Protein or peptide sequencing method

A method and apparatus for sequencing polypeptides, including a continuous flow reactor which may include a sample bearing membrane strip.

A. FIELD OF THE INVENTION 
The invention relates to apparatus and methods for protein or peptide 
sequencing. In particular, the invention relates to continuous flow 
reactors for peptide sequenators, and to sequencing methods and apparatus 
in which such reactors are used. 
B. DESCRIPTION OF THE PRIOR ART 
Practical automated peptide sequencing dates from the 1967 introduction of 
the liquid phase spinning cup sequenator in which the reactions proceed in 
a thin liquid film formed on the inside wall of rotating reaction cells. 
See Edman, P., and Begg, G., A Protein Sequenator, European Journal of 
Biochemistry, 1:80-91 (1967). A focal problem associated with the spinning 
cup sequenator is sample loss, particularly of short peptides. An 
alternative solid phase degradation method entails passing reagents and 
solvents in an appropriate program through a column packed with porous 
material such as a macroporous polystyrene matrix or preferably porous 
glass beads to which a peptide is attached covalently or by adsorption. In 
another, known type of automatic sequencer the peptide to be degraded is 
covalently linked to a gel-type of solid phase support contained within a 
tubular reaction chamber. Both the reaction chamber and the tubing by 
which it is connected to the sequenator may be formed from glass or from 
polytetrafluoroethylene, e.g., "Teflon". See Laursen, R. A., A Solid-Phase 
Peptide Sequentaor, European Journal of Biochemistry, 20:89-102 (1971) and 
Shively, "Methods of Protein Characterization", Humana Press, Clifton, 
N.J. (1986), Chapter 9. 
A sequenator that employs gas phase reagents instead of liquid phase 
reagents at critical points in the Edman degradation was proposed in 1981. 
See Hewick, R. M., Hunkapillar, M. W., Hood, L. E., Dreyer, W. J., A 
Gas-Liquid Solid Phase Peptide and Protein Sequenator, The Journal of 
Biological Chemistry, 256:79907997 (1981), U.S. Pat. No. 4,603,114 and 
Shively, suora, Chapter 8, Section 314, p. 229. This device includes a 
two-part glass cartridge assembly which houses a miniature continuous flow 
reaction chamber in which the peptide sample is presented as a dispersion 
in a thin film of a polymeric quaternary ammonium salt supported on a 
porous glass fiber disk. Means are provided for disconnecting the 
cartridge from its mounting base each time the sample is loaded. The 
cartridge is connected to a sequenator by Teflon tubing at its inlet and 
outlet ends. 
A modification of the Hewick sequenator is described by Hawke, Harris and 
Shively in Analytical Biochemistry, 147, 315-330 (1985), and Shively, 
supra, Chapter 7, page 210, et. seq. This modification replaces the glass 
reactor cartridge assembly of Hewick with an all Teflon cartridge of 
similar design, thus providing an all Teflon delivery and reaction system. 
The sample is presented within the reaction chamber on trimethylsilyated 
glass fiber disk. Hawke, et al., noting that Teflon is "self-sealing", 
report lower background levels and increased yields deemed to be 
consequent from a better seal achieved in the all Teflon design as 
compared to the seal observed with the Hewick glass cartridge. See 
Shively, p. 217. 
A multi-purpose sequenator constructed in units which are interchangeable 
for easy conversion to a spinning cup, column, or cartridge operational 
mode has been described. A polyfluorochloro (Kel-F) micro column unit 
filled with peptide bound glass support objects which has Teflon tubing 
inlet and outlet lines for attachment to a sequenator is provided. See 
Shively, p. 249 et. seq. 
SUMMARY OF THE INVENTION 
This invention provides an inexpensive continuous flow reactor column 
substantially free of unswept volumes in which accumulation of amino 
acids, by products and reagents is minimized. The reactor is inexpensive. 
Use of new and incontaminated reactors is facilitated by easy insertion 
into and removal from the sequenator. 
The reactor is formed from chemically inert synthetic resin tubing. A 
reaction chamber comprising a section of such tubing of relatively large 
internal diameter is provided with interference fitted inlet and outlet 
tubes of appropriate dimension. In a preferred embodiment of the 
invention, the reactor is formed from Teflon tubing. It may be packed with 
discrete articles, e.g., porous glass beads, or a porous synthetic resin 
such as polystyrene coated with the protein or peptide to be sequenced. 
