Note: Descriptions are shown in the official language in which they were submitted.
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SELECTOR BASED RECOGNITION AND QUANTIFICATION
SYSTEM AND METHOD FOR MULTIPLE ANALYTES
IN A SINGLE ANALYSIS
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. Provisional Patent
Application Serial
No. 61/594,193, filed February 2, 2012 (Atty. Docket No. PERF-P0006-US), and
is a
continuation-in-part of U.S. Patent Application Serial No. 12/721,173, filed
March 10, 2010
(Atty. Docket No. PERF-P0004-US) and International Patent Application No.
PCT/U52010/026819, also filed March 10, 2010 (Atty. Docket No. PERF-P0001-W0),
the
disclosures of which are hereby expressly incorporated by reference herein in
their entirety.
FIELD OF THE DISCLOSURE
[0002] The present disclosure relates generally to a multi-
dimensional analytical strategy
and system for analyzing antigens and particularly to an affinity selector
based recognition and
quantification system and method for analyzing multiple analytes in a single
analysis.
BACKGROUND OF THE DISCLOSURE
[0003] Determining a single analyte in a mixture of ¨100,000 other
components is a
formidable task. More than 60 years ago, analysts began to recognize that the
structural
selectivity of antibodies could be used to bind and purify antigens and
haptens from biological
extracts or blood on the basis of their chemical structure. This technique
became so important
that Rosalyn Yalow was awarded the Nobel Prize for radio-immunological assays
(RIA) in 1960.
Along with enzyme linked immunosorbent assays (ELISA), these two technologies
provided the
world with a simple method to measure antigens down to the pg/mL level.
[0004] A fundamental component of both RIA and ELISA is the use of
immobilized
antibodies to achieve antigen selection from samples in the first step of an
assay. Since the
inception of these approaches, analytical immunologists have understood that
binding antibodies
at a surface introduces significant kinetic limitations. For instance,
antigens have to travel
substantial distances in terms of molecular dimensions to reach surfaces,
thereby adding to the
amount of time needed for antigen binding to occur in a test tube or
microtiter well. Moreover,
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all antibodies used in an assay are bunched together at the surface of an
assay well or on a
particle, while antigens are uniformly distributed throughout the solution.
When an ELISA is
carried out in a microtiter well, incubation times of a day or more are
typically used to allow
time for the antigens to diffuse to the walls of the well where the antibodies
are bound. Efforts to
minimize the diffusion problems in the case of RIA involved using large
numbers of very small
inorganic particles to which antibodies were immobilized. Over the course of
the past several
years, many types of mixing, flow, heating, and even sonication procedures
have been used to
minimize the diffusion problem noted above. Despite these efforts, diffusion
problems still exist.
[0005] As analytical chemistry has evolved, it has been recognized
that better and more
complete answers can be obtained to various questions involving a sample if
multiple analytes
are determined simultaneously. This in turn has led to an increased interest
in "analytical
multiplexing", where large numbers of analytes are analyzed in a sample during
the course of a
single analysis. This is often done through immunological arrays.
[0006] Interest in immunological arrays stems from the popularity and
success of micro-
electro-mechanical-systems (MEMS). While several types of antibody arrays have
been used for
large scale multiplexing, including high throughput and parallel processing
techniques, other
approaches have focused on large scale multiplexing with smaller numbers of
samples, such as
would be needed in a clinical laboratory that is concerned about minimizing
the total analysis
time and cost per assay. No matter what approach is utilized, immunological
array systems still
face challenges with respect to antibody immobilization and kinetics. There
are also issues with
respect to whether antibodies will retain full activity after immobilization,
particularly if they are
improperly oriented at the surface. Steric issues must also be considered,
particularly in terms of
the orientation of the antibody to the surface, as well as its packing
density. Finally,
reproducibility is also a factor, particularly as it is very difficult to
reproduce immobilized
antibodies from picoliter volumes of solution deposited on a surface.
Evaporation, as well as a
myriad of other phenomena, also diminishes reproducibility from that
experienced at the titer
plate level of immobilization.
[0007] Although the distance antigens must diffuse to reach surfaces
is smaller in an
immunological array than in a microtiter well, kinetic issues are still a
serious limitation with
immunological arrays. This is particularly true at low antigen concentrations.
A substantial
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amount of time is required for an antigen to diffuse from all points in the
solution to one of the
array elements. Because molecular docking in antigen:antibody complex
formations requires
precise spatial orientation, antigens generally strike surfaces many times
before establishing the
correct capturing orientation. For instance, if an antigen is not captured
after colliding with the
surface on a 128 element array, it has a lot of space to navigate before
striking the surface a
second time.
[0008] As noted above, particle based assays began with the RIA and
Yalow approaches.
Currently the Yalow approach has evolved into two types of assay systems: 1) a
particle
approach used in flow cytometry assays (e.g., the Luminex system) where the
fluorescence of
individual particles is examined; and 2) an approach where antibodies are
placed on a magnetic
particle where the immune complex is formed, and the particle is then pulled
out of solution and
the antigens released for further measurements (e.g., the SISCAPA system of
Leigh Anderson).
Multiplexing requires the preparation of a different set of immunosorbent
beads for each antigen
being determined. This means that 20-50 different sets of antibody carrying
beads would need to
be added to the sample solution, thereby causing even larger kinetic
limitations of the antigens in
terms of finding the appropriate antibody particle. In addition, the solution
becomes crowded
with so many particles that the antigens must diffuse around particles not
carrying their antibody.
One proposed solution for dealing with these limitations is to immobilize
multiple antibodies on
a single particle. However, this solution does not completely address the
issues of diffusion and
stoichiometric control. Moreover, the dilution of antibody concentration on
the particle surface
means that the antigens can strike particle surfaces, while not contacting
their antibody. In
addition, the total surface area, and thus the total number of particles
required, would still remain
high.
[0009] With respect to the flow cytometry strategy (such as the
Luminex system),
immune complexes must be formed on the particle surfaces before they can be
analyzed by flow
cytometry. While this system is very similar to the above, each bead carries a
single antibody
targeting a single antigen. Again, there is the diffusion problem in antigen
capture.
[0010] It is interesting that the function of mammalian immune
systems is to deal with
thousands of antigens, albeit not all of them simultaneously. As immunity to
foreign substances
develops in an individual mammal, antibodies to thousands of immunogens are
produced. These
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antibodies are contained within the immunoglobulins circulating in blood where
at any time,
hundreds of antigens are being sequestered as antigen:antibody complexes are
formed. Upon
analyzing mammalian immune systems, it can be concluded that antibodies have
evolved to
function in solution as they form immune complexes. In addition to functioning
in solution, they
also sequester large numbers of antigens at the same time and have few of the
limitations seen
within immobilized antibody assay systems.
[0011] The above-noted observations of mammalian immune systems are
extremely
important, particularly as they clearly suggest that the formation of immune
complexes in
solution are naturally efficient, while the formation of complexes on
immobilized surfaces are
not. Moreover, it is clear that several immune complexes can be simultaneously
formed in a
solution (such as blood), which is a necessary factor to achieve when
performing an analytical
multiplexing process. Finally, most of the problems that commonly impact
immunological
assays (e.g., loss of activity during immobilization, proper orientation of
antibodies, diffusion
kinetics and having sufficient surface area) are not prevalent within these
natural solution based
systems.
[0012] Despite the above-described advantages of naturally formed
immune complexes,
such immunological assays still require the addition of antibodies to the
samples, which can be
concerning. For instance, adding large numbers of antibodies to a plasma
sample may cause the
protein concentration to increase to such a level that analyte diffusion is
hampered. While this
issue may seem concerning on its face, upon taking a further look, it can be
concluded that this
issue is likely inconsequential. More particularly, the average concentration
of serum albumin in
plasma is present in the range of about 50 to about 100 mg/mL, while
immunoglobulins are
present in an amount of approximately 4 mg/mL, and that of any particular
antibody is probably
in the range of from about 1 to about 100 ug/mL. If it is assumed that the
concentration of an
antibody needed to carry out an analytical measurement is 10 ug/mL and when
100 antibodies
are to be added to a plasma sample, the total increase in protein
concentration would be about 1
mg/mL. Similarly, if the concentration of protein in the plasma sample were 75
mg/mL, the
increase in protein concentration would be about 1.3%. As such, it can be
concluded that the
addition of 100 antibodies to plasma to carry out a 100-fold multiplexed
analysis would have
almost no effect on protein concentration, solution viscosity, and ultimately
analyte diffusion.
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Moreover, adding a thousand antibodies would only add 10 mg/mL of mass, or a
14% change in
protein concentration; which again, would likely not be enough to impact the
analysis.
[0013] Prior to 1960, immunological assays generally targeted
individual antigens and
were carried out in solution through a process called the 'precipitin
reaction'. Subsequent to
immune complex formation, the polyclonal antibody mixture being used in the
assay either
formed a precipitate, or was induced to do so by the addition of a
carbohydrate or an ethylene
glycol polymer. Antigen concentration was determined by light scattering;
however, the lack of
sensitivity and linearity associated with this approach, as well as the fact
that the precipitin assay
only permitted one antigen to be assayed at a time, led to the demise of the
method, and
ultimately a transition to the far more sensitive RIA and ELISA methods.
Despite the failures of
the precipitin reaction method, it can still be reasoned that solution based
immune complex
formation approaches could be useful for immunological assays if selectivity
and detection
sensitivity were vastly improved.
[0014] Although the original precipitin and RIA approaches depended
on a single
method of selection (i.e. one antibody) in the execution of antigen
measurements, it is accepted
today that a sole antibody is not sufficiently selective to discriminate
between an antigen and all
the other chemical entities in a sample. Current immunological assays are
built on multiple
dimensions of selection and/or discrimination, and it is particularly ideal if
each of these
dimensions is of orthogonal selectivity.
SUMMARY
[0015] The present disclosure overcomes or ameliorates at least one
of the prior art
disadvantages discussed above or provides a useful alternative thereto by
providing a novel
multi-dimensional analytical strategy and system for analyzing antigens.
[0016] In one form thereof, a multi-dimensional analytical strategy
for antigen analysis is
provided. In accordance with this aspect of the present disclosure, antigens
in a sample solution
are sequestered by antibodies in a soluble immune complex during the first
dimension of analysis.
The antibodies being added to the solution at the beginning of the analytical
process are for the
specific purpose of binding antigens for qualitative and quantitative analyses
in subsequent steps
of the multi-dimensional process. Cross-reacting antigens, non-specifically
bound substances,
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and species that associate secondarily with antigens, may also adsorb to
immune complexes in
the first dimension and are eliminated in later dimensions of analysis. Unique
features of the
immune complexes formed in the first dimension are then exploited in the
second dimension of
analysis to resolve them from other components in samples on the basis of
their hydrodynamic
volume using a molecular sizing system or sorbent media that target a specific
feature of the
sequestering antibody. Fractionation based on either of these two features is
orthogonal to
epitope:paratope recognition, and discrimination in the third and fourth
dimensions is achieved
in a combination of ways ranging from analyte specific chemical modifications
(such as
derivatization or proteolysis) and size discrimination along with adsorption
and differential
elution from surfaces (such as those on reversed phase or ion exchange media)
or a combination
of sizing exclusion and hydrophobic adsorption (as with restricted access
media). The particular
discrimination mechanism chosen, and the order in which they are coupled,
depends on the
chemical nature of the analytes being targeted and the fractionation
mechanisms used in the first
two dimensions. Dimensions of analysis in the fifth dimension and beyond occur
within
detection systems ranging from mass spectrometry to fluorescence and
electrochemical detectors.
In accordance with certain embodiments utilizing a mass spectrometry detection
system, the
analytes can be resolved according to their mass in the fifth dimension,
collision induced
dissociation of analytes in a sixth dimension, and mass analysis of the
resulting fragment ions in
a seventh dimension.
