.5 was added. The tubes were incubated at
37°C
for 10 minutes. The reaction was stopped with the addition of 1.0 ml of ethyl
CA 02518555 2005-09-14
WO 2004/083405 PCT/US2004/008655
acetate: ethanol: trichloroacetic acid n a ratio of 5:5:1 (v,v,w). The tubes
were
centrifuged at 15,000 rpm for 1 minute before measuuing the absorbance at 4.~0
mn.
E~AT~~I! ~L~ 4~ AI'~1AL ~ ~T1~ ~h ~TIJBc~E~LULA~ 1~R~TE~I~E~ ~~
~RGANEL>LE ~RAcC°I'~~1~TS )~Y 2I~ ~E~.
ELE~'IC1~~~H~I~E~Tl~ ~I'~T~D T~IA~~ ~~lE~CTR~I~lE'1C~
B~~EAL~ 1'~T~~YEL ~ll~~°~~Ill'~T~
The subcellular proteomes of the organelles of the fractions provided by
Examples 1 and 2 were further analyzed by 2D gel electrophoresis and mass
spectrometry. To analyze the subcellular organelle proteomes, proteins were
separated by two-dimensional gel electrophoresis ("2D-GE"). It will be
appreciated
by one of ordinary skill in the art that 2D-GE is a powerful approach for
separating
complex mixtures of proteins. All proteins in an electric field migrate to a
defined
distance that is dependent upon their conformation, molecular size and
electric
charge. 2D-GE uses the latter two of these parameters to allow high-resolution
separation of proteins. In the first dimension, isoelectric focusing is used
to separate
proteins based on their isoelectric point. In the second dimension, SDS
polyacrylamide gel electrophoresis is used to fractionate proteins according
to their
molecular weights. The result is an array of proteins spots that are assigned
X and Y
coordinates.
Here, separation of organelle protein extracts subsequent to organelle lysis
was performed by 2D-GE and detection was with either Coomassie blue, silver
staining or Sypro RubyTM (MOLECULAR PROBES). Organelle protein extracts
were compared relative to unfractionated crude extracts fractionated on 2D-GE
gels,
all stained with Coomassie blue, silver, or Sypro RubyTM. Digital images of
the 2D
gels were generated and annotated using Z3TM software (COMPUGEN) or
ProgenesisT~ software (IVON LINEAR). Resultant images were superimposed to
identify common and new spots, especially low-abundance proteins.
The isoelectric focusing step was performed using Bio-Rad 7 cm IPG strips
over a full pH range (3-10). SDSPAGE was then performed using pre-cast
NuPAGE 4-12°1° Bis-Tris ZOOM gels with a molecular weight
standard. Samples
were run in duplicate with one gel stained with Coomassie and a second gel
stained
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with Silver. ~rganelle fractions and crude homogenates were subjected to mass
spectrometry using a 2-D gel intenmdiary and analyzed by I~ALDI.
FIG. 12 compares the protein spot patterns of a crude extract (A) of rat liver
tissue and the endoplasmic reticulurn fraction (E) of Example 1. Compared to
the
crude extract gel, the endoplasmic reticulum gel shows significantly greater
proteome content, i.e. a greater number of visible and/or detectable protein
or
polypeptide spots.
In addition to the 2D gel results shownn in FIG. 12 for the analysis of the
endoplasmic reticulum fraction, similar 2D gel analysis was carried out for
fractions
containing mitochondria, plasma membrane, and Golgi apparatus (data not
shown).
The resulting 2D gels were further analyzed by mass spectrometry. A number of
the
spots of the gel of FIG. 12(A), as well as the gels for the mitochondria,
plasma
membrane, and Golgi apparatus fractions, were analyzed by mass spectrometry.
The resulting peptide profile determined for each spot was compared against
known
peptide profile databases such as, for example, GENBANK and SWISS-PROT, to
determine the identity, if any, of the protein spot.
The results showed that many proteins could be detected in the mitochondria,
endoplasmic reticulum, Golgi apparatus, and plasma membrane fractions that
were
not present or detectable in the 2D gels of the crude extract. Further, the
proteins
fOlllld on the 2D gels of each of the organelle fractions were identified as
having a
broad range of molecular weight, namely a high molecular weight of about 80-
125
kD to a low molecular weight of about less than 20 lcD. Thus, the results
suggest
that the method of the invention is not biased or limited as to any particular
molecular weight. A number of protein spots that were not observable on the 2D
gel
of the starting biological material and were of low-intensity on the 2I) gel
of the
organelle-containing fraction were analyzed by mass spectrometry. Samples from
both IieLa cells (data not sh~wn) and rat liver tissue were e~~amined. ~f the
protein
spots examined for the rat liver tissue, about 50% were found to match
proteins
deposited in SWISS-PRGT. Also, the identity of about 50% of the protein spots
were ascertained through sequence analysis and comparison to known sequences
in
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CA 02518555 2005-09-14
WO 2004/083405 PCT/US2004/008655
GENBANI~. Where necessary, homology searching was carried out on non-rat
databases. Results from the rat liver tissue are shown in FIG. 21A and 21B.