When such packings are used, a porous support member, preferably of 
Teflon, may be provided. Pursuant to another embodiment of the invention, 
the sample may be borne by a hydrophobic membrane such as polyvinyl 
difluoride (PVDF) to which proteins adhere. Optionally the hydrophobic 
membrane may be coated with a polymeric quaternary ammonium salt such as 
1,5 dimethyl-1,5-diazaundecamethylene, polymethobromide or poly 
(N,N-dimethyl-3,5-dimethylene piperidinium chloride. One such product is 
available under the trademark Polybrene from Aldrich Chemical Company.

DETAILED DESCRIPTION OF THE INVENTION 
A continuous flow reactor according to the present invention is shown in 
FIG. 1 where it is generally designated by reference numeral 10. The 
continuous flow reactor 10 includes reaction tube 14 and interference 
fitted supply and drain tubes 12 and 16. Each of the tubes 12, 14 and 16 
is formed from a self-lubricating fluorocarbon such as a 
polytetrafluoroethylene. Numerous fluorocarbons are available. See, e.g., 
Plastics Engineering Handbook, Van Nostrand Reinhold Co. (1976), pp. 
60-62. 
Each of the tubes 12 and 16 may be of substantially the same size, and the 
reaction tube 14 may be larger than the tubes 12 and 16. For example, the 
tubes 12 and 16 may have an outer diameter of approximately one-sixteenth 
(1/16 or 0.0625) inch. The reaction tube 14 may have an inner diameter 
also of approximately one-sixteenth (1/16 or 0.0625) inch and an outer 
diameter of approximately one-eighth (1/8 or 0.125) inch. The inner 
diameter of the reaction tube 14 is slightly undersized relative to the 
outer diameters of the tubes 12 and 16. With these size relationships, 
leak proof joints are provided between the tubes 12 and 16 by interference 
or press fitting. 
Alternatively, the drain tube 16 may be larger and so dimensioned as to 
provide a press fit on the outside instead of the inside of the reaction 
tube 14 as shown in FIG. 1. A reaction zone free of unswept volumes is 
provided in this manner. 
A porous support member 18, such as a disk, is tightly fitted inside the 
reaction tube 14. Preferably, the support member 18 is positioned near the 
location of the upper edge of the tube 16 as in the embodiment shown in 
FIG. 1. The support member 18 has a porosity requisite to allow passage of 
fluids and yet retain discrete objects 20, such as beads packed into the 
upper portion of the reaction tube 14. Support member 18 is formed from a 
chemically inert synthetic resin, preferably a fluorocarbon. For example, 
the support member 18 may be made from polytetrafluoroethylene cut by a 
14-gauge needle to provide a circular disk slightly larger than the inner 
diameter of the reaction tube 14. The disk can be pressed into the 
reaction tube 14 where it will be retained in the desired position by the 
resulting press fit. 
Useful support members 18 may be formed from a synthetic resinous material 
sold under the trademark "ZITEX". This material is available from a number 
of suppliers including Norton Chemplast, 150 Dey Road, Wayne, N.J. in 
different porosities such as extra fine, fine and medium. Materials with 
all of these different porosities can be used satisfactorily in the 
continuous flow reactor 10. 
The reaction tube 14 is appropriately packed with discrete objects 20. 
Preferably, the discrete objects are made from a porous material such as a 
porous silica, microporous glass beads, or macrorecticular polystyrene. 
The discrete objects may be irregular or spherical. Suitable discrete 
objects having an irregular and porous configuration may be obtained from 
Electro-Nucleonics, 368 Passaic Avenue, Fairfield, N.J. Preferred 
irregular discrete objects have a mesh size between one hundred and twenty 
Angstroms (120 .ANG.) and two hundred Angstroms (200 .ANG.) and a pore 
size of approximately three hundred and seventy nine Angstroms (379 
.ANG.). Silica packing materials meeting these specifications are 
available as GC Porasils B and C from Waters Chromatography Division of 
Millipore Corporation, 34 Maple Street, Milford, Mass. See Waters 
Sourcebook for Chromatography Columns and Supplies (1986). 
Spherical discrete objects having a diameter of from about one hundred 
microns (100.mu.) to three hundred microns (300.mu.) are preferred. Such 
spherical objects when packed in the reaction chamber 14, provide spacings 
which assure that fluids will flow along substantially non-linear paths 
and will be drained without entrapment. The provision of non-linear flow 
paths is further facilitated by the use of packings which comprise a 
plurality of different sized spherical objects. For example, a mixture of 
spherical particles of different diameters in the range of one hundred 
microns (100.mu.) to three hundred microns (300.mu.) is appropriate. 
