Note: Descriptions are shown in the official language in which they were submitted.
This application is a divisional appLication from application Serial
No. 386,403.
This invention relates generally to an improved apparatus and method
for the performance of chemical processes and, more particularly, to an improvedapparatus Eor automatically performing the sequential degradation of protein
or peptide chains containing a large number of am:ino ~cid units ~or purposes
o-f determining the sequence of those U11its.
The linear sequence of -tlle amino acid units in prote:ins and peptides
is of considerable interes-t as an aid to understanding their biological
functiolls and ultimately syn-thesizing compounds performing -the same functiolls.
Althougll a variety of techniques have been used to determine thc l:inear order
of amino acids, probably the most successful is known as the Edmall Process.
Various forms of the Eclman Process and apparatuscs for automatic.1lly performing
the processes are described in the follo~iing publications:
Edman and Begg, "A Protein Sequenator," European J. Biochem. 1 (1967)
80-91; IVittmann-Liebold, "Amino Acid Sequence Studies of Ten Ribosomal Proteinsof Eschcrichia coli with an Improved Sequenator Equipped Witll an Automatic
Conversion Device," lloppe-Seyler's Z. Physiol. Chem. 354, 1415 (1973);
IYittmann-Liebold et al., "A Device Coupled to a ~lodified Sequenator for the
Automatcd Conversioll of Anilinothiazolinoncs into P~-l Amino Acids," AnalyticalBiochemistry 75, 621 (1976); Unitcd Statcs Patcnt ~o. 3,959,307 issucd to
l~ittmann-Liebold and Graffullder on l\lay 25, 1976, for "~lctllod to Dcterm~incAutomatically thc Sequencc of Amino Acids;" llun~apillcr and llood, "Dircct
~Iicrosequcncc of Polyl~cptidcs Using an Improvcd Scquenator, A .~'onl)rotcill
Carricr (Polybrcnc), ~nd lligl~ Prcssurc Licluid Chromatograplly," Biocilcmistry212-~ ~1975); I.aurscn, R. A. Eur. J. Biochcm. 20 (1971); IYncIltcr, E., ~laclllcidt,
.~
.1~. I
Il., llofncr, Il., alld Otto, J., FEBS l.ett. 35, 97 (1973); United Statcs Patent
No. 3,725,0lO issued to Penllasi on ~\pril 3, L973, for "~pparatus for Auto-
matically Performillg Chemical Processes;" Uni-ted States Patent l~o. 3,717,~36
issued to Penhasi et al. on ~ebruary 20, 1973, for "Process for the Sequent:ial
Degradation of Peptide Cllaills;" United S-tates Pa-tent No. 3,892,531 issued
to Gilbert on July 1, 1975, for "Apparatus ~or Sequenc:illg Peptides and
Proteins;" United Sta-tcs Patent No. 4,065,412 issued to Dreyer on December 27,
1977, for "Peptide or Protein Sequcncing ~Icthod and Apparatus." A furtller
apparatus of note is dcscribed in l]n:ited Sta-tes Pa-tent Serial l~o. 4,252,769
filecl December 26, 1979 by Leroy E. Ilood alld Michael 1~. llunkapiller, two of
the applicants hereon~ on "Apparatus fo:r the Performallce of Chemical
Processes."
Briefly, as discussed in the above publications, thc Edman sequen-
tial degradation processes involve three stages: coupling, cleavage and
conversion. In the coupling stage phenylisothiocyanate reacts with the N-
terminal ~ amino group of the peptide to form tlle phenyltlliocarbamyl
derivative. In the cleavage step anhydrous acid is used to clcave tlle
phenylthiocarbamyl derivative to form the anilinothiazolillolle. After ex-
traction of the thiazolinonc the residual peptide is ready for -the next cyclc
of coupling and clcavage reactions. Aqueo-ls acid is used to convert the
thiazolinolle to the phenylthiollydantoill ~-~hich may be analyzcd in an appropri-
ate manner, such as by chromatogral)lly.
Thc automated apparatus of thc Penhasi 3,725,010 p;ltcnt, as modificd
in thc abovc-rcfcrcllccd articles of l~ittmanll-Licbold ancl thc patcllt Scrial .~o.
4,252,769 of llu~ pillcr alld llood, rclatcs to all automatcd scqucnator in
~hicll thc rcactiolls arc carric~l on in a thill film formcd on thc insidc ~all
_
of a ro-tating reaction cell wh:ich is commonly known as a sp-inning cup and is
located withill a closecl reaction chamber. Me ms a:re proviclecl for int-roducing
and removing controllc?d amowlts of liquid reagell-ts relat:ive to thc chamber for
reaction with a sample of a protein or pepticle in an incrt atmospherc. The
sample to bc analyzecl is initially ylacel in the spinninK cup followe~ by the
sequential introcluction and witllclrawal of -the various reagerlts and solvents
necessary for carrying ou-t the coupling and cleavage reactiolls. Tlle liquicl
reagents and solvents thclllselves form -films on the walls of thc cup which
pass over and interact wi-th the samplc film as the cup spins. The reagonts
dissolve the sample film and per:form the couplillg and clcavagc stagcs of the
Edman proccss. Upon completion of the couplillg and clcavage stagcs the
reaction chamber is evacuated to rcmove volatilc componellts of the reagcnts.
Following thc post-coupling evacuationJ the remaining sample film is extracted
witll solvent to remove non volatile components. Following the post-clcavage
evacuation the rcsulting thiazolinone is extractecl from the s~mplc film ~ith
solvcll-t and transferred either to a scparate flask for con~ucting tlle conversion
step or to an appar~tus for collection and drying of the various fractions.
In cases wllere the conversion process is not performed immcdiately in a
conversion flas~ the process may be performed later on a numbcr of fractions
simultalleously.
The introduction and witll~rawal of fluids relative to the spinnillg
cup has beer acllicvecl witll fluicl concluits passillg through a plug WhiCIl scals
all olcnillg in tllc upl)er ~all of tllc rcaction cllambcr and clcpcnds thcrcfrolll to
a location Witllill tllC CUp. I:luicls arc introducccl ~ircctly illtO thc spillnillg
CUp ;It ;1 pOillt ;Idj;lCCllt tilC bOttOIIl tilCrCOf, .111(l arc Wit~ r;U~Il from ;111 ;Illlllll;lr
groovc in thc cylilldric.ll intcrior surf;lcc of thc CUp. Thc flui~ to hc
withlr;lwll is forcc~ illtO tlle allllul;lr groo~c b~ ccntrifug;ll forcc \i'hCII tllC CUI)
is rotated at a higll rate, and is wi-thdrawn throug?l a conduit havil)g an inner
end projecting into the groove. This effluent condui-t -thus acts as a seoop
for removing the reaction products and by-produc-ts and the extracting solvents.
If the protein or peptide sample in a spinnillg eup deviee does not
llave suffieien-t mass to form a cohesive film by itself, it is sometimes ear-
ried on the inner wall of the cup durillg the solvent extractions within a
relatively thick layer oE nonE~rotein carrier material. Tlle carrier material
and the sample are then dissolved in the liquicl reagents during the reaction
stages to enable the coupling and cleavage reactions to -take place. A polymer-
ie cluaternary ammoni-lm salt having the chemical composition 1,5-climethyl-1,5-
diazaundecametllylene polymetllobromide has been used for this purpose. The
earrier must be applied in substantial quantities to securely retain the sample,
and the carrier and sample are both dissolved by the liquid reagents to permit
reaetion between the sample and the reagents.
Althougil devices of the spinning cup type call provide acceptable
experilnental results in many cases, they have several disadvantages. ~or ex-
ample, the expenses of obtaining a suitably large protein or peptide sample and
maintaining an adequate supply of the necessary reagents are quite high, pri-
marily because the reagents used are in liquid form ancl must be usecl in
substalltial qucantities. Liquid reagellts alld solvents tend to separate portions
of the sample from the film and ~ash them from the w.lll of the cup as tlley pass,
reducillg tlle yield of terminal amillo aeid wlits obtained in each suceesive
eycle of the apparatus. The ;nitial qu;llltity of sarnple must tllerefore be
great enough to insure that sui`ficient saml)le ~iill remain throllgll the last eycle
to produce useful results. [)evices of this t~c a1so havc ratllcr long cyclc
tilllCi duc to the collsi-lerable volume of the reactioll ch~lllber all~ tllC IICC~ to
3.~.~D'~
reyeatedly remove semi-volatile liquicl reagents and solvents by vacuum drying
the sample therein. In addition spinning cup seq-lenators are quite cornplex
ancl expensive both to manuEacture and rep~lir.
A differellt type of sequencing device is clisclosed in the above-
cited Laursen and Wachter papers wherein the sample is immobilizecd by covalent
linkage to the surfaces o-E a plurality o-E small beacls. 'I`he beads form a porous
packing within a reaction column and the column is flooded witll liquid
reagents to perform the chemical procosses. 13ecause the cleavllge reagents used
are e~eellent solvents for proteins and peptides the covalent linkage must
be comple-te in order to hold the sample in place. Ilowever covalent linkage
is dlfficult to obtain in practice. The packed column is also dif-ficult to
wash and the beads thereill tend -to disintegrate during use.
There have heretofore been proposed sequencing devices designed to
overcome the deficiencies of these apparatuses by contaillillg a sample witllin
a stationary reaction chamber and subjecting the sample to at least one reagent
in gas or vapor form. The Gilbert and ~reyer patents cited above disclose
t~o such devices neither of which operates entirely satisfactorily.
The device of the Gilbert patent provides a closecl finger-shaped
extension within a reaction chamber for holcling a peptide or protein sample at
a controlled temperature during sequential exposure to gaseous reagents and
solvents. Eacll time a reagent is introduced the extension is cooled inter-
nally to produce condensatioll thereon. The eYtensioll is then ~armed -causing
the salllple to dissolve in the liqui-l ~nd the reactioll proceeds. .~fter
reacting witll the s~mple the ull~;lllted semi--voltile chemic.lls may either be
dried from the salllple by a combin~tio1l o~ he;lt alld .L stream of inert gas or
be w;lslle(l from the e.Ytension alollg ~ith the termill~l amillo ;lci~l hy a solvent
whicll :is condensed on -tlle extensioll until it clrips therefronu
In the clevice ancl metllocl of the Dreyer patent, a yro-tein or peptide
sample is applied -to both the inner and outer surfaces of many sma11 macro-
porous beads within a reaction column by chemical coupling or direct adsorp-
tion thereto. Various reagen-ts and solvents are passed sequentially through
the packed column in either gaseous or liquid form to produce the desired
degradation reactions. The flow of reagents and solvents -to the column is
controlled by a ten position rotary face soal valve.
Unfortunately, the devices of tlle (,ilbert and Dreyer patents do not
provide a sufficien-tly contamination-free environment to achieve acceptable
results through a large number of degrad.ltion cycles. For example, it is
difficult to efficiently wash the protein or peptide samp]e in the Gilbert and
Dreyer devices. The Gilbert method of washing the sample by condensation of
solvent thereon to the point at which solvent drips from the sample would
tend to leave traces of the various reaction products on the sample, contaminat-
ing future chemical reactions. Likewise, the packing used to retain thc sample
within the reaction column of Dreyer is difficult to wash because tlle various
chemical products must be transported entirely through it and away from the
colulM to avoid contamination. This is not easily done even w}lell large amounts
of solvent are used, because the solvent tends to pass through the spaces
between the beads rather than througll the small pores inside the beads wllere
most of the protein sample is located. The fluid feed lines and flow~valves
of the Gilbert aod Dreyer devices are also difficult to fully evacuate and are
prone to trapping chelnical residues wllic}l call interfere with tlle intellded
chemistry of furtller reaction cycles.
The glass or pl.lstic bC;ldS uscd ;15 p:lCkill~ in the rcaction colullm of
~ ~ ~3~d~ ~
Dreyer also have a tenclency to disintegrate over a number of degradation
cycles, clogoillg thG system to the point at whicll the ~)assage of :fluids there-
through is hindered. It tllen becomes virtually impossible to wash the system
between cycles and the chemistry within the column becomes hopelessly
con-taminated.
The contamination caused by the several factors described above has
a culllulative effect over the duration of a sequential degrcldatiorl process.