[0017] In accordance with another form of the present disclosure, a multi-
dimensional
method for simultaneously analyzing multiple analytes within a sample solution
is provided.
According to this aspect of the disclosure, the method comprises adding
affinity selectors to the
sample solution to form immune complexes between the affinity selectors and
the analytes;
providing a first separation means to partially or totally resolve the formed
immune complexes
from other non-analyte substances within the sample solution; providing a
second separation
means to partially or totally resolve the analytes from one another within the
sample solution;
and resolving the analytes according to their mass-to-charge ratio.
[0018] In accordance with still other aspects of the present
disclosure, a multi-
dimensional method for simultaneously analyzing multiple analytes within a
sample solution is
provided. In accordance with this aspect of the disclosure, the method
comprises adding affinity
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selectors to a sample solution containing analytes to be measured, the
affinity selectors having an
affinity for one or more of the analytes within the sample solution; allowing
immune complexes
to form between the affinity selectors and the analytes; partially or totally
resolving the formed
immune complexes from non-analyte substances within the sample solution;
dissociating the
resolved immune complexes; separating the analytes and the affinity selectors
of the dissociated
immune complexes from one another by capturing the analytes through a surface
adsorption
process; transferring the captured analytes to a detection means; and
resolving the analytes with
the detection means in accordance with their mass-to-charge ratios.
[0019] In certain aspects of the present disclosure, the formed
immune complexes are
totally or partially resolved by separating the analytes or formed immune
complexes according to
their hydrodynamic volume, targeting a unique structural feature of the
affinity selectors or the
analytes to capture antibodies of the analytes or formed immune complexes,
hybridizing
oligonucleotides or adsorbing and capturing biotinylated affinity selectors
with immobilized
avidin.
[0020] In yet other aspects of the present disclosure, the analytes and
affinity selectors
within the sample solutions are separated from one another by separating the
analytes or formed
immune complexes according to their hydrodynamic volume, adsorbing and
differentially
eluting the analytes from a hydrophobic surface, targeting a unique structural
feature of the
affinity selectors or the analytes to capture antibodies of the analytes or
formed immune
complexes, hybridizing oligonucleotides, capturing biotinylated affinity
selectors with
immobilized avidin, adsorbing and differentially eluting the analytes from a
charged surface,
adsorbing and differentially eluting the analytes from an immobilized metal
affinity chelator, or
adsorbing and differentially eluting the analytes from a boronic acid rich
surface.
[0021] The analytes under analysis in accordance with the present
teachings may include,
but are not limited to, at least one of analyte fragments, analyte derivatives
and analyte
isotopomers. Moreover, the affinity selectors of the present teachings may
include, but are not
limited to, at least one of an antibody, an antibody fragment, an aptamer, a
lectin, a phage display
protein receptor, a bacterial protein and an oligonucleotide. In accordance
with certain
embodiments, the bacterial proteins can include at least one of G proteins, A
proteins and
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proteins that are produced by an organism targeting a protein from another
organism, while the
oligonucleotide can include at least one of RNA, DNA and PNA.
[0022] In accordance with certain embodiments of the present
disclosure, the analytes
and the affinity selectors are separated from one another by targeting a
unique structural feature
of the affinity selectors with an immobilized antibody. In accordance with
these specific
embodiments, the unique structural feature being targeted may include, but is
not limited to, at
least one of a distinctive natural structural feature of the affinity
selectors, a hapten that has been
conjugated to the affinity selectors and an immunogen conjugated to the
affinity selectors.
[0023] In accordance with some aspects of the present disclosure, the
analytes under
analysis include ionized analytes that are detectable using a mass
spectrometry analysis that
specifically detects parent ions of the analytes that have been separated in
accordance to their
mass-to-charge ratio.
[0024] In certain aspects of the present disclosure, the analytes are
resolved by using a
detection means that is configured to perform the integrated steps of: (a)
ionizing analytes that
have been separated according to their mass-to-charge ratio; (b) generating
fragment ions of
parent ions from step (a) by utilizing a gas phase fragmentation process; (c)
separating the
generated fragment ions from step (b) according to their mass-to-charge ratio;
and (d) recording
a produced relative ion current as the generated fragment ions from step (c)
collide with a
detector surface. In terms of the detection means that is used to resolve the
analytes, in
accordance with certain embodiments, the analytes are detected by one or more
of the following
techniques: mass spectrometry, absorbance, fluorescence and electrochemical
analysis.
[0025] In accordance with further embodiments of the present
disclosure, isotopically
coded internal standards can be used to achieve relative or absolute
quantification of the analytes
under analysis. Moreover, sequential addition, competitive binding assays can
also be used to
achieve the relative or absolute quantification of the analytes.
[0026] In still other aspects of the present disclosure, antibody
concentration can also be
considered to collect an aliquot of an analyte from the sample solution. In
accordance with these
aspects of the present disclosure, the collected aliquot is configured to fit
an optimum detection
range of a device that is being used to resolve the analytes.
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[0027] In accordance with yet another form of the present disclosure,
a method for
analyzing multiple analytes within a sample solution using a plurality of
orthogonal separation
dimensions is provided. According to this aspect of the disclosure, the method
comprises adding
affinity selectors to the sample solution to form immune complexes between the
affinity
selectors and the analytes and to independently sequester one or more antigens
and interfering
substances within the sample solution; using a selective adsorption technique
to remove in a first
or second orthogonal separation dimension, irrespective of order, the
sequestered one or more
antigens and interfering substances; providing a first separation means to
partially or totally
resolve the formed immune complexes from other non-analyte substances within
the sample
solution; providing a second separation means to partially or totally resolve
the analytes from
one another within the sample solution; and resolving the analytes according
to their mass-to-
charge ratio.
[0028] In accordance with still another aspect of the present
disclosure, a method for
simultaneously analyzing analytes within a sample solution is provided, the
method comprising
adding affinity selectors to the sample solution to form immune complexes
between the affinity
selectors and the analytes; separating the formed complexes from other non-
complexed
substances within the sample solution by providing a first chromatographic
column; dissociating
the formed complexes; separating the analytes from the affinity selectors by
capturing the
analytes through surface adsorption of a second chromatographic column;
transferring the
captured analytes to a third chromatographic column that is coupled to the
second
chromatographic column; and analyzing the captured antigens or fragments
thereof as they elute
from the third chromatographic column.
[0029] In accordance with certain aspects of the present disclosure,
the non-complexed
substances comprise substances that do not have an epitope targeted by the
added affinity
selectors.
[0030] In yet other aspects of the present disclosure, the
chromatographic column used to
separate the formed complexes from other non-complexed substances within the
sample solution
is selected from at least one of a size exclusion chromatography column, a
restricted access
media column packed with a sorbent, an antibody column that is configured to
target a general
class of the added affinity selectors, a protein A or G column that is
configured to target all of the
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added affinity selectors, a DNA oligonucleotide column having immunobilized
DNA that is
complementary to an oligonucleotide attached to one or more of the added
affinity selectors, an
avidin column that is configured to target biotin attached to one or more of
the added affinity
selectors and a chromatography column that is configured to select a naturally
occurring or
synthetically created feature of the added affinity selectors.
[0031] In accordance with still other aspects of the present
disclosure, the
chromatographic column used to separate the analytes from the affinity
selectors by capturing
the analytes through surface adsorption is selected from at least one of a
reversed phase
chromatography column, a restricted access media column, an immunosorbent, an
immobilized
metal-ion affinity chromatography column, an ion exchange column and a
chromatographic
retention mechanism.
[0032] In accordance with certain aspects of the present disclosure,
the affinity selectors
include, but are not limited to, aptamers, protein A, protein G, phage display
proteins, natural
receptors, lectins, DNA, RNA, synthetic affinity reagents, or some other
species that shows an
affinity for the analyte to form a plurality of intermolecular complexes
between the affinity
capture agents and the analyte.
[0033] In accordance with still another aspect of the present
disclosure, a multi-
dimensional method for simultaneously analyzing multiple analytes within a
sample solution
includes: adding affinity selectors to a sample solution containing analytes
to be measured, the
affinity selectors having an affinity for one or more of the analytes within
the sample solution;
allowing immune complexes to form between the affinity selectors and the
analytes; partially or
totally resolving the formed immune complexes from non-analyte substances
within the sample
solution in a first dimension of separation using a selective adsorption
technique; dissociating the
resolved immune complexes; separating the analytes and the affinity selectors
of the dissociated
immune complexes from one another in a second dimension of separation using a
selective
adsorption technique; and resolving the analytes in accordance with their mass-
to-charge ratios.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] The above-mentioned and other advantages of the present
disclosure, and the
manner of obtaining them, will become more apparent and the disclosure itself
will be better
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understood by reference to the following description of the embodiments of the
disclosure taken
in conjunction with the accompanying drawings, wherein:
[0035] FIG. 1 is a multi-dimensional scheme showing the simultaneous
analysis of
multiple analytes based on an affinity selector complexing with an analyte to
form a complex in
accordance with the teachings of the present disclosure;
[0036] FIG. 2 is a restricted access media (RAM) particle in
accordance with the
teachings of the present disclosure;
[0037] FIG. 3 is an illustration of a semipermeable surface (SPS)
support in accordance
with the teachings of the present disclosure;
[0038] FIG. 4 is an analytical protocol for direct mass spectrometry (MS)
analysis of
proteins captured from a complex sample matrix by an affinity selector in
accordance with the
teachings of the present disclosure;
[0039] FIG. 5 is an illustration of a restricted access column in
which the interior is a
hydrophilic gel and the exterior is coated with immobilized trypsin in
accordance with the
teachings of the present disclosure;
[0040] FIG. 6 depicts the synthesis of the coating involved in the
trypsin-RAM column in
accordance with the teachings of the present disclosure;
[0041] FIG. 7 is an analytical protocol for a mass spectrometry
analysis of proteins
captured from a complex sample matrix by an affinity selector and then
subjected to proteolytic
digestion before further analysis in accordance with the teachings of the
present disclosure;
[0042] FIG. 8 is an exemplary valving system that allows high
pressure to be applied to
an immobilized enzyme column in the stopped flow mode of analysis in
accordance with the
teachings of the present disclosure;
[0043] FIG. 9 is an illustration of a protocol used in the analysis
of haptens and peptides
captured by a polyclonal antibody affinity selector in accordance with the
teachings of the
present disclosure;
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[0044] FIG. 10 is an illustration of a protocol used in the analysis
of haptens and peptides
captured by a biotinylated affinity selector in accordance with the teachings
of the present
disclosure;
[0045] FIG. 11 is an illustration of a protocol used in the analysis
of haptens and peptides
captured by a DNA, RNA, or PNA affinity selector in accordance with the
teachings of the
present disclosure;
[0046] FIG. 12 is an illustration of a protocol used for
fractionating immune complexes
based on molecular size in accordance with the teachings of the present
disclosure; and
[0047] FIG. 13 is a liquid chromatography component of an instrument
platform that is
capable of carrying out a high resolution analysis of an affinity selector
based process for
simultaneously analyzing multiple analytes in accordance with the teachings of
the present
disclosure.
DETAILED DESCRIPTION OF THE DRAWINGS
[0048] The embodiments of the present disclosure described below are
not intended to be
exhaustive or to limit the disclosure to the precise forms disclosed in the
following detailed
description. Rather, the embodiments are chosen and described so that others
skilled in the art
may appreciate and understand the principles and practices of the present
disclosure.
[0049] As mentioned above, the present disclosure is generally
related to a multi-
dimensional analytical strategy and system for analyzing antigens. As will be
explained in more
detail below, one discriminating feature of the present teachings is the
ability to form large
numbers of inter-molecular complexes with analytes in sample solutions in a
first dimension,
followed by some type of inter-molecular complex separation in a second
dimension that will
generate fractions for further fractionation or chemical reaction in a third
dimension followed by
very specific types of separation or chemical reactions in a third dimension.