Silica derivatives, such as octadecyl silica and octyl silica, may also be 
used for the discrete objects 20 and may be preferred for the sequencing 
of certain peptides or proteins. Various other silica derivatives 
currently available for reverse phase high performance liquid 
chromatography may be used. Such derivatives may be either specifically 
prepared for use in the reactor of this invention, manufactured or 
purchased. Waters GC Porasils B and C and Waters GC Bondapack C18 material 
may be used as a starting material for the preparation of silica 
derivatives. 
The discrete objects may be coated with a peptide 22 (see FIG. 2) which is 
to be sequenced. For example, one microgram or less of the peptide may be 
provided for each milligram of the discrete objects. In a specific case, 
1-3 micrograms of sperm whale apomyoglobin may be added to 5-10 milligrams 
of the discrete objects. 
The peptide sample size depends on the purity, molecular size and the 
desired number of amino acid residues to be determined. It can be as low 
as 0.1 to 10 picomoles. Ten to twenty milligrams of discrete objects is 
generally adequate to provide 0.1 to 10,000 picomoles samples. However, 
the required quantity of such objects may vary by as much as tenfold for 
certain applications. 
Alternatively, a first coating material 24 (see FIG. 3), such as Polybrene 
which has affinity both for the objects and the peptides 22 is applied. A 
Polybrene coating is particularly appropriate when porous discrete objects 
are utilized. Preferably, at least one milligram of Polybrene per 
milligram of discrete objects 20 is applied. However, the amount of 
Polybrene may vary approximately fifty percent (50%) above or below this 
amount. 
The polymeric quaternary ammonium salt such as Polybrene may be applied 
before or after the discrete objects 20 are placed in the continuous flow 
reactor. In the case of a reaction tube as illustrated at 14 in FIG. 1 
which may have a length of about 3 cm, a discrete object bed of from about 
0.5 to 1.0 cm in length is appropriate thus providing 5 to 10 mg of 
silica. Approximately 5 microliters (5 .mu.l) of 100 mg/ml of Polybrene 
solution is then applied to the discrete objects in the reaction tube. 
When applied outside the reaction tube, a quantity of Polybrene or the like 
sufficient to wet the surfaces of the discrete objects completely is 
preferred. For example, either discrete objects providing about 10 mg of 
silica, or a Polybrene solution containing approximately 30 mg/ml is 
appropriate. 
FIG. 4 depicts a closure member 26 is disposed in the reaction tube 14 at a 
position near the end of the inlet tube 12. It may be constructed in a 
manner similar to the support member 18. It encloses the top of the 
reaction tube 14 to confine the discrete objects 20. 
FIG. 5 shows chromatograms which provide a comparison of sperm whale 
apomyoglobin sequencing results in cycles 1 through 4 and 7, 9, 10 and 12, 
obtained with a cartridge reaction chamber as shown in Hawke (1985), and 
with a continuous flow reactor 10 of this invention. 
The upper panels A-H in FIG. 5 show Edman degradation cycles for the 
sequencing of 200 picomoles of sperm whale apomyoglobin using a cartridge 
reaction chamber made from polytetrafluoroethylene as described by Hawke 
(1985). Before the sample could be applied, it was necessary to coat the 
glass fiber disk in the reaction chamber with Polybrene (1 mg) and 
precycle the disk for two 45 minute cycles of Edman chemistry. 
The amino acid derivatives obtained from the cartridge reaction chamber 
were analyzed by reverse phase high performance chromatography by a method 
similar to that of Hawke (1985). 
The lower panels, A'-H' in FIG. 5 are the chromatograms obtained from an 80 
picomole run of sperm whale apomyoglobin using a continuous flow reactor 
10 as of this invention connected to the same sequencing apparatus as 
described above for the prior art cartridge reaction chamber. 
Chromatographic analysis of the derivatives was conducted in the same way 
and at the same attenuation settings. 
The large offscale peaks labeled DEA and DPTU in the panels of FIG. 5 are 
common background peaks observed in Edman chemistry. The DEA peak is the 
phenylthiocarbamyl derivative of diethylamine (DEA), a trace contaminant 
of triethylamine (TEA). The DPTU peak is diphenylthiourea, which is formed 
from the reaction of phenylisothiocyanate (PITC) with aniline formed by 
the base catalyzed destruction of PITC. 