Based on the identity of the proteins having matches to known proteins in
existing databases, the method of the invention detected a variety of
proteins,
including metabolic enzymes, proteosome components, translational factors,
receptors, immunological components (complement), and ribosomal proteins.
E PLE 5. ANALYSIS OF SIJBCELLiJLAR PROTEOMES OF
GOLGI AND
PLASMA MEMBRANE FRACTIONS BY 2D GEL
ELECTORPHORESIS AND MASS SPECTROMETRY
DEMONSTRATES DETECTION OF POST-
TRANSLATIONAL OR OTHER VARIANTS OF
PEPTIDYL-PROLYL CIS-TRAMS ISOMERASE
(CYCLO-SPORIN A-BINDING PROTEIN)
Separate fractions containing Golgi apparatus and plasma membrane isolated
from HeLa cells according to Example 2, as well as HeLa crude extracts, were
analyzed by 2D gel electrophoresis and mass spectrometry. The fractions were
lysed to release organelle-contained proteins. A Bradford assay (Bio-Rad, #500-
0006) was used to determine the concentration of the protein in Golgi sample,
the
plasma membrane sample, and the crude extract sample. Next, as outlined above,
2D gel electrophoresis was carried out on equal quantities of protein from
each of
the samples. As described previously, the 2D gel was stained appropriately to
visualize the protein spots and then imaged by ProgenesisTM software (NON
LINE).
FIG. 13B shows the results of 2D gel electrophoresis of the crude extract, the
Golgi fraction, and the plasma membrane fraction. Each are provided in
triplicate
from three individual 2D gels. FIG. 13A shows a close-up of the Golgi sample 3
and points to protein spots 12, 13, and 14. Spots 12, 13, and 14 appear to be
visible
in both the Golgi and plasma membrane fractions; however, the same spots do
not
68
CA 02518555 2005-09-14
WO 2004/083405 PCT/US2004/008655
appear evident in the crude extract sample. As such, spots 12, 13, and 14.
likely
represent lo-w-abundance proteins.
IVIass spectrometry was can-ied out on spots 12, 13, and 14. to identify the
polypeptides therein. FIG. 14 shows the mass spectrometry data for each of the
peptide spots. The tables list for each spot both the sequence of the peptide
fragment detected (indicated from left to right in the hT-terminal to C-
terminal
direction) and the average molecular mass for each fragment. Upon inspection
of
FIG. 14, it can be noticed that the same or substantially overlapping peptide
fragments are detected, which is consistent with each of the spots 12, 13, and
14
being the same protein. Thus, each of the proteins is the same or
substantially same
molecular mass, which is consistent with their equivalent migration distances
from
the top of the gel. However, since 2D gel electrophoresis resolves proteins in
two
dimensions, namely in one direction based on molecular mass and in another
based
on charge, the overall charge of the proteins must be different to the extent
that they
are resolved by the electrophoresis. Thus, this observation suggests that the
protein
spots 12, 13, and 14 are three different post-translational or other variants
forms of
the same protein. Perhaps, one spot represents the unmodified protein product
and
the remaining spots represent two unique post-translationally modified or
amino
acid substituted variants. Perhaps all three represent distinct variants.
The results demonstrate two advantages of the present invention. First, the
results show enhanced sensitivity in the detection of low-abundance proteins,
e.g.,
proteins that are not detectable in the crude extract but which are detected
in the
organelle fractions prepared by the method of the invention. Second, the
results
demonstrate that the fractionation method of the instant invention provides
for the
enhanced separation and detection of different variants of a low-abundance
protein,
which is an advantage given that much of the complexity of a proteome is
derived
from a multitude of modifications of proteins occurring during or following
protein
translation, - which act to alter protein characteristics, such as, for
example,
enzymatic activity, solubility, and stability.