In certain aspects of
the present teachings, higher dimensions of analysis can then be utilized
after the third dimension,
and particularly such that at the end of the multidimensional analytical
process, it will be the case
that either 1) each substance (analyte) being examined can be individually
identified and
quantified, or 2) a closely related family of analytes can be determined
together.
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[0050] Moving now to FIG. 1, a scheme for simultaneous analysis of
multiple analytes
based on an affinity selector (S*) (such as an antibody, aptamer, lectin,
protein G, protein A,
phage display protein, or binding protein) complexing with an analyte (A) in
the first dimension
of analysis to form a complex (S*:A) in accordance with the present disclosure
is shown. While
a specific affinity selector will generally be used for each analyte in
accordance with certain
aspects of the present disclosure, in other aspects, there can also be
affinity selectors that form a
complex with multiple analytes, such as would be seen with an antibody
targeting the Lewis x
antigen that is coupled to many glycoproteins.
[0051] With respect to the first dimension (or the "affinity
selection dimension"),
processes in this dimension are based on complexation of the affinity selector
(S*) with an
analyte. The symbol (*) on S indicates a unique structural feature of the
affinity selector, or
complex that may be exploited in selecting it later in a higher dimension.
[0052] Analyte analysis in this first dimension starts with the
formation of individual
complexes in solution as described in Formula 1 below:
S* + AI ____________ S* :A1
Formula 1
where S* is an affinity selector such as an IgG or IgM antibody, a lectin, a
binding protein, a
DNA specie, an RNA specie, or any type of specie with a binding affinity for
an analyte (A);
S*:A is the non-covalent complex formed by the association of the affinity
selector (S*) and a
specific analyte (A). A specific affinity selector S* can form a complex with
a single analyte or
with multiple analytes depending on the selectivity of the affinity selector.
An analyte can be an
antigen, hapten, or any analyte being measured that has an affinity for 5*. An
S*:A complex will
be formed for each analyte. Placing S* or A in brackets indicates their
concentration in moles per
liter.
[0053] It will generally be the case that:
[s*:Aii
Kb = _________________________________________________________________ (1)
1 [Sl[Aii
where Kbi is the binding constant of a specific affinity selector for a
specific analyte (A1). The
binding constant is equal to the rate of complex formation divided by the rate
of dissociation.
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Each type of complex formed in solution will have such a binding constant and
can be
represented by the general equation:
[S*:Ani
Kb =
(2)
n [S*1[An]
where An is any specific analyte.
[0054] While not required herein, in certain embodiments, it is beneficial
to have an
affinity selector with high affinity for the analyte and a large binding
constant (e.g., greater than
about 106). Moreover, in certain embodiments it is beneficial when the off-
rate is low such that
the complex S*:A will not dissociate during the subsequent dimensions of
analysis unless it is
the intent during a specific dimension to dissociate the complex by changing
the incubation
conditions. In addition, having the complex stay intact during separation from
other, non-antigen
components in the sample is an important factor to be considered. It should
also be noted that
while complexes with low binding affinities can be analyzed in the second
dimension, it is
helpful if the separation is achieved quickly and before the complex has time
to dissociate.
[0055] In accordance with certain aspects of the present disclosure,
it is desirable that
complexes formed between an affinity selector (S*), such as an antibody and an
analyte (A), not
precipitate. To discourage such precipitation, affinity reagents that do not
promote high levels of
cross-linking, or perhaps any cross-linking at all, can be used in accordance
with these
embodiments. When the analytes are of low molecular weight, this will
generally not be a
problem, particularly as it has been determined that the valence of the
affinity selector typically
becomes an issue with higher molecular weight analytes. In the case of
antibodies, it will be
possible to use either polyclonal or monoclonal antibodies when targeting low
molecular weight
analytes because they are much less likely to form cross-linked precipitates.
With proteins and
other macromolecular analytes, monoclonal antibodies are less likely to form
precipitates. The
fact that Fab fragments of antibodies are monovalent makes them a good
candidate for an
affinity selector agent in accordance with the teachings of the present
disclosure. Moreover, ionic
strength, pH, and additives will also play a role in keeping complexes in
solution.
[0056] In accordance with yet another aspect of the present
disclosure, a sample
undergoing analysis is fractionated before immune complex formation or before
affinity capture
of the immune complex on a solid phase sorbent. The function of the optional
step is to remove
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cross-reacting species, differentiated between natural complexes of which the
antigen is a
component, differentiate between isoforms of the antigen, or recognize
fragments of an antigen.
Removal or cross-reacting species before affinity capture of the S*:A complex
(shown in
Formula 1) greatly facilitates analyte analysis and can be achieved in two
ways. First, when the
cross-reacting species have some distinguishing feature (e.g., a unique
epitope, charge property,
or hydrophobicity), they can be selected from the sample either before S*:A
complex formation
or after S*:A complex formation, but before capture of the complex on an
affinity sorbent.
Precluding formation of a complex between S* and the cross-reacting (CR)
species would be one
case while in another the S*:CR complex is removed before S*:A complex is
captured.
[0057] In yet other embodiments, the antigen is in several different
complexes such as
S*:A:P 1, S*:A:P2, S*:A:P3, and S*:A:P4, and P is one more non-analyte
protein, as is common in
the interactome or when an antigen is partially or totally complexed with an
auto-antibody. This
is sometime the case with thyroglobulin in plasma. Moreover, one of these
forms may interfere
with analysis of the form of antigen associated with a disease.
Differentiation between these
complexes is achieved by targeting non-analytes in the complex with an
immunosorbent that
removes one or more forms of the complex before S*:A complex formation or
before the S*:A
complex is captured. Removal of interfering complexes is achieved with either
an
immunosorbent targeting the non-analyte in the complex or some other type of
adsorbent such as
an ion exchanger, an immobilized metal affinity chromatography sorbent, a
hydrophobic
interaction column, or a size exclusion chromatography column than
differentiates between the
interfering non-analyte and analyte. Still another use of this step is to
differentiate between the
native form of antigen and fragments that are of lower molecular weight. It
can be the case that
either a fragment or the native form of the antigen is the desired analyte
being targeted for
detection. Again, it should be understood herein that differentiation can be
achieved either with
an immunosorbent or a size exclusion column, and antigens can exist in post-
translationally
modified isoforms. As such, this optional step may be used to remove
interfering isoforms.
[0058] Moving now to FIG. 1 and following complex formation, all S*:A
complexes are
separated from non-analytes in the second dimension of analysis. This
separation step can be
carried out in a number of ways, ranging from a highly specific selection
process targeting the
tag (*) on the affinity selector (S*) to electrostatic or hydrophobic
adsorption, electrophoresis or
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even a size based separation; all of which will be described below. An
essential feature in this
dimension of analysis is that complexes must be partially or totally resolved
in some way from
the rest of the solution. Achieving this step by adsorption is illustrated in
Formula 2 below,
wherein a matrix (M) surface adsorbs the S*:A complex as it passes.
M+S*:AI ¨> M:S*A1
Formula 2
[0059] This step can be achieved in many ways. Ideally, the matrix is
of high specific
surface area (area/unit volume) and the distances at any point in the solution
to the surface are 10
um or less. The surface of the matrix should also be rich in functional groups
that react readily
with derivatizing agents to allow covalent attachment of reagents such as some
type of binding
agent that targets the tag (*) on the affinity selector. The nature of these
binding agents is
described below. Examples of matrices are silica and organic resin particles
used in
chromatography columns to support stationary phases and monolithic
chromatography columns.
Chromatography particles with desirable properties will be <20 um in size,
have pores of about
100 to about 500 nm in size, and a surface area exceeding about 40 m2/mL. The
material in a
monolith will be silica or organic resin with through pores of <10 um and a
second set of pores
between about 100 and about 500 nm.
[0060] The symbol (*) on S* indicates a unique structural feature of
either the affinity
selector or complex that may be exploited in binding it to a sorbent matrix.
The feature may be a
structure element in S* alone or a new feature created as a result of S*:A
complex formation.
This unique structural feature can occur in S naturally or it can be
chemically conjugated to S as
a tag. An example of a natural feature would be amino acid sequences in a
mouse, rat, rabbit,
bovine, porcine, or equine antibodies that are uniquely different than those
found in human
immunoglobulins. Using an anti-mouse IgG immunosorbent attached to the matrix
M would
make it possible to select mouse antibodies from human plasma along with the
antigens to which
they have formed complexes.
[0061] In certain embodiments, semi natural tags can be added through
genetic
engineering that would allow the generation of amino acid sequence tags during
expression of a
polypeptide affinity selector. One such example in accordance with this aspect
of the disclosure
is a single chain antibody with an added peptide tail.
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[0062] In accordance with other aspects of the present disclosure,
affinity selectors can
be extracted from samples either alone or with analytes to which they have
complexed by
covalently attaching a biotin, a hapten, a highly charged group, or an
oligonucleotide tag to a
selector (S*). In terms of the oligonucleotide tags, it should be understood
and appreciated herein
that it is possible to individually tag large numbers of different affinity
selectors if desired.
[0063] In further aspects of the present disclosure, the affinity
selectors can be separated
from the analytes by one or more of the following non-limiting techniques:
separating the
analytes or formed immune complexes according to their hydrodynamic volume,
adsorbing and
differentially eluting the analytes from a hydrophobic surface, targeting a
unique structural
feature of the affinity selectors or the analytes to capture antibodies of the
analytes or formed
immune complexes, hybridizing oligonucleotides, capturing biotinylated
affinity selectors with
immobilized avidin, adsorbing and differentially eluting the analytes from a
charged surface,
adsorbing and differentially eluting the analytes from an immobilized metal
affinity chelator, and
adsorbing and differentially eluting the analytes from a boronic acid rich
surface.
[0064] In another non-limiting example in accordance with the teachings of
the present
disclosure, anti-antibody immunosorbent media is used to capture mouse or
rabbit antibodies that
have been added to human plasma samples. During the course of recapturing
these antibodies
from the samples, any substance with which they had formed a complex will also
be captured as
well. After capturing the S*A complex, extensive washing of the surface bound
complex is used
to remove all other weakly bound components from the sample. This technique
can be referred to
more generally as the anti-affinity selector approach. In accordance with this
illustrative
embodiment, it should be understood that an antibody targeting any type of
affinity selector can
be used to pull complexes out of samples.
[0065] In terms of samples that do not contain large amounts of
immunoglobulins,
protein A, protein G and/or some other immobilized, antibody targeting protein
can be used to
isolate S*:A complexes in which S* is an immunoglobulin. This method is very
similar to the
anti-antibody strategy discussed above.
[0066] In accordance with another non-limiting example of the present
disclosure, avidin
sorbents are used to select biotin tagged affinity selectors and their
complexes back out of
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samples. Again, extensive washing is used to remove substances from the sample
that bind with
low affinity.
[0067] It is envisioned that affinity selectors could also be tagged
with a hapten for which
an antibody has been prepared. When this hapten targeting antibody is
immobilized, a second
dimension capture matrix is used in the isolation of S*:A complexes. Moreover,
when the
affinity selector is an aptamer it can be selected from samples through the
use of an
oligonucleotide sequence immobilized on a second dimension matrix (M). The
sequence on the
aptamer targeted by the immobilized sequence is not used in complex formation
and must be free
to hybridize with the complementary sequence on the sorbent surface.