The sample and the reagents ~ithin the reaction cell thus become more and more
contaminated, hinderillg tlle desired coupling and cleavage reacti.ons and causing
a number of undesired reactions to take place. The yield from each complete
cycle oE the apparatus is thus clecreased and a series of contaminants is
introduced into the fractions.
The yield is further decreased by direct loss of the sample due ~o
a variety of reasons, ineludillg the disintegratioll of the paclcing, solubilityof the sample in the flushing solvents, and failure of the sorptive bonds
bet~een ~ac~ing and sample.
~hile these effects may be overloo~ed in some cases wllere large
amounts of the protein or peptide sample are available or where the ehain llas
a relatively small number of units, they become devastating in cases where -the
chain has a very large number of ~mits or only very small amoullts of the
partieular protein or peptide are avail;lble. Both of tllese eircumstances are
present in the case of interferon, a small protein made in hum;ln eells in
response to certain viral infections. Interferon has recently caused a grea~
deal of e.ccitemellt in the world of clinical medicille because it prolllises tobe an effective agellt for arresting viral infectioTIs ;Ind it apl)e;lrs to offer
consideral)le nope as a1l allti-c,lllcer reagent. Interferon is l)roduced alld,
~f~
accordillgly, is uvailable only in very small quantities. Currently, virtually
the entire worll s production of the two types ot` human interferon originates
in the relatively few world centers that have access to large quantities of
human whi-te blood cells (leukocyte interferon) or certain human cells in
tissue culture (fibroblast interferon). Because of this limited productive
capacity of interferon, i.-t has been diffi.cult to carry out well controlled
clinical studies and :fundamental analyses of how this molecule fllnctions. To
further complicate -the pictllre, in-terferoll is composed of a chaill of approxi
~na-tely 150 amino acid units, which must be individually cleaved from the cllain
for analysi.s. Contamination losses of tl-e -types clescribed above can prevent
the sequencing of any but the first few amino acid units oE interferon with the
very small quantities of the protein available. Beyond the first few cleavage
eycles, the small sample can become contaminLted to the point at wllicll positive
results are unobtainable.
Tlle most sophisticated prior device known to the applicants herei
for eonverting the various thiazolillolles e].eaved from tlle sample into tlle
more stable phenylthiollydalltoins is the conversion flask described in the
above-refereneed articles of ~ittmann-Liebold, as modified in tlle patent
Serial ~o. 4,252,769 of llunkapillar and llood. Ilowever, applicants have found
that this flask suffers from inefficient waslling of its inner walls when
reagents and solvents are introduced tllrough the ~ppropriate capillary tube.
It has been suggested tllat the reagents and solvents can be deliverel-~itll a
stream of inert gas to ~.~ash the flask walls l)v splatterillg the liquid thereon,
however, this techTliqlle causes an erratic flow of liqui~ to the flas~ and
makes it very difficult to control tle volume of liquid delivered.
i~pl~licants h;lvc also foun(l tllat wllcll tl~c prior convcrsioll flasli is
~ ~3~ '7
~ , . ~
scalecl down appreciably in size to accommodate lower volumes of liquid, it is
difficult to obtain the optimum degree o~ dispersion of inert gas bubbles
within the liquid contents of the flask to agitate the con-tents during the
conversion reaction and evaporate the semi-volatile components thereof. Iner-t
gas introducecl to the bottom of the Elask for these purposes tends to rise to
the surface of the liquid in rela-tively large bubbles which do not uniformly
agitate the liquid and instead promote splattering of the liquid onto the top
of the flask.
Therefore, in many applications it is desirable to provide an
apparatus for performing chemical processes sucll as the se~uencing of proteins
or peptides WlliC]I operates efficiently and with a minimum of system contamin-
ation to enable the maximum number of sequencing cycles to be successfully
performed with a very small amount of sample.
The invention of application 3S6,403 comprises chamber means having
an interior surface defining a reaction chamber, the chamber having inlet and
outlet means for conduction of fluids therethrougll in a pressurized streann,
and solid matri~ means permeable by diffusion to a plurality of fluids and
loeated witllill the ehamber, sueh tllat a saml)le embedded in the matri~ means
is immobilized and e.Yposed to any of a plurality of fluids passed through the
ch.lmber for chelllical interaction therewith.
The ehamber means may inelude surfaee means supporting the solid
matri.Y~ means as a thin film thereoll, and the matri.Y means may eomprise a
polymeric ~uaterllary ammoTIium salt sueh as 1,5-dimetllyl-1,5-diazaundeeamet}ly-
lene polylnetllobrolllide or poly (~ -dimethyl-3~5-dimetl-ylelle peperidinium
chloridc).
Tlle surface me.llls m;ly comprise all or a portioll of tilC interior w;lll
o:E the chiamber mea.ns, or may comprise porous sheet means extencling substan-
tially transversely across the chamber and permitting passage of the :Eluids
theretllrougll. The shee-t means may compri.se a sheet made of a plurality of
glass fibers.
The chamber means may comprise a pair of abutting chamber elements
having first and second cavities, respectively, on opposecl mating surfaces
ther of, the first and second cavi.ties being aligned l~ith each nther to ~orm
-the reaction chamber. The first ancl second cavities may be tapered in clirec-
tions away from the mati.ng surfaces to locations at ~hich they communicate withthe inlet and outlet means, respectively. Ille porous sheet means, :if used,
is received ~ithin a recess in at least one of the mating surfaces for
retention within the chamber in an orie-ltation substalltially separating the
: first and second cavities. The chamber means may include at least one sheet
of yielding material s mdl~iched bet~een the mltillg surfaces in a sealing
relationship, the yielcling material being permeable to the plural:ity of fluids.
At least one of tlle chamber elements may tllell include a raised portion on a
mating surface thereof ~illich extends about the cavity tllereill to compress the
sheet of yielding material against the mating surface of the chamber element
and thus enhance the sealing relationsllip.
The inlet and outlet means may comprise a pair of capillary passages
e.Ytending tllrougll tlle chamber elements, respecti~ely, and communicating at
inner ends thereof witll the reaction chamber on opposite sides of the porous
sheet means. Tllc capill;lries may be coa.Yial ~ith tlle reaction chamber ancl
e.Ytelld therefrom to outer capillary openings at substalltially flat outer sur-taces of tlle cllamber elements.
rhc mc;llls for se~luentially passillg a pl-lr;llity of fluids tllrough the
1 ()
chamber may comprisL valve block means hIving a plurality of subs-tantially
flat valving sites on -the surface -thoreof, the valve block means defining a
primary yassage continuous be-tween two ellds thereof anLi communicat:ing through
primary openings with each of -the valving sites and a plurality of secondary
passages each communicating througil a secondary openil1g with one of the valving
sites; and a plurality of resilient substantially impermeable diaphragms
covering the respective valving sites, eacl1 of the diap11ragms being actuable
between a first sealing condit-ion in wi1ich i-t is forced against one of the
valving sites to cIose off the primary and secondary openil1gs commun:icating
with that site and a second condition in which it i.s drawn away from the site
to provide a flui.d flow path between the primary and secondary openings over
the exterior of tl1e valve block means; whereby fluid flow betl~een the primary
passage and the secondary passage can be selectively controlled. The connect-
ing means may comprise at least one tapered ferrule closely received in sliding
engagement over a tubing member and urged against a differel1tly tapereLl recess
in the valve block means communicating witll one of the passages thereill sucl
that an inward force applied to said ferrule is focused on a relatively small
area of contact between the ferrule and the recess to produce a fluid seal
therebetween. The recess is preferably tapered .~t a greater angle than said
ferrule. The conl1ectiIlg means may furtl1er include a fitting at one end of the
primary passage for connectiIlg the primary passage to anotl1er portion of ti1e
apparatus such that the primary passage serves as a manifold 1.hicll can be
flusheLI by a flow of fluid between the fitting allLI the secondary passage
furthest aw.ly from the fitting. Ihe primary pass;lge may comprise a plurality
of straigl1t passages connecteLI end to enLI to form a conduit h:Ivillg a sal~tootII
configur.ltiol1 al1LI communic.ltil1g at alterll;ltillg intersectio1ls t11creof with the
respec-t-ive valving sites.
Tllus this :invention provides valve apparatus for use in an apparatus
for the sequential performallce of chemical processes to control the flow o-E
fluids therein, comprising: valve block means having a plurality of substan-
tially Elat valving sites on a surface thereo-E, sa:id valve block means defining
a primary passage continuous between two encls thereoE and communicating through
primary openillgs with each of said valv:ing sitesl and a plurality of secondary
passages each communicating through a secondary opening with one oE said
valving sites; and a plurality of resiliellt, substantially :impermeable
diaphragms covering said respective valving s:ites, each of said diaphragms being
actuable between a first sealing condition in which it is forced against one
of said valving sites to close ofE -the primary and secondary openings, communi-
cating with said site and a second condition in which it is drawn away from
said site to provide a fluid flow path between said primary and secondary
openings over the exterior of said valve block means; whereby fluid flow between
said primary passage and said secondary passages can be selectively controlled.
Conveniently the apparatus may include a conversion flask llaving a
plurality of capillary tubes extending into the interior thereof for the intro-
duction and withdrawal of various fluids, at least one of the capillary tubes
having an inner end at which the bore is closed and which is provided with a
plurality of restricted radially-spaced orifices, such that passage of fluids
through the capillary tube produces a spray onto the interior walls of the
flask to wash them down. A capillary tube terminating at a point adjacent the
bottom of the flask may also have a closed end with a plurality of restricted
radially-spaced orifices adjacent thereto, such that passage of a gas inwardly
through the capillary tube produces a plurality of small bubbles agitating
any liquid within tlle flask and accelerating the drying thereof.
3 ~'13~
The metilod of the present invention for sequen-tially performing
chemical processes on a sample of chenlical ma-terial comprises embedding the
sample in a solid matrix which is permeable by di-Efus:ion to a plurality of
fluids, enclosing the solid matrix wi-thin a closed cllamber having an inlet a.lld
an outlet, and sequentially passing the plurality o:E fluids through the chamber
as a pressurized stream from the inle-t to the outlet thereof such that the
sample is exposed to eac]l of the :Ell:lids, whereby the sample is immobilized and
chemical interactlon between -the sample and the fluids is obtained. The solid
matrix may be supported as a thin :Eilm on the inner walls of the closed chamber.
Al-ternatively, the solid rnatrix may be supported on a porous sheet which ex-
tends substantially transversely across the chamber at a location between
the inlet and outlet such that fluid passed from the iillet to the outlet must
pass through the sheet. The step of embedding the sample in a solid matrix
may comprise the steps of applying the solid matrix to a support surface as
a thin film and then applying a solution containing the sample to -the film
such that the sample solution dissolves the matrix. The liquids are then eva-
porated from the solution, leaving behind a film with the sample embedded
therein.
The present invention to provide an apparatus including a valve
block~ and method -for the sequential performance of chemical processes on a
sample of chemical material with a minimum of sample loss and a minimum of
system contamination.
The present invention also seeks to provide an economic appa.ratus
and method for thesequential performance of chemical processes on a sample of
very small size through the use of minimum amounts of reagents and solvents.
Particularly the present invention seeks to provide an improved
apparatus and method for the sequential performance of chemical processes
~- 13 -
haviTIg a very shor-t cycle time.
Further, the present invention seeks to p:rovide arl lmproved apparatus
and method for the sequential performance o:E chemical processes on a sample
wherein the sample is more effectively washed between cycles.
The apparatus and method of the present invention solves a number of
- 13 a -
the problems of -the yrior sequenators by immobilizing the protein or peptide
sample within a solid matrix formed as a thin film permeable by diffusion to
both the reagents and solvents used in the degradation process The difficult
problem of attaining complete covalent linkage is thus avoided, as is the
problem of sGample loss experienced when -the samyle is directly adsorbed onto
a suppor-t surface and fully exposed to the mechanical she.lring Eorces of the
mobile liquid phase. The sample is securely held in place by the matrix, while
the smaller reagonts, solvents and arnino acid derivatives are able to diEfuse
through the matrix, in effect dissolving in the matrix to a sufficient concen-
tration to carry on the various steps in the delradation process. The solicl
matrix retains the sample so effectively when exposed to gaseous reagents that
virtually any shape of sample support surface can be used without causing
sample loss. The inner walls of the reaction chamber itself may be a suf-
ficientsupport surface.