These peaks, plus a number of smaller, unidentified peaks, constitute the 
background noise which interferes with the identification of the 
phenylthiohydantoin (PTH) amino acid derivatives. In each cycle, the 
single letter labeled peak corresponds to the assignment: V=valine, 
L=leucine, S=serine (S'=a breakdown product of serine), E=glutamic acid, 
W=tryptophan, and H=histine. The peak labeled "std" is an internal 
standard (the PTH derivative of aminoisobutyric acid). The peaks labeled 
in parentheses in the panels are the carryover signals from the previous 
cycle. Its appearance on a chromatogram is normal. 
Even though less than 50% of the amount of sperm whale apomyoglobin was 
sequenced on the continuous flow reactor 10, there is adequate sensitivity 
compared to the results obtained with the cartridge reaction chamber. This 
is seen, for example, by comparing the signals for valine (V) in panels A 
and A' or the signals for leucine (L) in panels B and B'. An improved 
signal-to-noise ratio is provided in addition to substantial sensitivity 
by the reactor 10. The improved signal-to-noise ratio is directly apparent 
from a comparison of the signal magnitudes for DEA, V and DPTU in panels A 
and A'. In panel A the signal magnitudes for the DEA and DPTU background 
noise signals are greater than that for V, the sought-after assignment. 
This relationship of signal magnitudes is reversed in panel A' where the 
results using the continuous flow reactor 10 are shown. Such improved 
signal-to-noise ratios are repeated in all of the panels. 
The continuous flow reactor 10 of this invention considerably facilitates 
the sequencing of the small sample. In addition, the sample was directly 
analyzed, without precycling, after the addition of Polybrene with 
consequent savings in time. 
A second embodiment of the continuous flow reactor of the present invention 
is shown at 32 in FIGS. 6 and 7. The reactor 32 includes reaction tube 34, 
and interference fitted adapter caps 36, and adapter plugs 38 into which 
are interference fitted supply and drain tubes 12 and 16. Each of the 
reaction tube 34, adapter caps 36, adapter plugs 38, supply and drain 
tubes 12 and 16 is formed from chemically inert, pliable synthetic 
resinous material, preferably a self-lubricating fluorocarbon such as 
polytetrafluoroethylene. 
The leak tight joints are provided by close interference or press fittings 
between the reaction tube 34 and the adapter caps 36, and between the 
adapter plugs 38 and the supply and drain tubes 12 and 16. The 
interference fitted parts may be chamfered to facilitate assembly. The 
fittings between adapter caps 36 and adapter plugs 38 can be made so that 
the inner surfaces of the adapter caps 36 against which the outer surfaces 
of the adapter plugs 38 contact are conical in shape. These surfaces can 
be roughened so that frictional contact is increased to prevent breakage 
of the leak-tight joints between the reaction tube 34 and tubes 12 and 16. 
A third embodiment 40 of a continuous flow reactor shown in FIGS. 8 and 9. 
It includes a reaction tube 42 with adapter plugs 44, and supply and drain 
tubes 12 and 16. The adapter caps 36 of the second embodiment have been 
replaced by conical surfaces at the ends of the reaction tube 42 for 
mating with the adapter plugs 44. To provide leak proof joint, all of the 
parts for the reactor 40 are preferably formed from chemically inert, 
pliable synthetic resinous material such as polytetrafluoroethylene. 
Porous support members 18, such as disks, may be tightly fitted inside the 
reactor tubes 34 (of the second embodiment, see FIGS. 6 and 7) and 42 (of 
the third embodiment, see FIGS. 8 and 9). If used, the support members 18 
are preferably positioned inside the reaction tubes 34 and 42 to allow 
about 4 mm. of length for the interference fits between the supply and 
drain tubes 12 and 16, and the reaction tubes 34 and 42. As shown in FIGS. 
6 and 8 respectively, the support members 18 have a porosity requisite to 
allow passage of fluids and yet retain discrete objects 20, such as beads 
packed into the reaction tubes 34 and 42. 
The continuous flow reactor 40 is connected by the tube 12 to an apparatus 
(sequenator) for introducing reactive fluids, e.g., Edman reagents, for 
N-terminal sequencing, or Stark reagents for C-terminal sequencing. 