[0068] Affinity selectors tagged with DNA, RNA, or peptide nucleic acid
(PNA)
oligomers will be capable of binding to a complementary DNA, RNA, or PNA
sequence on the
matrix M through base pair hybridization. Each affinity selector (such as an
antibody) is tagged
(coded) individually or in groups with a unique DNA sequence of 8-12 bases. In
addition, each
coded affinity selector that contacts M to which a complementary
oligonucleotide sequence has
been attached will bind by hybridization, while complementary DNA sequences
immobilized on
the sorbent can either be distributed homogeneously or spatially grouped,
depending on the
manner in which they will be eluted. Complementary oligonucleotides are placed
at different
locations in a column when eluted sequentially by either denaturing the
complex, dissociating the
complementary hybrid, or both. When they are grouped together, a more
elaborate sequential
release procedure must be used involving thermocycling.
[0069] In terms of size, the matrix shows differential permeability
to the S*:A complex
instead of adsorbing it. After a small analyte has been complexed by a
macromolecular affinity
selector it behaves as a macromolecule, allowing it to be separated from other
small molecules in
the solution by a size discriminating separator such as a size exclusion
chromatography (SEC)
matrix, restricted access media (RAM), or semipermeable surface (SPS) media,
field flow
fractionation (FFF), hydrodynamic chromatography (HDC), or a membrane
filtration system.
The macromolecular S*:A complexes will have a much higher molecular weight
than the low
molecular weight components in samples and will be easily differentiated by
size separating
systems, including restricted access media (RAM) and semipermeable surface
(SPS) columns.
Low molecular weight, hydrophobic substances entering the pores or the
semipermeable surface
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of these media would be differentially adsorbed. This means the RAM and SPS
columns are
separating molecules by both a size and hydrophobic interaction mechanism.
[0070] Moving now to FIG. 2, an illustration of a restricted access
media (RAM) particle
is shown. In accordance with this illustration, RAM supports are generally an
aggregate of
submicron silica particles in which the interior surfaces of the support are
covalently derivatized
with stearic acid and the exterior surfaces of the support are derivatized
with a glycerol ether of
the structure --OCH2--CH(OH)--CH2OH. The average pore diameter between the
submicron
particles is about 6 nm on the average, which precludes entry of most proteins
exceeding
between about 20 to about 40 kD in molecular weight. In contrast, peptides and
haptens of less
than about 3 kD readily enter the interior of a RAM support where they are
adsorbed from water.
[0071] Electrophoretic separations of complexes can be achieved by
either differences in
electrophoretic mobility or isoelectric point. In accordance with certain
exemplary embodiments,
it is desirable that the electrophoretic properties of the affinity selector
be very different than
those of the other species in the solution. While this would generally be true
of DNA or RNA
based selectors relative to the proteins in the solution, peptides and
proteins in a complex with
DNA or RNA could also be easily separated from other peptides and proteins in
the solution as
well.
[0072] Field flow fractionation (FFF) separates molecules differing
in size by a
mechanism that exploits size-related differences in their diffusion
coefficients. HDC separates
macromolecules by a slightly different mechanism that also produces size
related differences in
elution properties.
[0073] In FIG. 3, an illustration of a semipermeable surface (SPS)
support is provided.
This sorbent is prepared by attaching a 300 dalton polyoxyethylene (POE)
oligomer to an
average of 1 in every 15 octyl (C8) silane groups attached to the surface of a
silica support. The
POE oligomer associates weakly with the C8 residues on the sorbent surface,
blocking contact
with proteins. Haptens and peptide in contrast can penetrate this coating and
bind to the C8
groups through a hydrophobic interaction mechanism.
[0074] The final component of the second dimension separation process
requires that
either the S*:A complex or the analyte alone be released from a sorbent in the
second dimension
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and/or transported into the third dimension for further analysis. In the case
of size selection of
SEC, RAM, SPS, or FFF separations, the analyte can either be transported
directly into the third
dimension or the complex dissociated before the analyte is transported into
the third dimension.
Dissociation in the case of molecular sizing separations can be achieved by
adjusting the pH at
the exit from the molecular sizing column with a relatively acidic (pH 2.5) or
basic (pH 12)
mobile phase. Moreover, dissociation of immune complexes from immunosorbents
is achieved
either by relatively acidic (pH 2.5) or basic (pH 12) conditions. In addition
to dissociating the
Ab:Ab complex, Ab:Ag complexes are dissociated as well.
[0075] Biotinylated affinity selector:analyte complexes can be
released from a mono-
avidin affinity column in several ways. One is by adding biotin to the
solution above the capture
agent or to the mobile phase in the case of chromatography columns. This will
release the
biotinylated S*A complex. Mono-avidin columns can be used for many cycles when
eluted in
this manner. An alternative elution strategy is to apply an acidic solution
having a pH of about
2.5. This will reversibly denature mono-avidin and release both the affinity
selector and probably
the analyte from the affinity selector. Moreover, other types of S*:A affinity
selectors that are
proteins can be dissociated from S* capture selectors in much the same way
(i.e. with acidic
solutions in the range of pH 2 to 2.5), and the analyte will generally be
released at the same time.
[0076] In accordance with certain aspects of the present disclosure,
the affinity selector
can be a polynucleotide or have a covalently attached oligonucleotide. In
accordance with this
aspect of the present disclosure, complementary oligonucleotide sequences on
some type of
sorbent matrix (MDNA or MRNA in the Formula above) are used to adsorb the S*:A
complex from
the sample. Dissociation and/or release of the complex or the components of
the complex from
MENA is achieved with eluents that i) are of low ionic strength, ii) are very
basic, or iii) contain a
denaturant. Dissociation and release can also be obtained by elevating the
temperature to melt
the DNA:DNA, RNA:RNA, DNA:RNA, PNA:DNA, or PNA:PNA hybrid. Still another way
would be to add a solution in the dissociation step that contains the same DNA
(or RNA)
sequence as in the tag on the affinity selector and then stopping the flow of
the reagent across the
surface of M. After flow is arrested, temperature of the solution is elevated
to the point that the
DNA:DNA, RNA:RNA, or DNA:RNA hybrid is melted. Thereafter, the temperature is
allowed
to return to room temperature so that rehybridization can occur. When the
concentration of the
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complementary oligonucleotide added is high, it will out-compete the
oligonucleotide tag on the
S*:A complex, thereby leaving it in solution. The released complex may then be
transported into
the second dimension for further analysis.
[0077] Analysis in the third dimension in accordance with the
teachings of the present
disclosure depends on the ultimate detection method to be used at the end of
the analytical
process, as well as whether the analytes are i) small molecules such as
haptens or peptides ii)
proteins, glycoproteins, lipoproteins, polysaccharides, polynucleotides, or
iii) some other
macromolecular species. In terms of small molecules (i.e., haptens and
peptides), when analytes
are identified and quantified by electrospray ionization mass spectrometry
(ESI-MS) or matrix
assisted laser desorption ionization mass spectrometry (MALDI-MS), detection
is more easily
achieved if the analytes have a molecular weight less than about 2 kD. Larger
molecules, on the
other hand, can be detected as shown in FIG. 4; however, it should be noted
that ionization
efficiency is often higher with smaller molecules.
[0078] FIG. 4 is a multi-dimensional "analysis option tree" (AOT) for
the analysis of
proteins beginning with affinity selector binding of analytes in solution.
Proceeding on from
affinity complex formation, in accordance with certain embodiments of the
present disclosure,
the number of options for analysis can increase to eight, or even more by the
fifth dimension of
analysis. AOTs generally begin with affinity selection in the first dimension,
proceed on through
a series of chromatographic and/or chemical modification steps in the
intermediate dimensions of
analysis, and conclude with detection by mass spectrometry, fluorescence, or
electrochemical
means. The type of analyte being detected, sensitivity requirements, and
available
instrumentation dictate the branch of the tree taken for analysis.
[0079] An AOT for direct MS analysis of proteins sequestered from a
complex sample
matrix by some type of affinity selector, generally an antibody, Fab fragment
of an antibody,
single chain antibody, or phage display protein is seen in FIG. 4. After the
intermolecular affinity
selector:analyte complex is captured from the solution in the second dimension
by a sorbent
targeting the affinity selector, the anti-selector:selector:analyte complex is
dissociated and the
components further fractionated by either size exclusion chromatography (SEC)
or reversed
phase chromatography (RPC) in dimensions 3 and 4 before being sent to either
an ESI-MS/MS
or MALDI-MS/MS for direct analysis of the intact protein. To this end, it
should be understood
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and appreciated herein that it can be helpful to convert proteins,
glycoproteins, and lipoproteins
to mixtures of peptides to identify and quantify them by mass spectrometry.
This approach will
be described below.
[0080] The outer branches of AOT for direct analysis of protein in
FIG. 4 shows that the
final choice is detection by either electrospray ionization (ESI) or matrix
assisted laser
desorption ionization (MALDI) mass spectrometry (MS). The choice between these
two depends
to a large extent on the instrumentation available and complexity of the
sample. With complex
mixtures, MALDI-MS may be a particularly useful option, particularly as MALDI-
MS generally
provides single charge states of a protein or peptide, thereby making it
possible to examine 5-20
polypeptides (or even hundreds of species) at a time. ESI-MS, on the other
hand, more frequently
produces multiple charge states of a polypeptide that require a deconvolution
algorithm to
recognize the molecular weight of a protein. Although the molecular weights of
proteins in a
mixture may be different, multiple charge states of the protein fall in the
same mass-to-charge
ratio of the MS spectrum. The algorithm is generally unable to handle more
than two proteins
simultaneously. Another problem with ESI-MS detection of proteins is that
distribution of the
protein across multiple charge states reduces detection sensitivity. It should
be noted that while
detection is possible with ESI-MS, it is often best done with MALDI-MS.
[0081] Another element of the AOT is whether the affinity selector
(Ab) will be removed
from samples in dimension 3 before they are sent to the MS or left in the
sample. Still referring
to FIG. 4, while antibodies are removed in analytical routes A, B, C, and D,
they are left in the
samples of routes E, F, G, and H. As should be understood and appreciated
herein, whether
antibodies are removed from samples before the analysis depends on several
issues. For instance,
as antibodies are typically at least about 160 kD in size, when the molecular
weight of the
analytes is <100 kD a MALDI-MS analysis would have no problem differentiating
between the
two. A non-limiting advantage of MALDI-MS/MS is that it identifies primarily
only the
molecular ion and is able to differentiate between multiple protein species
simultaneously on the
basis of their differing molecular weight. This is particularly useful when
examining a small
number of antigens where the total concentration of antibody would probably be
no more than
ten times greater than antigen concentration. With large numbers of antigens,
the total antibody
concentration could be one hundred times larger than antigen concentration.
Moreover, large
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amounts of antibody could potentially suppress ionization of low abundance
antigens. The
presence of antibody in samples would be more problematic in the ESI-MS mode
of detection
where multiple charging would cause ions from the antibody to overlap ions
from antigens. With
simple samples this mode of detection is certainly possible; however, as
sample complexity
increases, ESI-MS becomes less useful as a detection option.
[0082] With antibodies being 160 kD or larger in molecular weight,
SEC on a 100-150
angstrom pore diameter column readily separate antigens of 80 kD or less from
antibodies in the
3rd dimension of routes A, B, C, and D of FIG. 4. Antibodies elute at or near
the exclusion
volume and are sent to waste. Antigens are directly transferred to
chromatography columns
having hydrophobic surfaces where they are captured and reconcentrated. When
it has been
decided that antibodies will be sent to the MS along with antigens, the two
species are co-
captured and fractionated by hydrophobic chromatography matrices in the 3rd
and 4th
dimensions of routes E, F, G, and H. The options in the 4th dimension are
whether a high
resolution, gradient eluted analytical column is needed in routes E and F or
whether a short, low
resolution column would suffice as in routes G and H. When large numbers of
analytes are being
fractionation, routes E and F would be chosen. With small numbers of analytes,
routes G and H
are likely used.