A support surface whicll greatly facilitates complete washing of the
system is a porous sheet made of a plurality of overlapping glass fibers and
extending transversely across a flow-tllrough reaction chamber. The porosity
is provided by spaces between the fibers. This structure possesses a relatively
higll total surface area with a minimum dimcnsion in the direction of fluid
flow. The soli~ matrix forms a thin film on the surfaces of the fibers,
enabling reagellts and solvents to readily diffuse into the film to interact
with the sample embcdded therein. Tllis en~bles chemical processes and wasll
cycles to be performed Oll the sample witll a minilllum ot` reagents alld solvents
alld in a relatively short peliod of time. Tllc reagellts all~ solvents, some of
wllicll are in the form of a g;lS or vaE70r, pcrme;lte the thill film to colltact
thc s;~mple alld interact therewitll ~s comE~lctcly alld cfficicntl~ ;IS l)ossiblc~
`3 ~ d ~
The relatively thin profile of the porous sheet disclosed herein
forther enhclnces the ability o~ the sample to be thoroughly washed of residual
reagents and reaction products with a relatively small amount of solvent. The
solvent need only move the reagents and reactiorl p:roclucts the relat:ively short
distance beyond the surface of the sheet -to remove thcm from the system. ~'rom
a point outside the surface of the sheet they may be easily conducted out of
the chamber to leave the sample in conditioll for the next reaction step 'I'he
low solvent usage not only represen-ts a savings in the cos-t of solvent, but
also reduces the tendency o-E the sample to be washed from the react-ion chamber
and lost.
The use of reagen-ts in gas or vapor form also contributes to complete
exposure of the sample to the reagents, minimizing the amount of reagents
required. Lo~ reagents usage is important because the reagents used in the
Eciman degradation technique must be extremely free of contamination and tllere-
fore are very expensive. Further, the sample and the solid matrix containing
it are not dissolved by the gaseous reagents, eliminating the problem of sample
loss due to separation of the sample from the support surface
The reaction chamber of the present invention is constructed to allo~
passage of both gaseous and liquld reagents through the porous slleet holding
~0 the sample Witllout allol.ing the sample to become contaminated ~iith external
impurities or ~ith chemicals carried over from one reaction step or cycle to
another. The abutting chamber elements thus combine to form a lo~ volume
reaction chamber made up of first and seconcl cavities on opposite sicles of tlle
porous sheet A pair of capill;lry pass.lges cxtencling opposltely through the
respective ch.lmber elements from tlle cll;llllber itself en.lble a plur;llity of fluids
in gas or li(lu.i-l forln to be p;lssed as a pressuri-ed stream throllgh tlle cllami)er
- 15 -
and pas-t the sample ma-trix. rl`he low volume of the chamber and the passages
minimizes the volumes of reagents ancl solvents reciuired, and facilltates
vacuum drying of the system between cycles. Tlle two chamber elements are seal-
ed at mating surfaces -thereof against a shee-t o:f yielding material sandwiched
between the mating sur-faces. The yielding material is permeable to the plural-
ity of fluids passed through the chamber, and in ~Eact, improves the flow of
gases through the chamber by disbursing the gases to more ~miformly contact
the porous sheet.
~n altern.ltive embodiment of the reaction ehamber is a single capil-
lary tube or eapillary-type passage having a solid matr:ix formed as a thin
filn~ on the interior surfaee or bore -thereof. The protein or pel)tide sample
is embedded in the matrix, as in the case of the porous sheet, and the rea.gents
and solvents arè passed sequentially througll the tube to interact with the
sample. The chamber structure described above ean be used for this purpose
without the porous sheet element. The sample-containi.ng matrix is tllen formed
on the surfaces of the first and second cavities. Similarly, the surface
supporting the solid matrix ean be construeted in virtually any way wllieh
enables the reagent and solvent fluids to be passed over the matrix.
The novel valve assemblies of the present invention for eontrolling
the flow of fluids to and from both the chamber and the conversion flask are
especially eonstrueted to eliminate cross-contamination of the fluids. Each of
the valve means interfaces a single conduit with a plurality of other'conduits
for selectively connectillg the single conduit to eaeh o-t' the others. Tlle single
eonduit is made to communieate witll one end of tlle primary passage in the valve
bloc~ wllile eacll of the other conduits is conllected to one of thc SCCOll~ary
p;lSS.lgCS. In tllC nor.mal closed COllditiOII, C;lCIl of tllC di.l~)llraslns covering the
- 1 (j -
3'7
various valving sitcs is forcecl by gas pressure ag~ainst the surface of the
valve block to prevent communicntion of the primary passage with the secondary
passage leading to the particular valve site. Fluid communication between the
single conduit and the other conduits may be selectively provided by applying
vacuum to one or more of the diaphragms to draw the diaphragms away from the
valving sites and allow ~fluid to pass over the surface of the valve block lying
between the openings to the respective passages. Thc secondary passage leading
to the valve site at the remote enc1 of the primary passage may be connected
to a pressurized source o-E a flushing fluid such as inert gas for the purpose
of completing the delivery of each fluid througll thc valve. Thusl after a
particular reagent or solvent is introduced into thc rcaction chamber by
applying a vacuum to the corresponding diaphragm o-f the delivery valve, -the
diaphragm at the remote end of the primary passage may be opened to complete
the delivery by forcing any of the reagent or solvent remaining ~ithin thc
primary passage to the chamber. This is possiblc due to thc continuity of the
primary passage and results in thc manifold formed thercby bci11g purgcd of a
particular reagent or solvent before delivcry of the next reagent or solvcnt
is commenced. In the case of thc valvc at the outlet to the reaction chambcr
the primary passage is connected to thc outlct ~hilc the sccondary passages
are conncctcd to the conversion flask vacuum and ~aste respcctively. The
continuity of thc primary passagc cnablcs it to be thoroug111y evacuatcd and
virtually elimin.1tcs thc possibility that scmi-volatilc substances ~iil be
trappcd therei11 bct~ccn cycles.
It ~ill bc u11dcrstood th~t thc s.u~toot11' configulation of thc pri-
mary valve p:155~gCS discloscd hcrcin has prcviously bccn uscd in valve asscm-
blics of othcrs in tllc sc(lucl1ator ficld. Ilo~cvcr~ thc prior s~tooth v.11vc
- 17 -
3 ~
assemblies of which applicants are aware have incorpora-ted a plurality of in-
dividual blocks mourlted iaga:inst valving sites on a main valve bloclc to slide
back and forth between conditions of communication and noncommunication oE
passages within -the main block. The sliding blocks tend to wear, causing leaks
both to the atmosphere and between the passages. The novel valve assemblies
disclosed herein solve tlle problem of ~ear by combining the prior sawtooth
manifold with a series of diaphragms for es-tablishillg and cutting off` flow be-
~ween pairs of openings communicating ~ith the respective passages. The
diaphragms can be made of substantially inert material such as commercially
available fluorocarboll pol~ners "~hieh will function indeEinitely without
deterioration. ~loreover, the prior means for connecting the valve passages
to external conduits tend to produce excessive pressure on the sides of the
valve bloek, promoting distortion of the upper sealing surface of the valve
block and loss of its ability to form a seal. In particular, the prior sawtooth
valves of wllich applicants are aware corlnect external conduits to the valve
passages by pressing substantially flat flange surfaces associated with the
various conduits against the sides of the valving block to produce a series of
seals between pairs of flat surfaces. Beeause each of these components is made
of materials such as fluorocarbon polymers which are very difficult to accur-
~0 ately maclline, a considerable amount of pressure must be applied to conform
the respective sealing surfaces to each otller and form tlle required seals. The
pressure is borne by tilC valve block, causing distortion of its uyper sealing
surface .
The valve assemblies of the present in~entioll incorporate a plurality
of tapered ferrules receivable parti.llly ~ithill differently tapered recesses
in tlle valve bloc~ to etfect a se;ll witllout the a~)plicatioll of undue pressure
to the valve l)loc~ relatively small se;llillg force is focused on a l)arti-
3 ~ 7
cular portion on the ferrule -to seal the ~errule against the corresponcling
tapered recess without distort:ing the ~lock.
The provision of interfaces at opposite ends of the ferrules between
surfaces having different tapers fur-ther enha1lces -the seals obtainecl. Inter-
faces of this type between differently -taperecl surfaces are preferably provicled
on opposite sides of the ferrules to ob-tain optimum sealing chaIacteristics.
The conversion flask of the present invention enables reagents and
solvents to be introduced in the form o~ a spray im~ lging on the interior walls
of the flask to ~.ISh any chemicals ~llicll rnay have been condensed or splat-tered
thereon do~n the walls and illtO the body o~ licluid ~jitllin the flask. A major
source of cross-contaminatioll of the system between cycles is thus eliminated.
The conversiorl flask also enables gases to be passed upwardly through the body
of liquid in the form of small bubbles which uniformly agitate the liquid and
aid in drying semi-volatile components thereof "~ithout causing sample loss
due to excessively vigorous bubbling ancl splattering. This promotes rapicl,
gentle remov~l of liquid from the sensitive ~nino acicl derivatives. Solvent
used to carry the amino acid derivatives into the conversion flask can thus be
removed in a mucll sllorter time than in the l~ittmann-Liebold pater1t cited above
(l to 2 minutes rat11er than 5 to lO minutes) a1ld at a lo~er temperature (40
Z0 to 50C rather thln 50 to 80C). Tl1is significantly improves yields of the
most unstable amino ~cid derivatives, such as those of serine, threonine,
histidine, arginine, and tryptoph.ln. Reagents usecl in the conversion flask
can be removed by a combination of fine streams of inert gas bubbles and lo~
vacuwn in ~ to 5 minutes rather tll;ln thc ~0 to 40 mi1lutes recluirecl uncler tlle
l~ittmall1l~Liebolcl p;1tC11t. In ti1C process ol tlle l~ittm;11ln-~iebold patent, clrying
of tl1e reage11t must colnmence in11ncdi;1tely UpOIl its introduction to ti1C amino acicl
- 19 -
residue in the con~ersion ~lask in orcler to meet the requirement that the totalconversion flask cycle -time be no greater than the ~otal cycle time of the
primary reaction chamber. Since tlle ac:id component of the eonversion reagent,
trifluoroacetic or hyclroellloric acid~ is mueh more volatile than the water in
whieh it is dissolved, the acid component tends to be removed early in the
drying yrocess of Wittmann-Liebold leaving the amino aeid derivative in nothing
but a water solution for a signifieant portion of the eonversion s-tage. This
eauses ineomplete conversion of the derivltives of glyeine and proline and
deeomposition of other derivatives. In the present eonversion apparatus, the
conversion reagent ean be left in eontaet with the amino acid derivatives for
a time sufficient for eomplete conversion, 30 to 40 minutes, and then dried
rapidly, in 3 to 5 minutes, ~ithout splattering the sample throughout the
interior of the eonversion flask.