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WO 2004/083405 PCT/US2004/008655
E~AP~IPILE ~. A1~1A~~1~~~ ~1F ~TIl~~E~~~Tf.,AR ~~~~'lE~T~lL~~ ~P"
~Af~T~~J~ ~1~~~A1~'JETLILE ~"~A~ ll~~~l~T~ l~~ ~~D ~E~
~ILEWL"~1~.1h~1~ 1T~E~fI~ A1~TID IV~IA~~ ~1~~~'hl IL~GI~111E'f"ll~~I
Two-dimensional gel electrophoresis was carried out on various organelle
fractions prepared according to the method of the invention. The resulting
gels were
appropriately stained and imaged by ProgenensisTM software as described
previously. Mass spectrometry was carned out as before on a plurality of
protein
spots. The resultant peptide fragments identified for each protein spot was
compared to the sequences of proteins contained in existing databases,
including
GENBANK and SWISS-PROT.
FIG. 15, FIG. 16, FIG. 17, and FIG. 18 show the results for endoplasmic
reticulum, mitochondria, Golgi, and plasma membrane, respectively. In each
figure,
Panel A shows the complete ZD gel image of the resolved subcellular proteome
for
each of the organelle fractions. The complete crude extract gel is not shown.
Circles indicate the location of the protein spots detected by mass
spectrometry.
Also for each figure, Panel B shows a localized portion of the gel in Panel A
in
triplicate for three individual 2D gels. The top row of Panel B shows the
corresponding localized panel of the crude extract 2D gel, also shown in
triplicate
from three individual 2D gels.
From a comparison of the localized images of the crude extract and organelle
fraction 2D gels, it can be seen that numerous protein spots are visible in
the
organelle fraction panels but absent from the crude extract panel. In
particular, a
protein spot, which is absent from the 2d gel of the respective organelle, is
circled
for each of the organelle fraction gel images.. Thus, this suggests that the
protein
spots occurring in the organelle fraction gels are proteins which are not
detectable in
the crude extract samples. In Panel A, each of the spots of the gels of each
of the
FIGS was analyzed by mass spectrometry, as described previously.
This Example demonstrates that the fractionation method of the invention
provides for the detection of proteins in subcellular fractions prepared by
the method
of the invention, said proteins not being detected in the corresponding crude
extract.
CA 02518555 2005-09-14
WO 2004/083405 PCT/US2004/008655
E~'~1D~P~E 7. 1PA~AILIdE~ ~~~LA~°~~T~~T9 ~ILT1F~~A'ICf~1''~ A1~~T~
~~° lEI'~T~~~ILA~T~I1~~C 11~'lI"T1EC~JI~~I.TI~1 A~~T~
f°~LA~IY~tA
T~fEI~B'~E
1F~~I'~I 1FIEAIL~CEII~ Al'~T11D ~~~1~A~EID 1~A1'~T~CREA~'f~
'1I'f~~flTlE ~E°~I1~.
FIJ~7Cfil~~ 1P~~TE~I~~LE ~C~I~BAS~I~~T ~T~1~~E~.
Pancreas Ho~2ogenizatio~z. For these experiments, twenty healthy and
diabetic Wistar rats (150-2008 each) are fasted overnight prior to
decapitation,
dissection and pancreas harvest. Pancreases (100 grams in total) are
homogeiuzed
in five volumes of homogenization buffer and subjected to homogenization by
mechanical shear method utilizing blaring blender.
After homogenization, the post nuclear supernatant is obtained by
centrifuging the homogenate at 4-10,000 X g for 10-20 minutes. The supernatant
is
then adjusted to isotonic conditions by addition of an equal volume of
dilution
buffer supplemented with protease inhibitors.
Cohtirauous flow cerat~ifugation. The rat pancreas homogenate is fed into the
PI~3-800 rotor having a pre-established sucrose gradient therein. A flow rate
of
approximately 10-30 ml/min is used and the PI~II is operated initially at
15,000-
25,000 rpm for the first pass and then at maximum speed, 40,500 rpm for the
second
pass. At the end of centrifuge run, the rotor contents are unloaded from the
bottom
of the rotor in 25 ml fractions. Samples from each fraction are analyzed to
determine
the capture efficiency for the target organelles, such as ER and plasma
membrane.
The integrity and enrichment of the isolated organelles are determined by
Western blotting, enzymatic assays and electron microscopy. For these
experiments,
the fractions containing plasma membrane and ER are lysed and the protein
content
therein is determined by Bradford assay (Bio-Rad, #500-0006). Samples are
incubated with Coomassie reagent for five minutes at room temperature and the
absorbance is measured at 595nm. A standard curve is generated using BSA
(Pierce, #23210).