[0083] Recent successes in proteomics are based on the fact that
proteins are reduced to
more easily identifiable fragments by cleavage with proteolytic enzymes, the
most popular being
trypsin (see Formula 3 below). Trypsin digestion is most widely achieved by
incubating the
protein mixture with a 50:1 mass ratio of protein:trypsin for a 24 hour
period. At the end of
proteolysis, the peptides generated will be analyzed in a manner similar to,
or identical to,
samples starting with peptide components. When more trypsin is used per mass
of protein,
trypsin begins to autodigest, thereby contaminating the sample with trypsin
fragments.
. trypsin
proteins peptides
Formula 3
[0084] It should be understood and appreciated herein that
proteolysis is greatly
accelerated by using trypsin immobilized on high surface area chromatography
particles or
monolithic media of the type described above. Moreover, protein samples are
often forced
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through an immobilized trypsin bed in the manner an analyte is
chromatographed. In this
particular case, it is possible to use more trypsin than protein, particularly
as trypsin is
immobilized and thereby cannot autodigest.
[0085] As is shown in FIG. 5, immobilized trypsin can also be used in
a chromatography
column, but in a different manner than previously described. In accordance
with certain aspects
of the disclosure, the pH optimum of trypsin is in a pH range of from about 7
to about 9.
Moreover, the catalytic efficiency with most proteins decreases a million fold
when going to a
pH in the range of from about 2 to about 3. Moreover, when proteins are eluted
from second
dimension capture columns with a mobile phase pH of approximately 2.5 and pass
directly into
trypsin columns the proteins will be in a solution that is too acidic for the
trypsin to function. As
such, the pH must be adjusted to about 8 by some type of buffer exchange
before trypsin
digestion will occur. This is accomplished by transporting the mixture of
proteins and acidic
elution buffer through a chromatographic matrix in which the outer surface of
particles is coated
with covalently immobilized trypsin and the pores are so small that molecules
exceeding 20 kD
can not penetrate but buffers freely enter the pore matrix of the particles.
This size excluding
column is filled with trypsin digestion buffer. As protein samples are
transported through the
column, proteins readily move ahead of acidic elution buffer into the trypsin
digestion buffer
where proteolysis begins to occur. With proteolysis, peptides are formed that
can enter the pores
of the restricted access column, but they and their protein parents have moved
beyond the acidic
elution buffer.
[0086] FIG. 5 shows an illustration of a restricted access column in
which the interior is a
hydrophilic gel and the exterior is coated with immobilized trypsin. The pore
diameter of these
particles is in the range of 6 nm or less. This column serves the function of
achieving buffer
exchange and proteolysis in the same operation. When a sample in acidic buffer
is introduced
into a column packed with these particles, protein in the sample is precluded
from entering the
pores and migrates ahead of the buffer entering the pores of the particles.
Proteins migrate into a
region in the column filled with a buffer suitable for proteolysis. At this
point, proteolysis is
catalyzed by trypsin covalently coupled to the particles.
[0087] When trypsin is immobilized on a porous particle medium
ranging from 6 to 30
nm, the column also functions as a size exclusion column. The pores are of a
size that will
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partially exclude many proteins from penetrating the immobilized enzyme
matrix. Starting with
6 nm pore diameter silica particles, these particles are coated with gamma-
glycidoxpropyl
trimethoxysilane and the attached oxirane hydrolyzed to yield a diol. Acrolein
will be
polymerized onto the diol matrix through Ce 4 catalysis to yield the
poly(aldehyde) matrix
shown in FIG. 6, Step 1. The poly(aldehyde) then partially fills the pores of
the silica support
matrix and the trypsin is brought into contact with the particles where lysine
residues on the
enzyme will covalently attach to the silica matrix through Schiff base
formation in the presence
of sodium cyanoborohydride (Step 2 in FIG. 6). NaCNBH4 added to the reaction
mixture reduces
Schiff bases as they are formed but does not reduce aldehydes. The pores of
the support matrix
are so small that trypsin can only gain access to aldehyde groups on the
exterior of the particle.
In other words, there will be no trypsin in the pores of the particle. After
trypsin immobilization
and aldehyde residues (--C=0) on the particles will be reduced with NaBH4
(Step 3 of FIG. 6)
thereby completing the synthesis of the immobilized enzyme matrix.
[0088] In addition to having trypsin on the exterior of the particle,
the exclusion of
proteins (and to a large extent, peptides) from the pores of the particle are
both unique features of
the present inventive matrices. This means that when an immobilized enzyme
column is prefilled
with a buffer having a pH of approximately 8 and connected in series with, for
example, an anti-
mouse Ab column that has a selected Ab:Ag complex in which the Ag is a protein
or an avidin
column that has selected a biotinylated Ab:Ag complex in which the Ag is a
protein and is being
eluted with a pulse of pH 2.5 eluent: 1) the acidic mobile phase will enter
the pores of the
immobilized enzyme column while 2) all proteins will be excluded and moved far
ahead of the
acid, and 3) as part of the exclusion process move into pH 8 buffer. Residence
time of proteins in
the immobilized enzyme column is controlled by either the flow rate through
the column or by
interrupting flow through the column at some point after proteins have entered
the immobilized
enzyme column and before they exit. This column will generally be used in the
3rd dimension of
analysis as seen in FIG. 7.
[0089] In contrast to FIG. 4, which showed the selection and analysis
of protein at the
whole protein level, FIG. 7 shows the capture and isolation of intact proteins
from mixtures after
which they are converted to peptides for identification and quantification by
mass spectrometry.
Immune complexes are again isolated from samples and enriched on an
immunosorbent that
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targets epitopes unique to the antigen targeting antibody or on an affinity
sorbent targeting a tag
on the capture antibody. After washing to remove substances bound to complexes
with low
affinity, non-covalent complexes are dissociated with an acidic (¨pH 2.5)
mobile phase and
antigens along with the capture antibody are transported to the immobilized
trypsin column. As
the proteins and acidic buffer pass through this column they are resolved as
proteins migrate into
a trypsin digestion buffer. During this process, proteins are converted to
peptide cleavage
fragments in the 3rd dimension of analysis. These fragments are reconcentrated
in the 4th
dimension, further resolved by some type of hydrophobic interaction
chromatography, and then
finally identified and quantified by either ESI-MS/MS or MALDI-MS/MS. Proteins
generally
yield thirty to several hundred peptide fragments, any one of which can be
used to identify and
quantify a protein parent. Peptides chosen for identification and
quantification are generally
those that have high ionization efficiency, provide a sequence that is unique
to the parent protein,
and are well retained by reversed phase chromatography columns.
[0090] The rationale for the type of mass spectrometry to use is
different with peptides
than was the case with proteins in FIG. 4. In this case, it is possible to
fragment peptides from the
first dimension of mass analysis by either collision induced dissociation
(CID), electron transfer
dissociation (ETD), or some other method that fragments gas phase ions from
the first dimension
of MS analysis. CID and/or ETD fragment ions are then resolved and recorded in
a second
dimension of mass analysis. This three dimensional process provides a sequence
bearing
signature unique to each peptide. An extremely attractive feature of this
approach is that the MS
data can be directly tied to sequences in DNA databases, allowing the
identification of genes and
parent proteins from which peptides were derived. ESI-MS/MS and MALDI-MS/MS
are more
comparable in capability in this case. With either approach, it is possible to
identify hundreds of
peptides in a single analysis. Because suppression of ionization occurs by
different mechanisms
in MALDI-MS and ESI-MS, some peptides are detected better with some types of
MS more than
others.
[0091] It should be understood and appreciated herein that the rate
of proteolysis in the
immobilized enzyme column can be accelerated by raising the temperature, by
sonication, or by
increasing the pressure inside the column to ¨10,000 psi. An exemplary set-up
for increasing the
pressure in this respect is illustrated in FIG. 8, which specifically shows a
valving system that
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allows high pressure to be applied to an immobilized enzyme column in the
stopped flow mode
of analysis. In accordance with this illustration, high pressure is thought to
facilitate proteolysis
by the partial denaturation of proteins. Proteolysis of a sample begins by
switching the valve into
a position such that the immunosorbent, trypsin column, and RPC concentrator
are connected in
series. In this position, proteins are desorbed from the Ab column and
transferred into the trypsin
column where the desorbing buffer and proteins are separated by a size
exclusion mechanism. At
this point the valve is switched into a position that arrests the flow of
mobile phase through the
column, thereby bringing the trypsin column into a high pressure incubation
mode. After
proteolysis, the trypsin column is switched back to the loading position and
the digested protein
mixture is transported to the RPC concentrator.
[0092] The immobilized enzyme column in FIG. 8 is in the 3rd
dimension of analysis in
FIG. 7. Antigens and antibodies released from the affinity column in the 2nd
dimension of FIG.
7 are transported directly into the immobilized trypsin column in FIG. 8 where
the acidic eluting
buffer and proteins are separated as the protein migrates into the trypsin
digestion buffer. Based
on the volumes of the columns in the 2nd and 3rd dimensions, the amount of
solvent that must be
pumped through the system to cause this separation is calculated. When
proteins have migrated
halfway through the trypsin column, the valve is switched to the position
indicted in FIG. 7. In
this valve position, very high pressure from the pneumatic pump can be applied
to the trypsin
column at zero flow rates. Flow from the immunosorbent column directly to the
RPC
concentrator continues in this valve position. After about 1 to about 10
minutes of stopped flow
in the trypsin column, the valve is rotated back to the position connecting
the affinity capture
column, the immobilized trypsin column and the RPC concentrator in series. The
proteolytic
digest from the trypsin column will then be transferred to the RPC
concentrator.
[0093] Following proteolysis, peptides from proteins that will be
identified and detected
by mass spectrometry are examined by one of the procedures described in FIG.
7. To this end,
dimensions 4 through 6 (as shown in FIG. 7) are the same as dimensions 3
through 5 for peptide
analysis, the reason being that peptides are being examined in both cases.
[0094] According to certain aspects of the present disclosure,
haptens and peptides are
reconcentrated after their generation in the analytical process and/or after
their separation from
non-analytes in the second dimension. This process, when it is carried out in
a chromatography
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column, is often referred to as refocusing since analytes are adsorbed in a
tight zone at the
column inlet. Reconcentration and further separation of analytes can be
achieved in multiple
ways ranging from some form of affinity selection mechanism to ion exchange or
hydrophilic
interactions mechanisms, but a hydrophobic interaction mechanism is
particularly useful because
it is the most universal adsorption method, and at this stage, most analytes
will be in water.
Moreover, water is the most favorable solvent for adsorption with the
hydrophobic interaction
mechanism. Separations by hydrophobic interaction (reversed phase
chromatography and RAM)
are seen in the third and fourth dimensions.
[0095] It should be understood and appreciated herein that the major
difference between
FIGS. 9, 10, and 11 are related to the manner in which the antibody used in
complex formation is
tagged and selected in dimensions 1 and 2. At the end of the first two
dimensions, analytes are
released from the selector(s) in a single group, or a small number of groups.
There could be 10 to
more than a thousand analytes in a group that must undergo further separation
before they can be
differentiated and detected individually. At least a portion of this
separation is achieved with
reversed phase chromatography.
[0096] Reconcentration of analytes released from affinity selectors
were achieved in one
of two types of affinity selector. One was the RAM (or SPS) column (FIG. 2).
The advantage of
this column is that it separates the affinity selector (generally an antibody)
from haptens and
peptides. Peptides and haptens penetrate into the pores of the RAM column or
through the outer
coating of the SPS column where they are adsorbed hydrophobically. Because the
antibody is
large, it cannot penetrate the pore or coating and is carried away to waste as
indicated in FIGS. 8-
10. An alternative concentrator is a short reversed phase chromatography
column of ¨1 cm in
length. Haptens, peptides, and protein are all captured on the column, in
addition to selector
antibodies. Because samples are being refocused at the column inlet, there is
no need for a sharp,
small, discrete injection of samples. In addition, large sample volumes can be
loaded
continuously.