BRIEF DESCRIPTION OF TIIE DRAI~INGS
The above and other objects of tlle present invention may be more
~; fully understood from the following detailed description taken together with
the accompanying dra~ings ~herein similar referenee eharacters refer to similar
elements throughout and in whicll:
Figure 1 is a perspective view of an apparatus constructed in aecor-
danee witll the present inventioll;
Figure 2 is a top plan viel~ of the apyaratus of Figure 1;
Figure 3 is a seilematic diagram of the apparatus of Figure 1;
Figure 4 is all enlarge-l exploded perspective view of a re.-ction
cllamber assembly construeted in aecordance witll the present invention;
Figure 5 is an enlarged vertical cross-sectioll.ll vie~i ta~en alollg
the line 5-5 of Figure 1;
-- _O --
Figure 6A is a ~ur-ther enlarged cross-sectional view of the reaction
chamber illustrated in Figure 5;
Figure 6B is a cross sectional vi.ew of a second embodiment of the
reaction chamber illustrated in Figure 5;
Figure 6C is a cross-sectional view of the reaction eha.mber of
Figure 6B witll the porous sheet elements removed therefrom, for use with a
sample-containing film applied to tlle i.nterior surfaee thereo~;
F:igure 7 is a vertleal eross-seetional view of a typical reservo:ir
of the present :invention for a reagent or solvent to be used in liqui.d form;
Figure ~ is a vertical cross-sectional view of a typical reservoir
of the present inventioll for a reagent to be used in the form of a gas or vapor;
Figure 9 is a vertical cross-sectional view of a diaphragm valve
assembly constructed in accordance witll the present invention for controlling
~he flow of 1uids to and from the reaction ehamber and the eonversion flask,
taken in a direction corresponding to the line 9-9 in Figure 11;
Figure 9A is a fragmentary enlarged cross-seetional view of one of
the eonneetor elements of the valve assembly illustrated in Figure 9;
Figure 10 is a vertieal eross-sec-tional view ta~en along the~ line 10-
10 of Figure 9;
Figure 11 is a side elevational view of the manifold bloek of the
valve apparatus illustrated in Figure 9;
Figure 12 is a hori~ont;ll cross-sectional view taken along the line
12-12 of Figure 11;
Figure 12,~ is a vertieal eross-seetional view taken alollg the line
12,~-12,~ of Figure 11;
Figure 1~,~ is all enlarged fragmellt;lry cross-sectional view of the
valving portion of the valve assembly of Figure 9 showing the diapllrlgm E>ressed
against the manifold block to prevent fluid communication be-tween the passages
at -that location;
Figure 13B is an enlarged fragment.lry cross-sectional view of the
valving portion of the assembly of Figure 9 sho~ lg the diapllragm drawn away
from the manifold block to permi-t fluid flol~ betweell the passages;
Figure 14 is a top plan view oE a conversion flask constructed in
accordance with the present invelltion;
Figure 15 is a vertical sec-tional view taken along the line 15-15
of Figure 1~;
Figure 16 is a vertical sec-tional view taken alorlg the line ]G-16
of Figure 14;
Figures 17A and 17B illustrate schematically the t~o primary prior
art methods of immobilizing a protein or peptide sample during degradation;
Figure 17C illustrates schematically the immobilization o-f a protein
or peptide according to the present invention;
Figure 18A is a fragmentary enlarged vertical cross-sectional vie~
corresponding to Figure S of a further embodiment of the reaction chamber
assembly of the present invention;
Figure 18B is a vertical cross-sectional vie~ showing the chamber
element of the embodiment of Figure lSA turned on its side for the purpose
of applying a sample-containing matri.Y to the interior ~alls thereof;
Figure lSC is a further enlarged fragmentary cross-sectional vie~i
of the chalDber element of Figure lSB ~ith a salllple-collt;linillg film apylied
to the interior ~alls tl~ereof; and
T;lble l is a listing of the vario~ls steps yerformed by the ayy.lr.ltus
of the presellt inventioll in a t~l-ical .Iegrad;ltion an(l conversioll cycle.
3~
DESCRIPTTON OF TIIE PREFERRED EMBODIMENTS
Referring now -to the clrawings there ls illustrated, in Figures I and
2 thereof, an apparatus embodying the present invention, generally designated
The apparatus 10 includes a chamber apparatus 12, a conversion flask 14
and a fraction collector 16, each of which is operl-ted through an automatic
control unit 18 An array 20 of pressurized solvent and reagent reservoirs
are connected through a bank 22 of diapllragm-type -Flo~ valves to the reaction
chamber 12 ancl the conversion Elaslc 14 A second bank of valves 24 regulates
the flow of liquids from the reaction chamber to the conversion flask 14 and
other locations A tllird bank of diaphragm valves 26 serves to connect the
conversion flask alld fraction collector to eithel waste or vacuum, cmd to
regulate fluid flo~ from the flas~ to the fraction collector
A filtered inert gas source 28 supplies the apparatus 10 with highly
purified inert gas, preferably argon, for pressurizing the solvent and reagent
reservoirs, purging oxygen-bearing air from the system and accelerating the
process of drying out the reagents and solvents wi~hin the system at various
times A bank 30 of pressure-regulating valves and gauges serves to individual-
ly regulate the pressure of the gas to eacll solvent and each reagent reservoir,
and to each of the other components of the apyaratus 10
Tile operation of tlle ayparatus 10 is depicted in Figure 3 The
ch~nber apparatus 12 is located \-~itllin a heated environment 32 and, in the
preferred embodiment, defilles a reaction chamber 34 communicating with inlet
and outlet passages 36 and 38, respectively The inlet passage 36 is connect-
ible througll a fluicl conduit 40 to a ylurality of reservoirs of the array 'O,
n.lmely, reagent reservoirs ~12, 4~ and 4S, and solvellt rc`scrvoirs SO, 52
alld 54 T11e reser~oirs 42 tllrough 5 4 arc yrcssuri-cd by the inert gas yres-
sure source 'S ~hrough ~n array of indivldll;ll gas pressure regul;ltors j~, and
- _3 -
solenoicl flow valves 58. Eacll o:E-thc reservoi.rs 42 -through 54 is also connect-
ib:Le at a poi.nt above tlle :Eluid level therein to a waste trap 60 through an
individual flow valve 62 and an indiv:idual :Elow regulator 64. Pressurized
i.nert gas introduced to the reservoirs from the source 28 can thus be vented
to the waste trap 60 at a rate controllcd by the :flow regulators 64. The
flui.d outputs of the reservoirs 42 through 54 are individually controlled
through diaphragm valves 66 communicating with a continuous manifold G8 which
is connected at one end to the ~luid condLIit 40. Fluicl from the ~ressurized
reservoirs 42 tllrough 54 can thus be passed through the conduit 40 and the
chamber inlet passage 36 to thc reaction chamber 34 by the selective actuation
of the valves 66. To aid in the delivery of tllc reagcnts and solvents and to
flush the maniEold 68 and conduit 40 after delivery, pressurized inert gas
from the source 28 may be introduced to tlle manifold 68 at the end opposite
to the conduit 40 through a pressure regulator 70 and a diaphragm valve 72.
Actuation of the valve 72 thus introduces pressurized gas at the remote en~ of
thc manifold 68 driving any reagents or solvents thercin through thc conduit
40 and the passage 36 to the reaction chamber 34.
The fluid outlets of the reservoirs 44, 46 and 48 are provided with
gas flow meters 74 in series with flow regulators 76 because t~e reagents stored
therein are used in gas or vapor form, whilc the remaining solvents and re-
agents are used in li~uid forms. The mcters 74 and flow rcgulators 76 are
necessary to accurately control tllc rate of gas dischargc througll tllc corres-
ponding valvcs 66.
Fluid flow from thc rcaction chamber ~t is controllcd by diaphragm
valvcs 78, S0 and 87 wllicll communicatc witll thc outlet passage ~8 throu~ll a
continuous manifold ~1. 'I'hc valvc ,8 is opcnc~l to pass tlle dcsircd product of
rC;lCtiOIl, typiC;llly thc .`~I-tcrmin;ll ;IlllillO .ICi~l Ullit of a protcin or pcpti~c
t
samplc, tllrougll the conduit 86 to the conversion flask 14. The valve 82 may
be opened to connect the outlet passage 38 to the waste trap 60 :for disposal
of unwanted reagen-ts, solvents and react:ion products, and -the valve 80 connects
the outlet passage 38 -to a vacuum trap 88 and a vacuum pump 90 for evacuation
o:E the reaction chamber 34, the outlet passage 38 and the manifold 84.
Similarly, the conversion flask 14 and the fraction col:lector 16
are connect:ible to tlle was-te trap 60 through valves 92 nnd 94, respectively,
and to vacuwn through the valves 96 and 98.
Reagent reservoirs 100 and 101 and a solvent reservoir 102 are pro-
vided to supply reagents and solvent -to the conversion flask 14 through a
conduit 104. TllC reservoirs 100, 101 and 102 are pressurized by the inert gas
source 28 through pressure regulators 106 and solenoid valves 108, and arc
connected to the waste trap 60 through incliviclual vent valves 110 and flow
regulators 111. The -fluid outlets of the reservoirs 100, 101 and 102 are
connected through diaphragm valves 112 to a con-tinuous manifold 114 which
communicates at one cnd Witll the conduit 104. The flow of fluid from the pres~
surized reservoirs may thus be produccd by selectively opcning the valves 112
to expel eithcr reagent or solvent into the manifold 114. Thc source of inert
gas is connectible througll a pressure regulator 116 and a diapllragm valve 118
to the end of the manifold 114 opposite the conduit 104 to propel the reagent
or solvent through the manifold and the conduit to the convcrsion flask 14.
Thc incrt gas pressurc source 28 is also connccted to the conversiGn
flasl; through ;3 prcssurc rcguLator 120) a di;lphragm valvc 122 and a conduit 124,
and to tllc fraction collector 16 througll a prcssurc rcgulator 126 and a valvc
128. I~hcn a particular fractioll h;ls becll convcrtcd in thc intcndcd manncr
within tllc fl;ls~ 14, it can bc c~pclled from thc flask by gas prcssurc throu~h
t~lC conduit 12-~ alld a valvc 1~1 to thc fractioll collcctor 16 for storagc ~ithill
a vail therein. ~fter tlle Eraction is expelled the flask 14 can be filled to
a relatively high level with solvent to dissolve any residual chemicals therein.
The solvent can then be expelled by gas pressure through the conduit 1.24 and
a valve 125 to the waste trap 60 flushing the flask in preparation for delivery
of the next amino acicl derivative.
The fraction collector 16 comprises basically a carrousel of vials
actuated by the con-trol unit 18 once during each cycle of the apparatus 10 -to
place an empty vial in position to receive -the next succeeding fraction o~
amino acid units from the flask 14.
The control unit 18 is preferably a fully automatecl unit controlling
the diaphragm valves 66 72 78 80 82 ~2 94 96 98 112 118 122 125
128 and 131 as well as the solenoid valves 58 62 108 and 110. The control
unit 18 also controls the mechanism for maintaining the heated environment 32
at the desired temperature (not shown) the fraction collector 16 the vacu~m
pump 90 and a variety of sensors througllo~lt the system. The various gas
pressure regulators and flow regulators described above are manually adjusted
upon set-up to establish the desired pressures and flows wi.thin tlle corres-
ponding fluid conduits.
The chamber apparatus 12 is shown in detail in Figures 4 and 5 to
eomprise a two-piece base 130 supporting a sleeve 132 wllicll contains first
and seeond ell.~nber elements 131 and 136 respeetively. The sleeve 132 is
provided witll an enlarged cylindrical portion 138 centered about the axis of
the sleeve alld closely received ~ithill a cylindrical recess 140 of tlle base
130. The cylindrical portion 138 is held in position by a retaining collar
142 ~lhicll is threadillgly engaged ~ith the b;~se liO.
Tlle ch;lllller elements lil and li6 are cylindlical gl.lss elements h;lving
oplosed m;ltillg faces 1-1~ alld llG resl~ectively .nd ~losely received in a~i;ll
~ ~,
q~
alignmen-t within the sleeve 132. The inlet and outlet passages 36 and 3~
described above extend axially through the chamber elements 134 and 136 res-
pectively and are preferably capillary passages having a diameter on the
order of 1 millimeter. The axially outer ends 148 and 150 of chamber elemenl:s
134 and 136 respectively are generally flat and are abutted with a pair of
thin resilient washers made of a substantially inert material to provide the
chamber elements wi-th a cushion in the axial cIirection relative to the sleeve
132. A metallic washer 154 located on top of tlle upper resilient washer LS2
is provided with opposed loc~ing ears 156 for engaging slots 15~ in the upper
end of the sleeve 132. The metallic washer 154 is held in position by a cap
160 threaded to tlIe upper end of the sleeve 132 to snugly hold the chamber
elements in place relative to the sleeve. The engagement of the ears 15G
with the slots 158 prevents the washer 154 from rotating when the cap 160 is
installed, thus preventing the cap from damaging the assembly by rotating
the chamber elements. The fluid conduit 40 is provided with a flared lower
end 162 which abuts the outer end 148 of the chamber element 134 such that
the bore of the conduit 40 communicates witIl the inlet passage 36. The con-
duit 40 carries a backup washer 164 and a fitting member 166 which is threaded
a~ially into the cap 160 to force the flared end 162 against the chamber
element 134 in a sealing relationsIlip. The conduit 40 may be made of any
resilient substantially inert material, such as commercial fluorocarbon poly-
mers. The alignment of the bore of the conduit 40 Witll the inlet passage 36
is assured by precision construction of the varlous interfitting components
about a common a~is.