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WO 2004/083405 PCT/US2004/008655
After determining the protein concentration of the plasma membrane and
ER-containing fractions, samples are mi~~ed with 4.x NuPAGE SDS sample buffer
(IN~ITR~GEN, #NP0007) and 50 mM DTT prior to being loaded into either 1.0
mm x 10 well or 1.5 mmxl5 well, 4-12% Bis-Tris gradient minigels
(INVITR~GEN#NP) 335 or NP0323) for polyacrylamide gel electrophoresis.
Samples are electrophoresed for approximately 40 minutes at 150 ~ using MES
SDS rurming buffer. For total protein analysis, gels are stained for 0.5 hours
in
Coomassie blue in 40% methanol, 10% acetic acid and subsequently distained in
a
10% methanol, 10% acetic acid solution.
The fractions are measured for enrichment of organelle composition by
screening each of the fractions by Western blot using anti-NaKATPase antibody
for
plasma membrane detection and anti-BiP/GRP78 for endoplasmic reticulum
detection. Fractions are characterized using ECL detection (#RPN2108, ECL,
Western Blotting Analysis System, AMERSHAM, INC) and quantified using Kodak
Digital Science 1D Image Analysis software.
To assess the integrity of the isolated organelles transition electron
microscopy (TEM) is employed.
To determine organelle intactness by electron microscopy, samples from the
fractionation procedure are collected immediately following the centrifuge run
to
avoid potential damage from further manipulation. Samples are selected based
on
the expected density range as reported in the literature for the ER and plasma
membrane. Selected fractions are pelleted and fixed in a solution of 4%
formaldehyde, 1% glutaraldehyde in O.1M phosphate buffer, pH 7.4 and stored at
4°C until further preparation. After selection, samples are embedded,
sectioned,
stained with uranyl acetate and lead citrate and observed using a ~eiss
electron
microscope.
Additionally, to determine organelle integrity and intactness, succinate
dehydrogenase enzymatic assay is perfornzed. For these experiments, a SOuI
sample
of organelle fraction is incubated with 0.3 ml of a O.O1M solution of sodium
succinate (Sigma, #52378) in O.OSM phosphate buffer, pH 7.5. Following
72
CA 02518555 2005-09-14
WO 2004/083405 PCT/US2004/008655
incubation at 37°C for 10 minutes, 0. lml of a 2.5 mg/ml solution of p-
Iodonitrotetrazolium violet (II~TT) (Sigma, #I8377) in O.OSI~I phosphate
buffer, pl=I
7.5 is added. The tubes are incubated at 37°C for 10 minutes. The
reaction is
stopped with the addition of l.Oml of ethyl acetate:ethanolarichloroacetic
acid in a
ratio of 5:5:1 (v,v,w). The tubes were centrifuged at 15,000 RPM for 1 min
before
measuring the absorbance at 490 nm.
To determine whether the insulin receptor is localized to the plasma
membrane or ER in the pancreatic tissue of healthy versus diabetic rats, the
isolated
organelles are lysed and the resulting proteins are subjected to the 2-D PAGE
analysis as described in the Example 9. The gels for the healthy and diabetic
rat are
then compared to ascertain the location of the insulin receptor.
EXAMPLE 8. ANALYSIS OF THE CELLULAR LOCALIZATION OF
INSULIN
RECEPTOR BEFORE AND AFTER ROSIGLITAZONE
MELEATE TREATMENT OF DIABETIC RATS.
Rosiglitazone ameleate (also known as Avandia, GSK) is a well known
drug given to patients with Type II diabetes for sugar control. The molecular
basis
underlying the action of this drug is unknown and recent studies implicated
the role
of rosiglitazone in improvement of insulin secretion and changes in insulin
receptor
abundance and signal transduction (Diabetes, volume 52, pages 1943-1948,
2003).
This example illustrates the use of the instant invention to further elucidate
the
molecular basis of rosiglitazone, specifically, the role of the drug to alter
the cellular
localization of insulin receptor.
For these experiments, adult Wistar rats are housed in groups of four
animals per cage with instant access to food and water. None of the drug
treatments
are designed to affect general well-being of the animals. The rosiglitazone
ameleate
is administered to rats in drinking water. At the end of the treatment, rats
are killed
by decapitation. The pancreas (100 grams in total) is harvested from
approximately
twenty diabetic rats, those with and without drug treatment. Diabetic rats
without
rosiglitazone treatment are used as controls.
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CA 02518555 2005-09-14
WO 2004/083405 PCT/US2004/008655
Payac~eas FI~raa~genizati~fz. Pancreases obtained from rats before and ~.fter
rosiglitazone treatment are homogenized by mechanical shear method utilizilzg
blaring blender. Following homogenization, the post nuclear supernatant is
obtained by centrifuging the homogenate at 4-10,000 X g for 10-20 minutes. The
supernatant is then adjusted to isotonic conditions by addition of an equal
volume of
dilution buffer supplemented with protease inhibitors.