[0097] FIG. 9 is an illustration of the protocol used in the analysis
of haptens and
peptides captured by a polyclonal antibody affinity selector in accordance
with the teachings of
the present disclosure. In accordance with this aspect of the disclosure,
haptens and peptides
release from the affinity selector that captures immune complexes in the 2nd
dimension and are
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then refocused and further resolved by a RAM or RPC column prior to ESI-MS/MS
or MALDI-
MS/MS. Refocusing and reversed phase separation occurs in the 3rd and 4th
dimensions of
analysis. While it is possible to go directly from the 2nd to the 4th
dimension, the RAM or RPC
concentrator preserves the analytical column. It is also possible, in many
cases, to separate the
Ab used in the first dimension from analytes in the 3rd dimension.
[0098] Release from the affinity selector in the 2nd dimension is
usually achieved with
an acidic aqueous mobile phase. This mobile phase is ideal for adsorption of
down stream RAM
or RPC columns in the 3rd dimension. Because analytes are refocused in the RAM
or RPC
column, large sample volumes may be used without adversely affecting
resolution or the RAM
or RPC columns in the 4th dimension. The same logic and elution protocols in
dimensions 2, 3,
and 4 apply for FIG. 10.
[0099] One of the decisions in the analytical option tree shown in
FIG. 9 is whether to
use a RAM column or an RPC concentrator. While the RPC concentrator is
simpler, has greater
binding capacity, and is less expensive than a RAM column, RPC columns
disadvantageously
capture peptides, haptens, and antibodies. In addition, all antibody species
elute together from
RPC columns late in the chromatogram during gradient elution. This means that
the antibody
peaks will be large relative to analyte peaks. This is not a problem in most
cases because
peptides and haptens elute long before antibodies. The large antibody peak
will not mask analyte
peaks when this is true. When samples contain analytes that elute at roughly
the same time or
after antibodies, RPC columns cannot be used. In this case, a RAM column
should be used.
Antibodies are too large to penetrate the pores of RAM sorbents and pass
through the column
without adsorption, while peptides and haptens, in contrast, enter the pores
of RAM sorbents and
are captured in the hydrophobic interior of the particles.
[00100] When the hapten or peptide mixture is relatively simple, the
RAM or RPC
concentrator can be gradient eluted directly into the ESI-MS or onto the MALDI-
MS analysis
plate. The peak capacity of these short columns is often no more than 50
components. There is
no need for a higher resolution separation column such as the "analytical RPC"
column. This is
shown as occurring in the fourth dimension in FIGS. 9-10, but it is the same
column that was
loaded with sample in the third dimension. In contrast, complex analyte
mixtures will require
much longer, higher resolution RPC columns. These analytical columns are from
10-50 cm in
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length and are packed with particles ranging from about 1.5 to about 5 um in
diameter with an
octadecyl silane (C18) coating. Analytical columns can have peak capacities of
up to 600
components when slowly gradient eluted and heated to enhance mobile phase
diffusion rates.
Because substances are eluted from the RPC column into a mass spectrometer,
coeluting peaks
can be differentiated in either a first or second dimension of mass
spectrometry.
[00101] The type of mass spectrometry used in the 5th and higher
dimensions depends on
the complexity of the initial sample matrix, the number of analytes being
analyzed, and analyte
concentration. When the sample matrix is simple and fewer than 10 analytes are
being examined,
single dimension mass analysis of molecular weight alone in either the MALDI
or ESI mode will
be adequate. CID followed by further mass analysis of fragment ions in a 6th
and 7th dimension
allow higher confidence levels in identification but is probably not
necessary. The type of mass
spectrometer with simple samples is determined primarily on the basis of what
is available when
sample concentration is in the ug/mL to mg/mL range. Much higher sensitivity
would be
obtained in the ESI mode by using a mass spectrometer such as a triple-
quadrupole (QQQ) or
quadrupole-ion trap (Q-Trap) instrument in the selected ion monitoring mode.
Instruments such
as these, which mass analyze a particular ion for longer periods of time and
accumulate greater
numbers of ion counts for a specific analyte, can have a one hundred fold or
greater sensitivity
than rapid scanning instruments.
[00102] With very complex samples, the ESI-MS/MS instruments are
particularly useful
for the protocol of FIG. 9 because they provide a high level of
discrimination. Again, when
highest sensitivity is needed QQQ or Q-Trap type instruments provide the best
solution.
[00103] The major difference between FIG. 9 and FIG. 10 is in the type
of tag placed on
selection antibodies and the manner in which immune complexes are captured.
The antibody tag
in FIG. 10 is either biotin or some hapten. FIG. 10 shows an illustration of
the protocol used in
the analysis of haptens and peptides captured by a biotinylated or hapten
tagged affinity selectors.
In accordance with this illustration, mono-avidin is used in the 2nd dimension
affinity selector
because less severe conditions are required to release biotinylated species.
Haptens and peptides
can be released in the 2nd dimension either by biospecific displacement or
affinity selector
denaturation. The most gentle is with a biotin displacer. The disadvantage of
this approach is that
desorption kinetics are slow, requiring slow flow rates during the elution
step. By contrast,
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partial denaturation with an acidic mobile phase, as described in FIG. 9, is
much faster.
Additional steps along the analytical option tree are identical to those
described in FIG. 9.
[00104] FIG. 11, on the other hand, is an illustration of a protocol
used in the analysis of
haptens and peptides captured using antibodies tagged with a DNA, RNA, or PNA.
As in other
examples discussed above, separation of the immune complex from other
substances in a sample
occurs in the 2nd dimension. Elution of captured antibodies from the 2nd
dimension occurs by
raising column temperature above the melting point of the oligonucleotide
hybrid. When
thermocycling occurs under flow, all dissociated species are swept from the
column.
Thermocycling under arrested flow in the presence of competing
oligonucleotides allows
differential displacement of specific antibodies. Elution conditions in the
2nd dimension are
sufficiently mild that the immune complex may still be intact as it passes
into the 3rd dimension
and is captured. When this is true the immune complex will dissociate in the
3rd dimension as
RPC or RAM columns are eluted with an acidic mobile phase containing
acetonitrile. Whether
the immune complex is intact or dissociated will not impact the net outcome of
the analysis. The
rest of the workflow is as described in FIG. 9.
[00105] With reference to FIG. 9, analytes are selected in the 1st
dimension by
complexation with an affinity selector such as an antibody. The unique feature
of the affinity
selectors used in this case is that they are tagged with an oligonucleotide
(ONTt composed of an
ordered base sequence of RNA, DNA, or PNA. All the antibodies can have the
same
oligonucleotide sequence or each antibody species can be tagged with a
different oligonucleotide
sequence. Subsequent to complex formation in solution, the soluble complex is
captured from
samples by passing an aliquot of the solution through particles or across the
surface of a solid
support containing an immobilized oligonucleotide selector (ONTs) with
sequences
complementary to those on the affinity selector. There will be one
complementary
oligonucleotide sequence on the solid phase surface for each nucleotide tag on
an antibody.
During the course of passing an aliquot of the solution across the
oligonucleotide (ONTs) bearing
surface, analyte:selector-ONTt complexes will be captured by hybridization,
forming a --
ONTt:ONTs:selector analyte complex. Oligonucleotides (ONTO immobilized on the
solid
surface may be comingled or each can be located at spatially different sites.
In accordance with
certain specific embodiments, it is particularly useful that the support
matrix for immobilizing
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ONTs is an organic resin, such as the pressure stable styrene-divinylbenzene
resins used in HPLC.
The POROS support matrices from Applied Biosystems are ideal examples of such
resins.
Advantageously, these resins withstand temperatures from about 80-90 C., as
well as wide
extremes in pH that may be used in analyte elution as described below.
[00106] Following the capture of analyte:selector complexes in the 2nd
dimension they
must be released and eluted into the 3rd dimension. The elution step can be
achieved in several
different ways in accordance with the teachings herein. One exemplary method
is to dissociate
the hybrid by raising the temperate of the solid phase adsorbent bearing the --
ONTt:ONTs:selector analyte complex above the melting point of the --ONTt:ONTs
hybrid while
the mobile phase is passing across the surface and being transported to the
3rd dimension. The --
ONTt:ONTs hybrid will dissociate and the ONTs:selector:analyte complex will be
transported to
the 3rd dimension. In most cases, the ONT,:selector analyte complex will
remain intact, but in
some cases it will partially or totally dissociate. Whichever the case may be
will be
accommodated in the 4th dimension.
[00107] A second exemplary method of elution is to use extremes in either
pH or ionic
strength to dissociate the hybrids. When the solid phase adsorbents are eluted
with distilled water,
the --ONTt:ONTs hybrid generally dissociates. Use of a pH 10 buffer at low
ionic strength
accomplishes the same thing.
[00108] Immune complexes can also be fractionated according to
molecular size (see FIG.
12). Immune complexes are fractionated in the 2nd dimension in this workflow
using size
exclusion chromatography. The pore diameter of the SEC column is in the range
of about 100 to
about 150 angstrom while the particle size of the exclusion matrix should be
about 3 to about 5
um. Immune complexes will elute near the exclusion volume of the column in the
case of the 100
angstrom pore diameter packing material and are directly transported to the
RPC or RAM
concentration or directly to the analytical RPC column. At the conclusion of
this transfer, lower
molecular weight species eluting from the SEC column are diverted to waste.
The RPC columns
are eluted in a linear gradient with a mobile phase ranging from 0.1%
trifluoroacetic acid or 1%
formic acid to the same concentration of acid containing 70% acetonitrile.
This mobile phase is
sufficiently acidic to dissociate immune complexes captured at the inlet of
the RPC columns.
The rationale for which type of mass spectrometry to use is the same as in
other cases.
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[00109] As noted above, proteins require the addition of a proteolytic
dimension beyond
that required in the analysis of haptens and peptides. This is generally
achieved in the third
dimension. FIG. 13 shows a liquid chromatography component of an instrument
platform that is
capable of providing the automated, high resolution separation of an affinity
selector needed for
high throughput and simultaneous analysis of multiple analytes. Sample
preparation is initiated
in an auto-sampler housed in a refrigerated chamber that minimizes microbial
growth of a
sample prior to analysis. Auto-sampler vials holding samples are of the
conical bottom type to
minimize sample volume. In addition to multiple samples, the auto-sampler also
holds antibody
solutions at a fixed concentration necessary for the analysis. The auto-
sampler can also be loaded
with additional reagents needed for reduction, alkylation, derivatization,
proteolysis, internal
standards addition, and diluents, any of which can be aliquoted into sample
vials in any order.
An analysis begins in the auto-sampler when a robotic syringe removes an
aliquot of antibody,
antibodies, or reagent from a reagent vial and adds them to a sample. Multiple
reagents can be
added sequentially to a sample vial as might be needed in reduction,
alkylation, and proteolysis
of a sample before analysis. A single antibody reagent vial may contain one
antibody or all the
antibodies necessary for a particular multiple analyte analysis. After
dispensing a reagent or
antibody into a sample the syringe goes to a vial in the auto-sampler
containing reagent free
buffer and is cleaned by fully loading the syringe with buffer multiple times
and dispensing the
buffer to a waste vial.
[00110] After an incubation time suitable to form a derivative and/or
immune complex(es),
the auto-sampler withdraws an aliquot of solution from a sample vial and loads
a fixed volume
sample loop on a high pressure valve in the sample loading position. The valve
is then rotated to
introduce the sample loop into a mobile phase flow path connecting it to a
down stream column.