TiIe outlet pass~ge 38 is pl;Iced in communic;Ition witIl the contiIluous
maIlifold 84 described aI)ove by a mass l72 of substaIlti;llly inert material
enc;Ised witIIin ~ steel sleeve 17~ hC sleeve l74 h;IS I smootII e.~terior re-
- _7 -
ceived witlliIl nligned axial openings 176 and 178 of the enlargecl cylindrical
portion 138 and the base 130 respectively. TIle mass 172 extends axially
in either direction beyoncL the steel sleeve 174 to engage -the outer end 1;0
of the second chamber element 136 and a tapered recess 180 o~ a valve block
182 which defines the manifold 84. An axial passage 184 within the mass 172
is precisely aligned with the outlet passage 38 and one end of the manifold
84 to provide a single continuous capillary passageway from the chamber elemellt
136 to the valve block 182. As in the case oE the conduit 40 discussed above
the accurate construction of the rclated componen-ts about a common axis insures
precise alignment and complete sealing between -the various passages. The
chamber apparatus 12 can thus be easily clisassembled and reassembled in a
very sllort time without compromising alignment of tlle various passages or the
integrity of the various seals.
The valve block 182 is of a novel construction l~hich will be des-
cribed in detail in relation to Figures 9 tllrough 13. It will suffice to note
at this point that portions of thc valves 78 80 and 82 are included ~ithin
the valve bloc~ 182 to control tlle flo~ of fluid from the chamber apparatus
12.
As seen most clearly in Figure 6.~ tlle chamber 34 is formed by
aligned cavities 186 and 188 in the opposed mating surfaces 14~ and 14G res-
pectively of the t~o chamber elcmcIlts. Tlle two cavities are arrangcd coaxially
witII the inlet and outlct passages 36 and 38 and arc preferably circular in
cross-section providing an axially symmetric patll for fuild passiIlg rrom the
inlet passagc to the outlet passage. A porous sllcct clcment 190 c.Ytcnds
transvcrscly across thc rcaction cllambcr ~4 an~ may be rcccivc~l at lcast par-
tially witllin a dcl7rcssion I92 of the cavity 186. Thc porous shect clcmcnt
190 tllus scp;lratcs thc inlct p.lSSIgC 3G from thc outlct passa~c 3S such tlult
-- _8
Eluids flowing Erom one to the other must pass throllgh the sheet elemen-t.
The porous sheet element 190 preferably comprises a sheet or mat made of a
compressed fibrous material, such as glass. Commercially available glass
fiber filters are suitable for this purpose and have a high resistance to de-
composition or o-ther damage during use. It has been found tllat a porous sheet
of this type provides a rather large sur-Eace Eor supporting a thin fi.lm in
which a protein or pepticle sample can be embeclded. l-E the film is made of a
fluid-permeable material, i.e. one which allows diffusion of liqukls and gases
into it, then the material can form n solicl matrix which is able to securely
hold the sample but permits chemical interclCtion oE reagents and solvents
with the sample. Polymer:ic quaternary ammonium salts, such as l~S-dimethyl-
1,5-diazaundecamethylene polymethobromide or poly~N,N-dimethyl-3,5-dimethylene
piperidinium chloride)l are ideal for this purpose. They permit diffusion of
fluids, are insoluble in the solvents used and are chemically stable to both
the reagents and solvents. In addition, they ma~e a cohesive film and carry
a positive charge which enables tllem to bond ionically to the glass support
surface.
The fundamental differences between the forms of sample retention
practiced in the prior sequenators and tllat of the present invention will be
understood most clearly in relation to ~igures 17,\, 17B and 17C. Figures 17~t
and 17B illustrate schematically the two most conunon prior methods of immobiliz-
ing a protein or peptide sample 300 relative to a sample support surfice 302
or 30~.
~igure 17,t illustr.~tes the C.15C in wllic}l the sample is chelllically
lin~ed to a glass s~lpport surface 30~ covalent bonds 306; ~or e.~ample, the
surface 302 IllaY I)C SPCCi;l11Y trC;ItCd SUCh that some of the silica sites of the
glass l1;1VC fUI1CtiOI1.11 ;IminO grrOUI)S ~OX e.~tending tllerefrom for reaction witll
, (~
L~:~7
carboxyl groups 310 on the sample chain. Urlder proper conditions some of the
groups 308 and 310 will react leav:ing thc sample covalently bonded to the
glass and releasing a number of ~ater molecules. Bonds of this type are very
strong and one or -t~o of them per molecule are sufficient to hold the sample
in place. ~lowever~ covalent bonding is difficul-t to achieve with protein
and peptide samples. Also, covalent bonds hold the chain througll a very few
isolated units in the chain. IYhen the degradation process reaches those Ullits
and cleaves them frorn the chain the remainder of the chain is left unbound
and can be washed from the chamber.
Figure 17B illustrates the case in ~hich the sample 300 is adsorbed
directly onto a suppor~ surface 304. The saMple is then held in place by a
very large number of relatively ~eak noncovalent interactions 312 bet~een.the
sample and the surf~ce. On the molecular level thc surface intcracts with
many different sitcs on the sample. This ~orks l~ell in the case of large
proteins and peptides but as thc sample gets sequenced clo~n to a much smallcr
size it becomes susceptible to being knocked or dra~in from the surfacc. This
results in drastic sample loss.
Figure 17C illustrates schematically the immobilization of the sample
300 relative to the support surface 314 by embedding it in a solid matrix 316
formed as a thin film thcreon. .~s describcd above, the rnatrix 316 may be a
polymeric quaternary ammonium salt ~hich has a positive charge. The matrix
will thus be firmly retained on an acidic glass surface by a very large numbc
of ionic intcractions and ~ill securcly llold tllc samplc in place bccause the
sample is embeddcd in it. .~'o reliance is placcd in dircct bonding intcr-
actiorls betl~ecll thc salllple aTld the surface alld the cffcctivëncss of tllc
immobiliz.ltioll is not affcctcd l)y diminislling samplc sizc.
Tllc prcscllt invcntioll rclics 011 diffusioll of rcasgcllts solvcnts
~ ~ )
and amino acid derivatives -through the solicl matrix 316 to effect chemi.cal
interaction with the embeclded sample. The matrix :is formed as a thi.n :Eilm
~,lhicll absorbs the reagents and solvents passed over it. Once dissolved in the
film, the reagents and solvents are able to readily di:Efuse across its thick-
ness to carry on the degradation process.
Returning now to Figure 6A, the reaction chamber 34 is sealed at the
periphery of the cavitles 18G and 188 by a-t least one sheet 194 o~E a yielcling
sealing material sand~iiched btween the mating surfaces l~kl and 1~16. A pair of
yielding shee-ts 194 are preferably used, one on eitller side of the porous sheet
element 190. The sheets 194 are very thin and are permeable to the plurality
of reagent and solvent flui.ds to be passed through the chamber 34. An annular
sealing ridge or bead 196 at the periphery of the cavity 188 bears against the
sealing sheets 194 to provide a more effective seal against the surface 144
adjacent the periphery of the cavity 186. The sealing sheets 194 may be made
of any substantially chemically inert material, such as a commercial fluoro-
carbon polyrner, to minimi~e the possibility of seal deterioration. They serve
not only to provide a seal for the chamber 3~ but also to support the porous
sheet element 190 and to diffuse gases and liquids passed through the chamber
such that flow of the gases and liquids will be more evenly distributed across
the element 190. Long system life and optimum chemical interaction ~l~ith a
sample are promoted in tllis ~.~ay.
An alternative embo(limer.t 3~' of the reaction chamber of the presen~
inventioll is illustrate(l in Figure 6L~"~Ilerein the aligned cavities 186~1nd
188' in the opposecl mating surfaces of the two ch;lml)er elemellts are some~lhat
narro~ier alld longer th;ln tlle cavities 186 all(l 188. Other~ise, the structures
3~ and 8-l' are identical, alld the vario-ls elemellts of the stl~cture 3~' in
the dra~illgs are numl)ere(l simil;lrl) to those of tlle structures 31 l~ith the
addition of "primes" (') -to distinguish them. 'I'he reaction chamber 34' per-
mits a somewhat more direct Elow of fluids from -the inlet 36' to the outlet
38' but restricts the diameter of the porous sheet element 190' therein.
Pigure 6C illustrates the chamber 34' of Figùre 6B with the porous
sheet element 190' removed therefrom. In addition the sealing sheets 194'
are replaced with a single annular sheet 316 of yielding material having a
central opening equal to the diame-ter of the chamber. In this embocliment, a
solid fluid permeable matrix 318 having a protein or peptide sample embedded
therein is formed as a thin film on the walls oE the chamber 34 9 for exposure
to reagents and solvents passed -through the chamber. The flow of fluids
through the chamber 34' is thus enhanced, wllile a substantial film surface area
is retained.
A further embodiment of the chamber apparatus of the present inven-
tion is shown in Figures 18A 18B and 18C wherein the two chamber elements
134 and 136 are replaced by a single capillary-type chamber element 320 within
the sleeve 132. The remaining elements of the chamber apparatus 12 are the
same as those described in relation to Figures 4 and 5 and are numbered
similarly. All s-tructures and connections external to the chamber apparatus
are also identical to those described above.
Tlle chamber element 320 comprises a cylindrical glass structure having
interior walls 322 defining an axial capillary-type cllamber 324. The ehamber
324 inereases uniXormly in diameter from its t~o ends 326 to~ard its middle
328 and the protein or peptide sample is earried witllin a solid matrix 330
formed as a tl~ film on tlle walls 322.
It will be understood ~h.lt the reactioll ch;llllber of the yresent
inventioll can ta~e virtu;llly ally form h;lving a samyle su~ ort surfaee ~ast
wilicll a plur;llity oE reagellt all(l solvent Eluids call ~e l~assed. For ex;llnl)le a
~, ~
single elongatecl capi.lla-ry tube (not shown) would suffice for the chamber
apparatus 12, with a fluid-permeable solid matri~ formed on the interior surface
or bore thereof. Fluids passed through the tube would interact with a protein
or peptide sample embedded in the film -to per-Eorm the degradation process.
A typical reservoir 198 of the present invention for storage and
delivery of a liquid reagent to the reaction chamber 34, 3~' or 324 is shown
in Figure 7. The reservoir 198 corresponds to the reservoirs 429 50, 52, 100,
101 and 102 of Figure 3. A pressurized inert gas is supplied to the interior
200 o:E the reservoir 198 by a conduit 202 communicating therewith at a point
below a level 20~ of -the liquid reagent or solvent therein. The pressuri~ed
gas thus introduced draws any dissolved oxygen from the liquid and can be
released at a controlled rate through a vent conduit 206 to produce a dynamic
equilibrium condition within the interior 200. A liquid outlet 208 is provided
for the controlled expulsion of reagent or solvents from the reservoir 198
by the gas pressure therein. The inert gas supply li.ne 202 of each reservoir
198 receives pressuri~ed inert gas from the source 28 through a pressure
regulator 56 or 106 and a solenoid valve 58 or 108. The release of gas through
the vent conduit 206 is li~ewise controlled by one of the solenoid valves 62
or 110 and one of the flow regulators 64 or 111. The flow of liquid reagent
or solvent through the conduit 208 is controlled by one of the valves 66 or
112. Each time one of the liquid reagents or solvents is to be delivered to
the reaction chamber or the conversion flas~ 14, the corresponding argon supply
valves and vent valves are opened to establisll a dynamic equilibrium condi-
tion witllin the particular reservoir. Reagent or solvent in liquid form ca
then be introduced by OpCnillg the valve in the con~uit 208 and the valve 82
to W;lste trap 60. Liquid is e~pelled from the reservoir at a constant rate,
enablillg the q-l;llltity of liq~lid delivered to be accul;ltely controlled by con-
trolIing the IengtII oE time the valvo in tho delivery line 208 is held open.
A typical reservoir 210 of the present invention for delivery of a
reagent in gas or vapor ~form is illustrated in Figure 8. An inert gas inlet
212 terminating in a glass frit sparging element 213 is provided ~Eor intro-
ducing iner-t gas to the interior 214 of the reservoir 210 at a poin-t adjacen
the bottom thereof and substantially below a level 216 of liquid reagent
therein. A vent conduit 218 and an output or delivery line 220 communi.cate
with the interior 214 at points above the liquicl level 216. The rese7voir 2]0
is typical of the reservoirs 44, 46 and 48 of Figure 3, witIl one of -the
pressure regulators 5G and one of the valves 58 controlling the f]ow of gas
througIl the conduit 212 from the gas pressure source 28. Likewise, the
escape of pressurized gas to the vent conduit 218 is controlled by one of the
flow regulators 64 and flow valves 62, and tlle delivery of reagent along the
line 220 is controlled by one of the diaphragm valves 66 in line witlI a flow
meter 74 and flo~ regulator 76.