The resultant SI homogenate is reprocessed to generate a second post-nuclear
supernatant using the same disruption and same centrifugation conditions as
described above. The second postnuclear supernatant is equilibrated to
isotonic
conditions and used as a feed material for the PKII (Alfa Wasserman)
centrifuge.
Continuous flow cejZtrifugation. For these experiments, the sucrose gradient
is established in the PI~3-800 rotor after which the rat pancreas homogenate
is fed
into the centrifuge. A flow rate of approximately 10-30 ml/min is used and the
PKII
is operated initially at 15,000-25,000 rpm for the first pass and then at
maximum
speed, 40,500 rpm for the second pass. Samples from the effluent are captured
and
further analyzed to determine the capture efficiency for ER and plasma
membrane.
These organelles are given additional time to reach their densities after all
the
homogenate had been fed to the system. The rotor is brought to a controlled
stop
and the contents are unloaded from the bottom in 25 ml aliquots.
After centrifugation, the intactness and enrichment of isolated ER and
plasma membrane are determined by Western blotting, enzymatic assays and
electron microscopy as described in Example 7.
To determine the differences in cellular localization of insulin receptor
before and after rosiglitazone treatment, the isolated organelles are lysed
and further
subjected to 2-D PAGE as described in Example 9.
E~~T~E ~. ~l'~TAIL~~~~ ~~' l~lL~~i~~ l~f! ~I'~1~~11~ AI'~1~ ~~
PR~TE~1~~IE~
I~~l ~li~ ~ETL, IElLE~'L~1'~PPl(~~~~
74
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WO 2004/083405 PCT/US2004/008655
The subcellular proteomes of the ER and plasma membrane of the fractions
provided by Examples 7 and ~ are fitrtl2er analyzed by 2D gel electrophoresis.
To
aalalyze the subcellular proteomes, proteins are separated by two-dimensional
gel
electrophoresis. Separation of ER and plasma membrane extracts, subsequent
to organelle lysis, is performed by 2D-PAGE and detection is by either
Coomassie
blue, silver staining or Sypro Ruby (Molecular Probes). Digital images of the
2D
gels are generated and annotated using ~3 software (Compugen) or Progenesis
software (Nonlinear). Resultant images are superimposed to identify spots
corresponding to insulin receptor.
Thus, the protein spot patterns of ER and plasma membrane are analyzed
and the insulin receptor localization in diabetic pancreatic tissue before and
after
rosiglitazone treatment is compared to the insulin receptor localization in
healthy
pancreatic tissue.
This example illustrates how combining subcellular fractions obtained by the
PKII system with 2D gel electrophoresis allows one skilled in the art to
achieve one
of the major goals of subcellular proteomics, namely, monitoring protein
translocation events.
EXAMPLE 10. ESTIMATATION OF THEORETICAL AMOUNTS OF
BIOLOGICAL MATERIAL NECESSARY TO DETECT
A PROTEIN RELATIVE TO ITS COPY NUMBER IN A
CELL.
FIG. 22A and 22B illustrate the advantages of using the continuous-flow
process of the invention. For example, the figures indicate the folds of
accumulation
required for a particular amount of starting biological material typically
needed to
reach the detection limit of 50 ng in relation to the copy number of a protein
in a
cell. In one embodiment, referring to FIG. 22A given 1X10(9) cells of starting
biological material, one would need to use an ~ 19-fold increase in cell
number to
reach the detection limit of 50 ng for a protein occurnng at a single copy per
cell.
***
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WO 2004/083405 PCT/US2004/008655
'Those skilled in the art will recogW ~e, or be able to ascertain without
undue
experimentation any of the numerous equivalents to the embodiments of the
invention described herein. All such equivalents are considered to be within
the
scope of the instant invention and are encompassed by the claims that follow.
Unless otherwise explained, all technical and scientific terms used herein
have the same meaning as commonly understood by one of ordinary skill in the
art
to which the invention belongs. Although methods and materials similar or
equivalent to those described herein can be used in the practice or testing of
the
present invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references mentioned
herein are
incorporated by reference in their entirety. In case of conflict, the present
specification, including explanations of terms, will control. In addition, the
materials, methods, and examples are illustrative only and not intended to be
limiting.
Although preferred embodiments of the present invention and modifications
thereof have been described in detail herein, it is to be understood that this
invention
is not limited to those precise embodiments and modifications, and that other
modifications and variations may be affected by one skilled in the art without
departing from the spirit and scope of the invention as defined by the
appended
claims.
76