The first down stream column (in the 2nd dimension of analysis) is generally
an affinity column
or size exclusion column that will partially or totally resolve the immune
complex(es) from other
components in the sample. When the immune complex(es) are captured by an
affinity column, it
is desirable that 20 or more column volumes of mobile phase be pumped through
the column to
remove substances bound to the immune complexes or column with low affinity.
When high
binding affinity antibodies are used in the analysis, all other bound species
will be non-analytes.
This washing step thus removes non-analytes and is part of the separation
process.
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[00111] The column used in the second dimension of analysis can either
be housed inside
the refrigerated chamber of outside at room temperature as illustrated in FIG.
13. When using a
high affinity antibody in the capture column, or an SEC column in the 2nd
dimension, the
column is operated at room temperature. Lower temperature operation is used
with affinity
columns when the capture agent is of low affinity and the binding constant of
the capture agent is
increased by going to lower temperature. At 5 C. the binding constant can be
double that at
room temperature.
[00112] The illustration in FIG. 13 shows that analysis in the 3rd and
4th dimension can
be carried out in an oven. The illustration shows direct transfer of analytes
from the 2nd
dimension to the high resolution, analytical RPC column in the 4th dimension
in FIGS. 9, 10,
and 11. In accordance with some embodiments, a 3rd dimension column can be
added before
valve B. The function of a heating column in accordance with these embodiments
is to diminish
the limitations known in the liquid chromatography literature as mobile phase
and stagnant
mobile phase limitations. By reducing mobile phase viscosity and increasing
the rate of analyte
diffusion, resolution is increased in RPC. This increases the resolution of
RPC columns and the
number of analytes that can be resolved.
[00113] The first pump (P1) in the system in FIG. 13 provides solvents
for the first two
dimensions of separation. Solvent switching is done on the low pressure side
of the pump,
meaning that gradients generated with this pump are of the step gradient type.
The pump P5
between the two chromatography systems is used to introduce mobile phases for
dissociation of
immune complex as would be needed when an SEC column would be used in the 2nd
dimension
and a RAM column in the 3rd dimension. The eluent stream leaving an SEC column
would be
merged with an acidic solution provided by P5 that would dissociate the
complex before it
reached a RAM column on valve B.
[00114] Valve C is used to uncouple chromatography columns from the
detectors and
preclude transport of undesirable reagents or analytes to detectors. For
example, it was noted
above that large amounts of antibody will accompany samples into the RPC
column and will be
eluted at the end of the mobile phase gradient after all the analytes. In
accordance with this
particular illustrative scenario, valve C can be switched to the position
where the
chromatography system is uncoupled from the MS and antibodies eluting from the
RPC column
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are diverted to waste. A UV absorbance monitor is seen in the illustration,
however, it should be
understood and appreciated herein that it is not required and may be
eliminated in certain
embodiments. Moreover, redundant measurement devices can also be occasionally
used to
quantify analytes in multiple ways to validate accuracy of quantification.
[00115] Analytical optimization trees to this point have emphasized
detection by mass
spectrometry. When analytes leave the first 4 separation dimensions pure or
such that the analyte
is the only species in the eluent stream carrying a particular chromophore or
electrochemically
active functional group, it is possible to detect analytes in other ways,
referred to below as non-
MS detection modes. Examples of such non-MS detection modes include, but are
not limited to,
absorbance, fluorescence, or an electrochemical means.
[00116] With respect to the affinity selector based process, the
simultaneous determination
of multiple analytes to discriminate between analytes and a very large number
of non-analytes
through several dimensions of orthogonal analysis is important. Based on the
very high level of
selectivity afforded by antibodies, the first dimension of discrimination will
generally be an
affinity selector:analyte complex formation in solution. Most generally the
affinity selector will
be an antibody; however, it should be understood and appreciated herein that a
variety of other
selectors may be used as well. In addition, complex formation is executed in
solution because it
circumvents the need to immobilize large numbers of selectors and the inherent
problems
associated therewith.
[00117] The second, third, and often fourth dimensions of analysis
discussed above have
shown how several chromatographic methods, and often a chemical reaction, can
be coupled to
provide still higher levels of analyte discrimination. The resolving power of
these dimensions is
such that captured antigens can be fractionated into a few hundred to a few
thousand individual
components. Many times this degree of resolution is sufficient, at which point
antigens can be
detected by fluorescence, absorbance, electrochemical, or some other means in
which all
analytes produce very similar characteristics for detection. These types of
detection can be of
very high sensitivity, yet still not discriminate between analytes. As such,
analytes must be
partially or totally resolved when they arrive at the detector.
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[00118] Unlike traditional detection methods, mass spectrometry
provides another, quite
different detection method that is orthogonal in its mode of analysis. There
are many types of
mass spectrometers, several of which could be used in affinity selector based
analyses of
multiple analytes. The size exclusion methods described above examined the
hydrodynamic
volume of a substance. Multiple analytes may elute from a RAM or analytical
RPC column
together. Following ionization by either the MALDI or ESI process, analyte(s)
are transported
into a mass spectrometer where they are mass analyzed. Time-of-flight (TOF),
quadrupole (Q),
ion trap (IT), and hybrid forms of these instruments are used in mass analysis
of analyte ions. As
the name implies, mass spectrometry fractionates molecules on the basis of
their mass, often to
less than one atomic mass unit difference. Analytes thus analyzed
(fractionated) are transported
to an electron multiplier that produces an electrical signal, generally
referred to as an ion current
that can be used to quantify an analyte.
[00119] It can be the case that analyte ions of the same mass, or
nearly the same mass,
elute from the RAM, RPC, or some other type of chromatography column ahead of
the MS elute
together. This makes it impossible to differentiate between analytes that are
up to this point in
the analysis identical in their properties.
[00120] As a still higher dimension of analysis, it is possible to
carry out some type of gas
phase chemical reaction that causes ions from the first dimension to fragment.
Collision induced
dissociation (CID) and electron transfer dissociation (ETD) are two types of
gas phase
fragmentation strategies, but it should be understood and appreciated herein
that there are others
as well that can also be used in accordance with the present teachings. It is
often the case in these
gas phase reactions that molecules fragment in a manner unique to their
structure. Fragment ions
thus produced will also have unique masses that when analyzed in a second
dimension of mass
spectrometry can be recognized (by their mass) and quantified. Analyte
quantification can be
achieved from a single fragment ion or the sum of all fragment ions unique to
an analyte.
[00121] Fragment ions from the second dimension of mass spectrometry
can be selected
and further fragmented in a third dimension of mass spectrometry to gain even
more specificity,
but the amount of ion involved relative to the origin is small and thus
sensitivity is poor.
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[00122] Mass spectrometers of choice in terms of sensitivity are those
in which ions are
produced and collected and/or analyzed continuously during the elution of a
chromatographic
peak and then further ionized and transported into the second dimension of MS
for quantification.
QQQ and IT-TOF instruments are examples. One of the great advantages of these
instruments is
that they collect many more ions for analysis.
[00123] Unfortunately, mass spectrometry based detection is not as
sensitive as enzyme
linked immunosorbent assay (ELISA) methods. For instance, current detection
limits are in the
range of 100 pg/mL, even when using 100 um internal diameter RPC columns
before MS
analysis. State-of-the-art ELISA, on the other hand, is 1000 times more
sensitive. The enormous
advantage of using mass spectrometry in the 4th, 5th, and 6th dimensions of
analysis, however,
is specificity. Moreover, the MS analyses are very fast. In fact, tandem mass
spectral analysis
can generally be carried out in a second or less with most instruments. Still
another advantage is
that chromatographic retention time is integrated.
[00124] When analyte mixtures are simple and/or very high detection
sensitivity is needed,
going to high sensitivity liquid chromatography detectors is also possible in
accordance with the
teachings of the present disclosure. The advantage of using regular liquid
chromatography (LC)
detectors over the mass spectrometer is that they are far less expensive and
can be of much
higher sensitivity, especially when used with ¨100 um ID capillaries. The
sensitivity of an LC
detector is inversely related to the square of column diameter. Going from the
normal 4.6 mm ID
column to a 100 um ID column of the same length increases detection
sensitivity over 2000 fold.
Using a 100 um ID RPC column and a laser induced fluorescence detector, the
limit of detection
with a 70 kD protein is approximately 1 pg/mL. This is in the range of ELISA,
yet one is
quantifying multiple antigens.
[00125] Still other detectors that may be used in accordance with the
teachings of the
present disclosure include, but are not limited to, laser induced fluorescence
(LIF),
electrochemical detection (EC), and absorbance detection (AB). Out of these
detectors, the
absorbance detector is by far the least sensitive. LIF and EC detection, on
the other hand,
generally requires that analytes be derivatized with a reagent that
facilitates detection. In the case
of LIF, this would be a fluorophore that exhibits excitation and emission
wavelengths amenable
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to detection in the detector being used. The fluorescence tag agent must also
react readily with
analytes to facilitate the derivatization reaction.
[00126] It has been a practice for more than 50 years to add an
internal standard to
mixtures at known concentration and use the observed ratio of instrument
response to the internal
standard and analyte to determine analyte concentration. The great advantage
of this approach is
that it minimizes random errors that occur during sample analysis. With
immunological assays,
these measurements are generally referred to as competitive binding assays.
The addition of an
internal standard within an immunological assay is related to well-established
RIA practices,
where tagged synthetic molecules (Ag*) similar to antigens were added to
samples. The function
of the tag (*) was to enable detection, as the native antigen (Ag) in the
sample could not be
detected. When the two antigens Ag i and Agi* were added simultaneously and
allowed to
compete for a binding site from a limited amount of antibodies, this was
referred to as a
competitive binding assay and can be represented by the following illustrative
equations:
[Agi]+[Agi*HAb] ¨>[Ab:Ag]+[Ab:Ag*HAgeNAgel and [Agi]+[Agi*]>[Ab]
(3)
where [Ag] is the initial concentration of sample antigen, [Agi* ] is the
concentration of tagged
antigen added to the sample initially in known concentration, [Age] is the
final concentration of
sample antigen after equilibration, and [Age*] is the concentration of tagged
antigen after final
equilibration.
[00127] Here, the present disclosure is generally directed to
differentiating between one
hundred or more antigens (Ag) and tagged synthetic molecules (Ag*)
simultaneously. This is
enabled by liquid chromatography-mass spectrometry, which differentiates
between all these
species in a single analysis. As noted above, a particularly useful mode of
running assays with
large numbers of antigens is in the sequential addition, competitive binding
assays with antibody
saturation. The first step is to add a known amount of antibody [Abi] (or
antibodies with multiple
antigens) to the sample that exceeds the amount of antigen [Ag] in the sample.
Such step can be
represented by the following equation:
[Agi]+[Ab1].¨>[Ab2:Ag]+[Ab3]
(4)
where [Abi]-[Agi]4Ab3]. It is important to note above that Abi, Ab2, and Ab3
are the same
antibody, however, at different points in the assay and under different
conditions.
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[00128] The second step of the assay is to add the tagged internal
standard [Agil to the
sample in known amount. The sum of the two antigens must exceed the
concentration of the total
amount of antibody targeting them, i.e. [Agi]+[Agi*][Ab] such that:
[Agi*]+[Ab3]¨>[Ab3:Ag*]+[Agel
(5)
[00129] Separation of Ab3:Ag* from Age* allows one to quantify [Ab3:Ag*]
alone. The
concentration of [Ab3:Ag*] in the sequential addition assay is inversely
related to the
concentration [Agi], i.e. Ab3:Ag* is at a maximum when Agi approaches zero.
Conversely,
Ab3:Ag* approaches zero as Agi goes to large values. The sequential addition
assay is linear as
opposed to other forms of the competitive binding assay.