Thus, although the reagents R2, R3 and R3A are deliverecl to the
reaetion chamber in gas or vapor form, they are stored as liquids and vaporized
wllen needed. Vaporization is accomplished by the bubbling of inert gas upward-
ly through tlIe liquid reagent. In this way, the inert gas in the interior
214 of the reservoir 210 becomes saturated with reagent vapor. Each time
reagent is needed, the valves connected to the conduits 212 and 218 are opened
to bubble inert gas througl~ the reagent ancl establish a cIynamic ecluilibrium
condition. The valve 66 witlIin the delivery conduit 220 is then opened for a
predetermined length of time to deliver the desired quantity of reagent vapoI
to tlIe reactiolI cell. TIIe flow regulator 7G in line ~ith the particular valve
66 causes the vaI70r to be delivered hy the reservoir at a constaIlt rate
indicated by thc ~lo~ meter 7~.
Figures 9 througil 13 illustrate the structure and operation of a
valve assembly 222 which embodies the valves 66 and 72 and -the continuous mani-
fold 68 of Figure 3. The valve assembly 222 includes a valve block 224 which
is seen mos-t clearly in Figures 11 and 12. The valve block 224 is an elon-
gated block of rectangular cross-section ilaving a continuous primary passage
226 in a sawtooth pattern Eormed by cross-drilling the valve block frorn a
surface 228 thereof. The primary passage 226 is thus a single continuous pas-
sage communicating at alternatillg intersections thercof with a plurality o-E
valving sites 230 on the surEace 228 throllgh corresponding oponings 232. A
tapered connector port 234 communicatcs witll onc cnd of the primary passage
226. A plurality of sccondary passages 236 extcnd from tapered conllector ports
238 at the opposite side of the valve block 224 to corresponding openings 240
in proximity to the openings 232 at the rcspective valving sites 230. Tlle
valve block 224 is received within a longitudinal slot 242 of a base 244 h.lving
a plurality of threaded openings 246 in alignment with thc conncctor ports 234
and 238 for reception of connector fittings 248. The fittings 248 are adapted
to compress resilient doubly tapered ferrules 250 against the connector ports
234 and 238, respectively, ~o sealingly join tubes 252 extending through tlle
ferrules with the various passages of the valve block 224. In this l~ay the
connector port 234 of the primary passage communicates witll the inlet passage
36 of the chamber apparatus 12 througll the fluid colld~lit 40 of iigure 3 and
thc first seven of thc eight secondary passages communicate with thc fluid
outlets 208 and 220 of thc rescrvoirs 42 through 54 rcspcctivcly. The secon-
dary passage furtllcst from thc conllector port 234 communicates witll thc incrt
gas pressure source 28 througll thc prcssurc rcgulator 70 ShO~II in i~igurc 3.
Thc structurc of tllc doubly tal-crcd fcrrulc CollllCCtiOIls to thc ports
of thc valvc l)loci~ 1 is sllo~n in grc;ltcr ictail in i~igllrc ')~ dcl-icting
3~ 3t~
the port 234 by way of example.
T?le ferrule 250 of Figure 9A is received at its inner side 243
within the connector port 23~ and a-t lts outer side 2~5 ~ithin a tapered recess
247 of the fitting 2~8 -the por-t 234 and -the recess 247 being tapered at
angles greater than the angles of taper of -the respec-tive sides oE the ferrule.
The ferrule is pre-ferably tapered at the same angle on both sides with the
port 23~ and recess 247 being -tapered at an angle three degrees () greater
than the ferrule. This fit betl~een diEferently tapered surfaces focuses the
forces of compressioll upon the tips 249 of the ferrules Z50 providing an
effective seal with a minimum of pressure on the side of the valve block 22~.
Excessive pressures on the valve block 224 ~hich can distort the valving
sites 230 are thus avoided.
A series of diaphragm retaining blocks 25~ are bolted against the
surface 228 of the valve block 224 with diapllragms 256 sandl~iched therebetween.
The upper end of each diaphragm retaining block is threaded to receive an air
connector 258 communicating with a recess 260 on the underside thereof and
extending generally over one of the valving sites 230. An 0-ring 262 may be
received within an annular groove surrounding the recess 260 to provide an
effective air seal.
Tlle diaphragms 256 are constructed of a substantially chemically
inert air-tight material enabling tllem to be alternately dra~n a~ay from and
pressed against the valve sites 230 by the ilpplication of vacuum and air yres
sure, respectively through the fittings ?58. The two alternate conditions of
the ~iapllragm 256 are sho~ll in ~igures 13.~ ;Illd 13B. In the condition of ~igure
13/~ air or gas pressure applied to the fitting 25S forces the ~iapllragm 256
against thc openillg ?3? of the pril~ ry pass.lge and thc opening 2~0 of the
second;lry pnss;lge at the p;lIticul;ll valvillg site 230. Tllc opcllings 232 nn~
- ~6
3~ 3"~
240 are thus closed by -the diaphragm 256 permitting no communication there-
between. This correcnonds to tlle closed position o-E the valve loeated at the
particular valving site. In the condition of ~igure 13B vacuum applied to
the connector fitting 258 draws the diaphragm Z56 away from the openings 232
and 2~0 permitting communication between -the primary and secondary passages
over the surface of the valve block a-t that point. This corresponds to the
open condition of the particular valve permitting fluicl from one of the
reservoirs or -from the inert gas pressure source 28 -to flow through the primary
passage 226 to the inlet passage 36.
The novel construction of the valve assembly 222 described above
enables reagen-ts and solvents to be delivered to the reaction chamber in
accurate amounts ~ith virtually no contamination bet~een the various fluids.
The continuity of the primary passage 226 and the connection at one Clld thereof
to the inlet passage of the chamber apparatus 12 are largely responsible for
this aclvantageous operation. Delivery of any one of the seven reagents and
solvents can be accomplished by applyillg a vacuum to one of the diaphragms 256
and positive gas pressure to the others, enabling the desired reagent or solvent
to pass into the primary passage 226 and through the fluid conduit 4~ to the
reaction chamber. ~hen the desired amount of fluid has passed beneath the
particular diaphragm gas pressure is again applied thereto through the fitting
258 so that each of the seven reagent and solvent valves of the assembly 222
is closed. Delivery of tlle fluid can then be completed by applying a vacuum
to the diapllragm associate~ ~itll the inert gas port at the remote end of the
valve bloc~ 22~ to flush tile entire primary passage 226 ~ith inert gas and
force the reagent or solvent fluid remainillg in the lines into the reaction
ch;llllber 51. Because tllere are no discolltilluities or de.ld-end brallclles in the
primary paSS;lgC 226 there i; no pl;lce for ;Illy of the reagellts or solv~nts to
become tral)l)ed bet~eell delivery procedures. I`he rea~ellts alld solvents d livered
~7
in suceeeclillg sequencing steps are thus as pure as possible allowing the
chemistry within the reaetion cell to proeeed as intendecl and without any un-
necessary loss of yield due to contaminatel reagents or solvents.
The valve assembly 222 is also illustrative of the valve design
incorporated in many other portions of -the apparatus to minimize eontamination
whenever a single port must be selectively connected to a plurality of other
ports. Thus the valves 112 and 118 for delivering reagent and solvent to
the conversion flask 14 Eorm a valve assembly in eombination with the eontinu-
Ol1S manifold 114 and the valve 78 80 and 82 for directing fluids Erom the
outlet passage 38 oE the chamber apparatus 12 form a similar valve apparatus in
combination with the continuous manifold 81. Likewise the valves for the
eonnection of waste and vacuum to -tlle eonversion Elask and the fraction
collector and the valves eontrolling flow from the conversion flow to the
fraetion eolleetor are eonstructed similarly to the valve assembly 222. The
prineipal differenee in each oE these valve assemblies is the number of valving
sites assoeiated therewith.
It will be understood that while the manifolds 68 84 and 114 are
illustrated sehematieally in Figure 3 as having a series of small diseontinu-
uities or branelles adjaeent eaeh diapllargm valve assoeiated therewith eaeh of
the manifolds is aetually a single continuous passage eonstrueted in the manner
of the primary passage 226 of Figures 11 and 12.
The vacuum and gas pressure for actuating tle diaphragm valves
betl~een tlle open alld elosed eonditions are omitted from tile sehematie diagram
of Figure 3 for purposes of simplieity alld are preferably separated from the
source 28 alld vaellum pullll 90 descrii)elllereill.
The eonversion fla;k 1-1 is sllowll in detail in Figure~ 14 through 1~.
Tlle flask 11 is oE the doui)le-w;llled gl;lss tvle h;lving a Sp;lCC 2fi1 betlieell the
~ 43~
walls Eor circulation of a heating fluid sucll as water. The hea-tirlg fluid
is passed to and from space 264 through a pair of nipples 266 adaptel to re-
ceive standard flexible tubing ends. A large bore tube 268 connectible to the
vacuum trap 88 and the vacuwn pump 90 through the valve 96 and to the waste
trap 60 through thc valve 92 communicates with the interior chamber 270 of
the flask adjacent its upper end. Capillary tubes 272 274 and 276 extend
through the upper end of the flaslc to points within the interior chamber 270.
The bores of the tubes 272 and 276 are closed at the inner ends thereof and
each of the tubes is provided Wit}l a pl~lr;llity o-E relatively restricted radial-
ly spaced orifices 278 or 280 adjacent its inner end.
Due to the relatively small amount of protein or peptide sample for
which the apparatus 10 is designed and the relatively low volurnes of reagent
and solvent used therein the conversion flask 14 has a much smaller interior
volume than any of the prior flasks known to applicants. The volume of the
flask 14 is slightly over one (1) milliliter while tlle prior automatic
conversion flasks known to applicants have all had volumes of one llundred (100)
milliliters.
The fractions cleaved from the sample in the reaction chamber 34 are
passed sequentially to the flask 14 through the valve 7~ and the capillary
274. Reagents and solvents enter the interior chamber of the flask through the
capillary 276 from wllich they are forced through tlle restricted orifices 280
as a spray impinging on the walls of the cllamber 270 to wash any residue thereon
to the bottom of the flask. During tlle conversion reaction an inert gas
may be introduced through thc capillary ~ube 272 to agitate the liquid and aid
in evaporatillg tlle solvent tllerefrom. Ihc g.lS e~its the tube 72 tilrough the
restricted orifices 278 ;IS vcry small buhbles ~roviding optimal dispersioll
of the gls tllrougll the liqllil regardless of the si~e of the flas~ alld the amoullt
"
of gas used. ~hen the conversion reaction is complete, the ~Eraction is forcedupwardly by positive gas pressure wi-thin the -flask through the long capillary
272 to the respective vlal in the fraction collector 16. This can be accom-
plished in two allquots to effec-t a more complete transference of the fraction
to the fraction collector 16. The first alicluot, of approximately 200 micro-
liters ~Q), is initially expelled to -the fraction collector. Then an addi-
tional 50 microliters (~Q) of solvent is introduced to clissolve any residue
on the lower walls or bottom of the flask. The second aliquot is then expelled.
In this way, a high yield of each fraction can be achieved. '!
After a particular frac-tion has been trans-ferred, an additional 750-
900 microliters (lJ~) of solvent may be introduced to dissolve whatever resiclue
from tlle previous cycle remains on the upper walls of the flask 14. Tllis
additional solvent is then expelled to waste, leaving the flask walls clean.
The outer ends of the tubes 268, 272, 274 and 276 are sealed to res-
pective flexible conduits 282 leading to the various other elements of the
apparatus 10 by interfitting screw thread connectors 284. The end of each
conduit 282 is provided with a radial flange portion 286 having a resilient O-
ring 288 wllich abuts and seals against the flat ground glass face of a radial
flange 290 on one of tlle glass tubes. An internally threaded collar 292 is
slidably positioned over the conduit 282 to receive the glass flange 290 and
engage a two-piece e.Yternally threacled fitting 294 wllic}l is placed about the
glass tube. ~dvancement of tlle collar 292 over the fitting 294 forces the flange
portion 286 against the glass flange 290 to procluce an extremely offective seal.
The various portions of the connectors 284 m;ly be ma(le of substantially chemi-
cally inert materials, such as comlllercial fluorocarboll polymers, to virtually
elimillate the possibility of deterior;ltioll allcl subsecluent lea~;lge.
All.llterll.ltive collstructioll of tlle tlas~ ll would elilnillate tlle space
-- ~O -
264 and the nipples 266~ yieldillg a single-walled vessel which could be main-
tained at an oleva-ted temperature by placement in the heated environment 32.
This structure woul~ have the advan-tage of maintaining the entire length of the
glass tubes and the connectors 284 at the elevatecl temperature, minimizing
condensation of semi-volatile fluids -therein, but would not enable the flask
and the chamber 12 to be main-tained at different temperatures.
The reagents and solvents useci in the apparatus 10 Eor the sequell-
tial degradation of protein or peptide cllclins are preferably as follows:
Rl phenylisothiocyanate (PITC) (17% solution in heptane)
R2 trimethylamine or triethylamine (25~o solution in water)
R3 trifluoroacetic or heptafluorobutyric acid
R3A water vapor
R4 trifluoroacetic acid (25% solution in water)
R4A hydrogen chloride (lN solution in methanol)
Sl benzene
S2 ethyl acetate with .1% acetic acid
S_ butyl chloride
S4 acetonitrile or methanol
In operation, the various valves and other mechanisms of the appara-
tus 10 are preferably controlled by the automatic control unit 18 -to perform
- an indefinite number of degradation cycles on a protein or peptide sample
Witllout humall intervention. 1he control unit 18 may ta~e the general form of
the programming unit disclosed in Pcnhasi Unitcd St;ltes ~atent ~o. 3,725,010,
witll alterations to provide for the particular sequence of steps required by
the apparatus 10, or may bc a Illorc sophisticated elcctronic control in the
niltllre of a speci.ll or gener;ll purpose digital coml-uter. ;~ltcrnatively, the
various steps in C;lCil degr;ld.ltion cycle call be pcrtorllle(l m;lllu;ll ly by an
q3't)
ope~ator according to a predetcrmlned scheclllle to achieve the same results.
Prior -to commencing the degraclation processl a sample of the protein
or peptide hein~ investigated must be mounted -to an appropriate sample
support surface. The matrix material is Eirst applied to the support surface
with the sample later embedded therein, as described in Examples 1 and 2 belo~.
When the element 190 or 190' is used as a sample support surface it is placed
between the chamber elements 134 and 136 along with at least one of the
sealing sheets 194 such that thc element ]90 is received within the reaction
chamber 34 at the location o-f the recess ]92. In the preferred embodiment of
the present invention, a pair of sealing sheets 194 aro used sandwiching the
element 190 therebetween. The chamber elemellts 134 and 136 arc thcn insertcd
into the sleeve 132 and asscmbled with the various other componcnts to form
the chamber apparatus 12.
l~hen the sample-containillg film is to bc carried by -the interior
surface of the chamber 34' or tlle chamber 324, it is applied thereto as des-
cribed in Example 2 below.
The chamber elements are initially assembled within the slceve 132
and held in place by the cap 160. In the case of the chamber 34' thc annular
sheet of yielding material 316 is positioned between the chambeI elements 13~'
and 136' upon assembly. The solid matrix mlterial and thc sample are thcn
applied to thc walls of the chamber 34' or 324 as describcd in Example 2 and
the slceve 132 is asscmblcd with thc other components to form a complcte
apparatus.
Ihc reactioll chamber 34 and thc associatcd fluid conduits may be
initially evacuated by opcning thc valvc 82 to thc vacuum trap 88 and vacuum
pump 90 in l~rcp.lration for thc .sc~lucntial introd-lction of rca~cllts Rl throu~h
R ~, solvents Sl throu~ll S alld incrt g.lS from thc sourcc ''3. ~acll timc onc of
I
"3~7
the raagents or solvents is to be introduced the corresponding inert gas
supply valve 58 and vent valve 62 are opened to pressurize the particular
reservoir and establish a dynamic equilibrium condition therein. Inert gas is
thus introduced and released from the reservoir simultaneously to maintain a
constant pressure within the reservoir. Ill the case ot the reservoirs 44 46
and 48 the flow of saturated gas therefrom during delivery is controlled by
one of the flow regulators 76.
Once the reservoir containing the particular reagent or solvent to
be delivered is pressuri~ed and placed in equilibrium as described above a
vacuum is applied by the auxiliary vacuum source (not sho~n) to the diapllragm
of the corresponding flow valve 66 to open the valve and produce a flow of
the reagent or solvent through the continuous manifold 68 and tlle conduit 40
to the chambcr inlet passage. The valve 66 is held open a predetermined lcngth
of time to allow precisely the desired amount of reagent or solvent to pass
and is then closed by the application of gas pressure to the diaphragm thcrcof.
Vacuum from the auxiliary vacuurn source is then applied to the diaphragm of
the valve 72 at the remote end of the continuous manifold 68 to flush the
manifold of any remaining reagcnt or solvent and complete delivery thereof to
the chamber. Thc valve 72 is then closed by the application of positive pres-
sure to the diaphragm tllereof lcaving thc manifold 68 free to solvent and
reagent i31 prcparation for the ncxt dclivery step.
Thc diapllragrn valve 80 is gcnerally held open during passage of the
various rcagcnts alld solvcnts througll tllc chamber ~4 to conduct thc cfflucnt
tllcrefrom through tllc chambcr outlct passagc and thc mallifold 84 to tllc ~astc
trap 60. Aftcr ~clivcry of a particullr rcagcnt or solvcnt thc clulmbcr nlld
tllc associ;ltcd conduits c~n bc CVaCU.ltCd by OpCllillg thc diapllr.l~ valvc 87 to
thc vacuulll pUlllp ~0. ~ltcrnativcly, tllc cll.lmbcr ;IIId thc S;llllplC thcrcill call bc
~1,,
dried at the appropriate times by passing inert gas through the chamber by
way of the valve 72. The gas exiting the chamber can then be passed to the
l~aste trap G0, or if desired, clrawn ou-t by the vacuum pump 90 to accelerate the
drying process.
After completion of the coupling and cleaving steps, the extraction
solvent S3 is delivered to the reaction chamber 34 to dissolve the cleaved
amino acid derivative produced during the particular cycle of degradation and
to deliver the solution to the conversion flask l~i through the conduit 86. Por
this purpose~ the valve 78 is open and the valves 80 and 82 remain closed.
The conversion reagents l~4 and R4A ~iE used) and solvent S4 may -then be deliver-
ed to -the conversion flask 14 at the appropriate times by opening the corres-
ponding valves 112 to pass the liquids through thc manifold 114 and the
conduit 104 to -the conversion flask. Each delivery is preceded by the prcssuri-
zation and vcnting operations described above in rclation to delivery of thc
other reagents and solvents, and Eollo~ed by opcning the valve 118 to purge
the manifold 114 and the conduit 104.
The fraction transferred to the conversion flask 14 is the anilino-
thiazolinone derivative of the N-terminal amino acid of the protein or peptide
sample and is converted automatically during thc next coupling and cleavage
cycles of the reaction chamber ~4 to the morc stablc pllc1lylthiohydantoin amino
acid, partly according to tlle articles notcd abovc by l~ittmann-Liebold. Bricf-
ly, thc amino acid fraction ~ithill thc convcrsioll flask may first bc evaporatl;d
by passagc of inert gas ovcr thc solution througll the short capillary 276 and
bubbling of incrt gas througll thc liquid by ~iay of thc capillary 272, follo~cd
by application of vacuulll througll thc tubc 268. Thc convcrsion rcagcnt R~
may thcn bc introduccd througll tllc capillary 276 by ~ay of thc cond-lit l04 and
onc of thc valvcs ll' ~cc ligurc J) ill thc dcsircd (lu;llltity. R;ll)id cvapor;ltio
~ ~c~ 3t~
of the conversioIl flask aEter the clesirecl conversion time may be accomplixhed
by simultaneously app1ying vacuum to the conversion flask throu~h the -tube 268
and inert gas througll capillaries 272 and 276. In order to further stabilize
the aeidic side ehains of Pth-aspartic and Pth-glutamic acids, one can further
dissolve the residue in the conversion flask by introduction of reagent R4A
through the capillary 27G by way of the conduit 104 cmd one of the valves 112
in the desired quantity. Evaporation of the eonversion flask is again aeeom-
plished by applying vaeuum through the tubes 268 and inert g.lS through
eapillaries 272 and 276. The Ptl~-amino aeid remaining l~ithin the eonversion
flask is then redissolved in the solvent S4 introdueed through the eonduit 104
for transfer of the fraetion to the appropriate vial in ~he fraetion eolleetor
16. The transfer of the fraetion is accomplished by opening the valve 131
eonneeted to the long eentral eapillary 272 of the conversion flask and admit-
ting inert gas through the eapillary 276 to foree the fraetion from the flask.
The vial earrousel of the fraction collector 16 is rotated through a
predetermined angle once during eaeh degradation eycle, SUC}I that eaeh ineoming
fraetion is eolleeted in a separate vial. At an appropriate point in the
degradation cycle, the fraetions ~ithin the fraction collector may be further
dried by opening the valve 98 to vacuum or opening the valves 128 and 94 to
pass inert gas from the source 28 over the fractiolls aIld finally to the ~aste
trap 60.
The various compoIlents of the apptrattls 10 are preferably constructed
of materials whicll are substaIltially inert and are higllly resistant to deteriora-
tion. Sueh materials include l)orosilicate glass, certain fluoroearbon ~nolymers,
aIld~ in SOMC C..tSCS, stailllCss steel aIld alumin-lm. The ~ealiIlg structures aIld
other elements of the app;lr;ltus 10 h;lVC beeIl clesigned sucll th;lt they can be
m;lIlufaeturecl allIlost e~clusively from these nt;Iteri;lls. It is felt that the
resulting appar.ltus :is the cleanest and most contamination-free system obtain-
able and would function in that conclition indefin:i-tely.
The various steps performed by the apparatus lO in a typical de-
gradation and conversion cycle are listed in Table l. The duration of each
step and the :Eunctional state of the apparatus during each step are also given.
The functions shown correspond to the conditions of the various valves of tlle
apparatus, and the marks in the columns signify when the appropriate valves
are open. For example, the columns "Rl", R"2", "R3", "R3A", "Sl", "S2" and
"S3" denote the conditions of the various pairs of valves 58 and 62 for
selectively pressurizing ancl establishing a dynamic equilibrium condition with-
in -the reservoirs 42 through 5~ herever a mark appears in one of these
columns, the valves 58 and 62 associated ~ith the particular reservoir are open,
either in preparation for or during delivery of the particular reagent or
solvent to the reaetion chamber. The valves remain elosed at all other times.
Likewise, the "argon" eolumn shows the condition of the valve 72 for delivery
of an inert gas sueh as ~rgon to the reaetion ehamber through tlle manifold
68, the "deliver" eolwnn sho-~s the condition of the valve ~6 corresponding
to any reservoir l~hieh is pressurized at the time, and the columns labeled
"l~aste", "eollect" and "vaeuum", show the conditions of the valves 82, 78
and 80, respeetively. .~s deseribed above, each step of solvent or reagent
delivery is preeeded by pressurizacion of the appropriate solvent or reagent
reservoir and follo~ed by the introduction of inert gas through the valve 72 to
complete delivery of the solvent or reagellt and flush the delivery lines.
The columns "R~", "R~" and "St" denote the conditions of the various
p.1irs of valves 108 alld 1lO for selectively pressuri~illg alld estal)lisllillg .-
dyll.~ ic equi1ibrium withill the reservoirs 1~0 through 102, alld the "-Ie1iver/
argoll" co1ulllll .sigllifies the contlitioll of both the valve l22 wllich admits inert
I (,
q~J
gas into the conversion Elask througll-the line L24 and the valve 112 corres-
ponding -to the rcservoir which is pressurize~ at the time. The columns
"argon", "waste 1", "vacuum", "collect" and "waste 2" show the conditions of
the valves 118, 92, 9G, 131 and 125, respectively.
Of the fraction collector func-tions listed, the columns "argon",
"waste" and "vacuum" show the conditions of the valves 128, 94 and 98,
respectively.
l~ith the exceptions notcd hereillabove, the sequence of steps listed
in Table 1 essentially conforms to the Edman degradation processes described in
the cited yublications and l~ill no-t be discussed in detail. Any deviations
from general practice ~ill be clcar from the names of the steps and the cor-
responding functional states tabulatcd in 'I'able 1.