[00130] It is worth noting that the concentration of antigen being
determined within the
above-described assay controls, to a large extent, the amount of antibody and
tagged antigen that
must be added to carry out the assay. In other embodiments, such as the
competitive binding Ab
saturation assay described below, this is not the case. As such, it should be
understood herein
that the present teachings are not intended to be limited. A second notable
point is that at the end
of the reactions described in equations 3 and 5, both Ab2:Ag and Ab3:Ag*
coexist within the
solution. Moreover, this is still true at the end of the separation step where
Age* is removed from
the assay solution. The means that in terms of the detection of Ab3:Ag*, it is
necessary that the
tag allow discrimination between Ab2:Ag and Ab3:Ag*. Finally, it is also worth
noting that
differentiating between all the Ab3:Ag* complexes from all the antigens is
important to the
above-described multiplexing processes, particularly as a different tag must
be used for each
antigen being determined. In the case of absorbance, fluorescence, and
electrochemical based
detection, however, this would probably amount to no more than the
determination of five
antigens.
[00131] In accordance with the competitive binding assays of the
present disclosure, all
the analytes should be structurally different to be detected for the
multiplexed assays, including
the internal standards added (e.g., isotopically coded internal standards to
achieve relative or
absolute quantification of the analytes) to the assay solution. As such, the
assays will either have
a different chromatographic retention time, a different molecular weight, or
will fragment in a
unique way in a mass spectrometer. In accordance with the above-described
antigens, Ag* will
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either be a 13C, 5 '4N, 180,or 2H labeled version of the antigen or
carry some combination of these
isotopes that give the internal standard antigen a uniquely different mass
than the unlabeled,
natural version of Ag. An alternative coding strategy will be to derivatize
antigens with some
moiety that does not alter their antigenicity but gives them mass spectral
properties that are
uniquely different than Ag and structurally different from any other substance
in the solution.
Labels in the case of derivatization will frequently be heavy isotope coded
and may be a
universal coding agent that in one isotopic form, is used to code all antigens
(Ag) and in an
isotopically different version, is used to globally code all internal standard
antigens added to the
solution, i.e. Ag* in equation 5. When the final assay mixture described in
equation 5 is
subjected to RPC-MS/MS analysis, it will be possible to discriminate between
thousands of
antigens in the same sample. Advantageously, the teachings of the present
disclosure make it
possible to identify and discriminate between thousands of antigens in a
single analysis, thereby
allowing thousands of immunological assays to be carried out simultaneously
without
immobilizing hundreds to thousands of antibodies or some other binding protein
at specific array
elements on a surface.
[00132] Focusing on Equation 3 above, the present teachings describe a
case in which Ag
and the internal standard antigen Ag* compete for a binding site on the
antibody. Taking this
general concept into consideration, the below described conditions also apply
in accordance with
certain assay embodiments of the present disclosure: [Agi]+[Agi*]>[Ab] for all
antigens being
assayed, and the concentration of [Agil added to the solution is within 5-fold
of [Ag]. Moreover,
the concentration of the internal standard [Agil added is known precisely, and
the
[Ab:Ag]/[Ab:Ag*] ratio is measured at the end of the assay. In accordance with
this embodiment,
large numbers of antigens are measured, i.e. Aga, Agb, Age, . . . Aga, and the
antigens are capable
of greatly varying in concentration, i.e. thousands of fold. In some
embodiments, the same
concentration is roughly used for all the antibodies, while in other
embodiments, the
concentration of antibody may be roughly and not precisely known. Finally, the
ratios of Ag to
Ag* will be determined in the manner described above, and is generally through
differential
isotope labeling or global internal standard labeling methods.
[00133] It should be understood and appreciated herein that in
accordance with some
teachings of the present disclosure, adding a known concentration of internal
standard antigen
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makes it possible to determine the ratio of antigen to internal standard at
the end of the assay. In
accordance with these embodiments, it is possible to determine antigen
concentration using the
well known internal standard method. Moreover, when antibody concentration is
matched to the
optimum sensitivity range of the detection system, it is also possible to use
the antibody as a
sampling tool. In accordance with this sampling scheme, an amount of sample
can be selected
that matches the sensitivity of the detector without taking into consideration
the antigen
concentration, as well as can be used to bring antigens into a concentration
range that varies no
more than ten fold, regardless of their initial concentration. In accordance
with these
embodiments, antibody concentration is being used to bring analyte and
internal standard
concentration into the detection range of the measurement device used for
quantification.
Regardless of the optimum detection range of the monitoring system, it should
be understood
and appreciated herein that it is possible to bring analyte concentration into
the optimal range of
any detector, including a mass spectrometer. Ultimately, these described
embodiments are
capable of using antibody concentration to sample analytes and bring them all
to a similar
concentration range that matches the detection system, while the actual
concentration is
determined by the analyte to internal standard ratio.
[00134] Detection systems ranging from mass spectrometry through
optical,
electrochemical, interferometry, and surface plasmon resonance have been
described above.
Other than mass spectrometry, quantification processes in accordance with the
detection methods
of the present disclosure are generally known in the art and therefore will
not be discussed in
great detail herein. In terms of mass spectrometry, however, quantification
can be achieved in
multiple ways depending on the ionization method. While ion current arising
from the detection
of ions following mass analysis is widely used for detection in mass
spectrometry, the ionization
efficiency of such methods varies widely between analytes. In fact, ionization
efficiency can
even vary in some cases with concentration and other unknown matrix components
in the sample.
A more detailed discussion of quantification by mass spectrometry can be found
in the following
journal article, the disclosure of which is incorporated by reference herein
in its entirety,
"Primary amine coding as a path to comparative proteomics." Regnier, Fred E;.
Julka, Samir.
Proteomics (2006), 6(14), 3968-3979.
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[00135] Fortunately, isotopomers of an analyte ionize in a nearly
identical fashion when
presented to a mass spectrometer together. This means that the relative ion
current seen for the
heavy and light forms of an analyte accurately reflect the relative difference
in their
concentration. When one of the isotopomers is an internal standard of the
analyte added at
known concentration, the absolute concentration of the analyte of unknown
concentration can be
calculated. This is true with both electrospray ionization (ESI) and matrix
assisted laser
desorption ionization (MALDI). It is important that all the isotopomers of an
analyte ionize
under identical conditions, particularly to avoid isotopomers separating in
RPC. When they
exactly coelute they are experiencing identical matrix inhibitors of
ionization. Relative standard
deviations will vary by 6-8% with this detection method.
[00136] In accordance with certain aspects of the present disclosure,
mass spectrometers
for isotopomer ratio quantification, which are capable of sitting on an ion
for long periods of
time (seconds) while eluting from the RPC column are particularly useful
because they allow
many more ions to accumulate than with instruments that rapid scan the mass
range of ions
emerging from the RPC and fail to accumulate large numbers of ions for
detection. Non-limiting
examples of such instruments include the triple quadrupole and quadrupole ion
trap instruments.
[00137] Direct quantification by ion current measurements are also
useful in accordance
with the teachings of the present disclosure, and particularly wherein larger
measurement errors
in the range of about 20% to about 30% can be accepted.
[00138] With mass spectrometry it is often the case that fragment peptides
are separated in
the gas phase prior to the first dimension of mass analysis. Ion mobility is
one type of gas phase
separation strategy but it should be understood and appreciated herein that
there are other gas
phase separation strategies that can also be used in accordance with the
present disclosure. In the
case of ion mobility, ionization by ESI or MALDI is followed by the separation
of ions based on
their mobility in a carrier buffer gas. The output of this separation is
characteristic of the ion
mobility distribution. This type of separation often occurs in milliseconds
and can be applied to
the strategy described herein as a means of resolving peptide fragments.
[00139] It is frequently asserted that 40% of all proteins exist as
complexes that can vary
in activity. Affinity selection of protein complexes results not only in
pulldown of the analyte of
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interest, but capture of non-specifically bound materials. It is often the
case that these materials
interfere with downstream analyses. Additionally, the protein:protein
interactions resulting in
complex formation often inhibit epitope:paratope recognition. As such it is
frequently beneficial
to break these complexes by chemical means resulting in denatured species.
Reduction and
alkylation is one means of breaking apart these complexes. It should be
understood and
appreciated herein that there are other modification strategies such as heat
denaturation,
microwave denaturation and others that can also be used in accordance with the
present
teachings. It also is important to note that it is frequently beneficial to
affinity purify analytes of
interest after the breakdown of protein complexes into denatured species.
[00140] Columns packed with the materials needed to perform the separation
strategy
described herein is one strategy, but it should be understood and appreciated
that there are other
hardware formats that can also be used in accordance with the present
teachings. It is often the
case that the separation strategies described above would be applied to
cartridges, pipette tips,
flow-through plates, or magnetic beads containing the necessary packing
materials. Cartridges
can snap into place and be readily exchanged depending on the analyte of
interest. The use of
pipette tips as a micro-bioreactor shaped as a pipette tip with a filter
containing packing materials
would enable a single use format for this application. The use of flow through
plates equipped
with a filter enables parallel processing and therefore high throughput
application of the
materials described herein. The use of spin columns as a micro-bioreactor
shaped as a tube with
a filter containing packing materials is another single use format applicable
to the process
described herein. The use of flow through plates equipped with a filter
enables parallel
processing and therefore high throughput application of the materials
described herein. Use of
magnetic beads containing the necessary packing materials enables direct
application of these
materials to the sample eliminating the need for transfer. Following the
completion of a step
these materials are then removed through the use of a magnet. Application of
the
multidimensional strategy described herein is through stepwise addition of
various materials.
[00141] Discrimination of analytes is achieved in a combination of
ways ranging from
analyte specific chemical modifications (such a derivatization or
proteolysis).
[00142] Application of analyte specific chemical modifications (or
proteolysis) using
trypsin is one strategy but it should be understood and appreciated that there
are other
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modifications that can also be used in accordance with the present teachings.
Other possible
enzymatic modifications include the use of lys c, glu c, pepsin, papain,
pronase, PNGase F,
glucuronidase or a plurality of other enzymes. It is also important to note
that discrimination of
analytes is often achieved using in a combination steps in various order.
[00143] Affinity based discrimination followed by chemical modifications
(such a
derivatization or proteolysis) is one strategy but it should be understood and
appreciated that
there are various orders that can also be used in accordance with the present
teachings. It is
frequently the case that affinity selection of peptides is performed in such a
manner that
enzymatic modification precedes the first dimension of separation. This
enables the use of
synthetic peptides to standardize the affinity selection process. In other
cases the first dimension
of separation is followed by enzymatic digestion prior to the second dimension
of separation.
This enables the detection of protein variants or modifications that exist
outside the epitiope. In
other cases the second dimension of separation is followed by enzymatic
digestion prior to a
third dimension of separation. In one application of the material described
herein this is
beneficial to the understanding of post-translational modifications. Enzymatic
removal of post-
translational modifications from corresponding peptides simplifies the
analysis by reducing the
heterogeneity normally associated with these modifications. Once peptides
containing a given
modification are identified a subsequent analysis wherein the post-
translational modification is
not removed is frequently applied.
[00144] In other cases two enzymatic modifications are used in tandem with
a separation
step between them. As an example, a protein is purified by means of affinity
selection, in a
subsequent step this affinity based discrimination is followed by proteolysis
performed using
trypsin, a resulting peptide of interest is further purified by means of
affinity selection. In a
subsequent step this affinity based discrimination is followed by
deglycosylation using PNGase
F.
[0001] While exemplary embodiments incorporating the principles of
the present
disclosure have been disclosed hereinabove, the present disclosure is not
limited to the disclosed
embodiments. Instead, this application is intended to cover any variations,
uses, or adaptations of
the disclosure using its general principles. Further, this application is
intended to cover such
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departures from the present disclosure as come within known or customary
practice in the art to
which this disclosure pertains and which fall within the limits of the
appended claims.
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