In practice, the following variations of the sequencing program of
Table 1 may be imylemented to adayt thc yrogram to the needs of a particular
user:
1) Steys 18 through 58 can be looyed one or two times on the first
sequenccr cycle to insure complete coupling of all amino groups on the protein.
2) Step 65 can bc followed by a 20 ~o 60 second delivery of ~ater
vapor (R3A) tllrough the reaction chambcr to reduce dehydration of sidc chaills
of scrille and thrc-ollille.
3) Step 65 can be followcd by dclivcry of .05 ml of 1.~ hydrochloric
acid in metilanol (R~t) to tlle convcrsioll fl~sL; to methylatc thc si~c cllains oE
asp;lrtic acid and glutamic acid. If this is ~one, S~ if prcfcrably mcthanol.
4) Step 7G Call bc ~ollowcd by dclivcry ot' .7 to I ml of S~ (accto-
nitrilc or mcthanol) th;lt is subsc(luclltly dclivcrcd to wastc to thoroughl-
clcall thc convcrsioll flas~.
5) Stcl~ 8 can l1C incrc.lscd in dur.ltioll (500 to lO00 SCCOI1~5) to pro-
- -~7 -
mo-te cleavage of amino terminal proline residues.
Tlle nature of the presellt inventioll will be further clarified by
the following specific examples of the practice of the invention. It should
be ~mderstood that the data disclosed serve only as examples of processes
~hich have been performed with the apparatus and method of the present inven-
tion and are not intended to limit the scope of the invention.
The amino acid sequences set forth in the following examples are
tabulated in the one-let-ter amino acid code, which is defined as follows:
A - alanine L - leucine
R - arginine K - lysine
N - asparagine ~1 - me-thionine
D - aspartic acid F - phenylalanine
C - cysteine P - proline
E - glutamic acid S - serine
Q - glutamine T - threonine
G - glycine 1~ - tryptophan
II - histidine Y - tyrosine
I - isoleucine V - valine
Example 1
A solid matrix of polymeric quaternary ammonium salt, suitable for
embedding a protein sample for sequencing, can be prepared on the fibrous sheet
element 190 and 190' described above, and a protein sample can be embedded in
the matrix as follo~s:
A glass fiber disc, 12 nun in diameter and .75 to .5 mm tl-ick, is
cut from ;l sheet of ~lass microfiber filter ~hich is ~vail;lble commerci;llly
from l~h.ltm.lll, Inc., Clifton, .\'el~.Icrsey. rhc disc is pl.nced in the depressio
192 of the chamber elelllellt l:j-l '5 microliters ot .ln .Iqueous solution
con-taining 1.5 mg of 1,5-dime-thyl-1,5-diazaundecamethylene polymetllobromide
and .0033 mg of glycylglycine is dropped onto the glass fiber disc rom a
syringe or pipette. The water is evaporated under vacuum or by heating in
a stream of warm nitrogen. The remainder of the chamber apparatus 12 is assem-
bled and installed in the apparatus 10. I`he protein sequencing program is
initiated at step 18, and ~ to 6 comple-te degradation cycles are accomplished
in order to remove impurities -from the 1,5-dilllethyl-1,5-diazaundecametllylene
polymethobromide that mi.ght react chemical.ly witll the protein sample or other-
IYise interfere ~Yith the Edman process. The chamber apparatus 12 is parti.ally
disassemb].ed and 25 microliters of a solution of the protein is dropped onto
the glass fiber disc. The protein solu-tion dissolves the 1,5-dimethyl-l,S-
diazaundecamethylene polymethobromide and the liquid is removed by evaporation
to leave behind a thin film of 1,5~dimethyl-1,5-dia~aundecamethylene polymetho-
bromide with the protein sample embedded therein. If the initial protein
sample volume is greater than 25 microliters, the sample can be applied in 25
microliter aliquots IYith the liquid being removed by evaporation bet~een ali-
quot applications. The chamber apparatus 12 is reassembled and reinstalled in
the apparatus 10. The protein sequencing program is then initiated at step 18
and carried through as many degradation cycles as desired.
Example 2
A solid matri~ of polymeric quaternary ammonium salt, suitable for
embedding a protein sample for secluencing, can be prepared on tlle interior ~Yalls
of a glass capillary vessel, and a protein sample can be embedded in the matri.x
as follo~Ys:
Tlle ch;lmber elemellt ~'-1 or tlle ch;lmber elements 1~' alld 1~6' are
initi.llly asselllbled ~ith the sleeve 182 and tlle cap 160, as illustrated in the
figures, to forlll;l co~ r.lct cartridgesnl--;lsselllbly ~licll is easily m;lllipul;lted
I )
:for introduction o-E the ma-trix and the sample. The sub-assembly is held hori-
zontally and 10 microl.iters of an a~Iueous soluti.on containing .6 mg of 1,5-
dimethyI-1,5-diazaundecamethylene polymethobromide and .0013 mg of glycyl
glycine is injected into the reac-tion chamber from a syringe.
The chamber 320 is preferably cons-tructed to have a diameter of 1/L6"
a-t its two ends and a diameter of 1/~" at its middle, producing a chamber
whic}l will accommoclate up to 25 mi.croliters (~Q) o~ solu-tion in the horizontal
condition Wit]lOUt spilling out at the ends. TIlis conf:i.guration of the chamber
324 holding a solution in the horizontal condi.tion is illus-trated in Figure 18B,
~ith the sleeve 132 and related structure omitted for simpl:Lcity. T}le sub-
assemI)ly is then rotated about its axis ~hile the liquid in the reaction chamber
is evaporated by directing a stream of air or nitrogen through the chamber,
leaving a thin filnI of 1,5-dimethyl-1,5-diazaundecamethylene polymethobromide
on the chamber walls. The sub-assembly is then installed in the apparatus
10, the protein sequencing program is initiated at step 18, and 4 to 6 complete
degradation cycles are accomplished. TIle sub-assembly is next removed from
the apparatus 10 and held llorizontally wllile 10 microliters of the protein
solution is dropped into the reaction chamber from a syringe. The sub-assembly
is again rotated about its axis wIlile the liquid in tlle reaction chamber is
Z0 evaporated by a stream of nitrogen directed through the chamber, leaving a
thin film of l,5-dimetIlyl-1,5-diazaundecaIllethylene polymethobromide Witil the
protein sample embedded thereiIl as illustrated in ~igure 18C. The sub-assembly
is theIl reinstalled in the apl)aratus 10 aIld the protein se(IuenciIlg program
is initi.lted at step 18 and carried tlIrough as man- degradatioIl cycles as
desired.
I.~ample 3
~ngi.oteIlsill, .On()5 mg, cont;liIled in '5 microliters of a(IueoIls '0~
- r~o -
c~ 7
Formic acid was embedded in a solicI matr-ix o~ precycled 1,5-dimethyl-1,5-
diazauncIecametIlyleIle polymethobromicle accorclilIg -to the method OL '~.~.LillplC ..
The angiotensiIl was subjected to eight cycles of Edman ciegradation, and the
pheiIyltIlioIlydalltoin amino acids produced at each cyclo were analyzed by high
pressure liquid chromatography according to the method described by Johnson
et al, "Analysis of PhenyltIliohyclantoin Amino Acids by IIigh Pressure Liquici
Chroma-tography on Du Pont Zorbax CN Columns", Anal. Biochern. 100, 335 (1979).
The Eollo~ing sequeIlce, listed below as "Experimental," was obtainecI. 'I'he Icnown
sequence is also listed below for compar:ison.
1 3 5 7
Experimental: D - R - V - Y - I - I-I - P - i~
Known: D - R - V - ~ - I - iI - P - F
This result is sigIlificant because it demonstrates that even small
peptides can be sequenced in the manner of the present invention. The ability
to sequence small peptides stems from the fact that the sample is embedded in a
film for retention, rather tllan being adsorbed directly onto a support surface,
and tllerefore can be helcl securely regardless of size. I~hereas sorptive
bonds eomprise a large number of non-covalent interactions betlieen the sample
and the surfaee and are heavily dependent on the size of tlle molecules, the
holdiIlg po~er of the film disclosed herein is relatively unaffected by sample
size.
Example 4
Sperm ~hale apomyoglobin, . 01 mg, contained in '5 microliters of
aqueous 20o acetic aeici was cmbcclclccl in a solid matri.~c of precycled 1,5-dimethyl-
1,5-diazaundecaIllethylelIe polyInetIlobrolllicle accorcIiIlg to the n~etiIoci of Example
1. 'I'he apomyoglol)iIl was subjected to ~0 cycles of Eclman Iegr.Ii;ltiol), and the
j)hCnyltlliOIlydalltOill .IlllillO ;ICiCis ~'CI`C a~ lyzecI accor(IiIlg to the metIIocI of
[.xaIlll)Ie 3 to give the se~Illence listeci helow ;Is ''I'.~periIllent;Il.ll TllC ~llOWn
- 51
3~ 3t~j~
sequence of sperm whale apomyoglobin is also lis-ted below for comparison.
Experimental: V-L-S-E-G-E-W-Q-L-V-L-II-V-W-A-
Known: V-L-S-E-G-E-W-Q-L-V-L-II-V-~\I-A-
16 20 25 30
K-V-E-A-D-V-A-G-}I-G-Q-D-I-L-I-
K-V-E-A-D-V-A-G-II-G-Q-D-l-L-I-
~0
R-L-F-K-S-il-P-E-I'-L-
R-l.-F:-K-S-II-P-E-T-L-
Example 5
A Drosophila melanogaster larval cu-ticle protein of previously un-
knowll structure, .01 IDg~ contained in 25 microliters of an aqueous solution of
.1% sodium do-lecyl sulfate and .05~l ammonium bicarbonate, was embeddecl in a
solid matrix of precycled 1~5-dimethyl-1,5-diazaundecamethylene polymetho-
bromide according to the method of Example 1. The cuticle protein was sub-
jected to 36 cycles of Edman dcgradation, and the phenylthiohy~lantoin amino
acids ~lere analyzed according to the method of Example 3 to give the sequence
listed below as "Experimental."
Experimental: N - A - ,~ - V - E - V - ~; - E - L - V -
11 15
N - D - V - Q - P - D - G - 1~ - V - S -
21 25
~ - L - V - L - D - D - G - S - A - S -
31 ~5
S - .~ - T - G - I) - I -
Examplc 6
,~ I)rosophila mclallogastcr larv;ll cuticlc protcin o~ prcviously un-
l~nowll structurc, .005 m~, COllt;linCd ill 10 microlitcrs of all ~Iq~lCOUS ~;olution
of .l~o sodiul1l dodccyl s~llf;ltc allcl .05~1 ammolliulll bic;lrboll;ltc, W;lS cmbcd~lc-l in a
,
solid matrlx of- precyclecl l,5-dimethyl-1,5-diazaundecamethylene polymethobromide
according to the method of Example 2. The cuticle pro-tein was subjected to
24 cycles o Eclman degraclation, and the phenyltlliohydantoin amino acids were
analyzed according to the method of Example 3 to give the sequence listed below
as "Experimental."
Experimental: N - ~ - N - V - E - V - t~ - E - L - V -
11 15
N - D - V - Q - P - D - G - F - V - S -
K - L - V - 1. -
The results achieved in i~:xamples 5 atld 6 demonstrate -tllat the appara-
tus and method of tlle present invention i.s suitable for sequencing proteins
and peptides dissolved in a solution of sodium dodecy]. sulfate (hereinafter
"SDS"), a potent anionic detergent. This is important because the most general
method of isolating small quantities of medium to large pro-teins or peptides
for analysis, known as polyacrylimide gel electrophoresis, produces samples in
a solution of SDS. This common detergent causes samples to wash out of devices
which rely upon adsorptive bonding of tlle sample to a support surface, however,
the solid matrix of the present invention is unaffected by the presence of SDS.
From tlle above, it can be seen that there has been provided an
improved apparatus and mettlod for thc sequential performance of chemical pro-
cesses on a sample of very small si~e through ttlC use of minimum amounts of
reagellts and solvents and relatively short cycle timcs.
The appended claims are intended to cover all variations and adapta-
tions fallillg within the true scope alld spi.rit of the prescnt invention.