The isolation proteins in the low picomole range in sufficient purity for 
microsequence analysis has been a continuing problem. One long sought 
after goal is to sequence samples isolated from gels, particularly sodium 
dodecyl sulfate (SDS) gels. Methods of electroblotting samples from SDS 
gels to positively charged glass fiber paper have been explored. 
Matsudaira.sup.1/ reports high yields on protein samples electrotransferred 
from SDS gels to PVDF stained with Coomassie Blue and subjected directly 
to microsequence analysis on an Applied Biosystems Model 470 Sequenator. 
The stained bands were cut out, centered on the Teflon cartridge seal and 
placed in the sequenator. See generally, FIG. 5 of U.S. Pat. No. 
4,603,114. 
FNT 1/ Matsudaira, P. J. Biol. Chem., 262:10035-10038 (1987). 
Microsequence analysis of a protein carried by small PDVF strips presents 
unique problems for such cartridge based microsequencers. The sample is 
likely to be present on two or more strips. It is necessary, as Matsudaira 
illustrates, to lay the membrane strips across the cartridge seal. If the 
strips move or the reagent or solvent flow is not uniform across the 
strips, poor or irreproducible chemistry is likely to occur. 
These problems are avoided by a fourth embodiment of this invention. 
Pursuant to that embodiment, the protein or peptide to be sequenced is 
placed on a PVDF or similar hybrophobic membrane and the membrane is 
stained, e.g., with Coomassie Blue, and cut into thin elongated strips for 
insertion into a continuous flow reactor of this invention. No porous 
support member 18 is utilized. Sequencing ensues in the same manner as 
when the sample is carried by discrete porous objects. 
FIG. 10 depicts a continuous flow reactor 45 including supply and drain 
tubes 12 and 16 interference fitted to a reaction tube 14 which contains a 
plurality of sample bearing PVDF strips 46. The depth of insertion of the 
tubes 12 and 16 into the tube 14 preferably correlates with the length of 
the strips 46 to minimize the space between the tube ends and the strip 
ends. 
FIGS. 11 and 12 depict a preferred reactor design 47 for use with sample 
bearing PVDF strips. Reactor tube 48 as shown in FIG. 11 is provided at 
each of its ends with a generally convex inner surface 49 which mates with 
the generally concave outer surfaces 50 of the supply or drain tube 
bearing caps 51 as shown by FIG. 12. 
The inner diameter of the reactor tube 48 is reduced over a length 52 of 
its central portion thus providing upper and lower shoulders 53 and 54 
within the tube 48. Sample bearing PVDF strips 46 are placed in the 
portion of the tube 48 which is of reduced inner diameter. Preferably the 
length of such strips is similar to the length of the portion 52 of the 
tube 48 which is of reduced inner diameter. 
Like supply and drain tube bearing caps 51, see FIG. 12, are externally 
contoured to provide essentially leak proof fittings with each of the 
curved inner surfaces 49 at each of the ends of the reactor tube 48. 
As shown in FIG. 12, a similar supply or drain tube 56 is carried by each 
of the caps 51. The outer surface of each supply or drain tube 56 is 
provided with opposed raised planar surface element 57 which may extend 
slightly beyond the tube 56 at the tube ends which are inserted into the 
reactor 47. Upon insertion of the caps 56 into the reactor tube 48, the 
ends 57 of these raised planar elements 57 seat against the shoulders 53 
and 54. 
The invention includes continuous flow reactors and associated supply and 
drain tubes for automated sequenators of various kinds including, among 
others, apparatus of the kind described in U.S. Pat. No. 4,704,256 for use 
when proteins or peptides are sequenced from samples on PVDF and similar 
hydrophobic membrane strips. Accordingly, the invention includes 
substantially cylindrical reactors for receiving elongated PVDF or similar 
sample bearing strips positioned parallel to the longitudinal axis of the 
reactor. Such reactors and associated supply and drain tubes accordingly 
may be fabricated from any desired chemically inert plastic, such as 
polyethylene or polypropylene. Particularly in the form of reactor shown 
in FIGS. 10, 11 and 12, the component parts are made from the same 
resinous materials as those described for the reactor tube 14 and supply 
and drain tubes 12 and 16 described with reference to FIG. 1 and assembled 
by interference fitting. 
Any porous hydrophobic membrane to which peptides adhere, e.g., by 
adsorption, and which is stable to the sequencer chemistry may be 
utilized. PVDF membrane is preferred. PVDF membrane is available, in 
various pore sizes including 0.45 .mu.m and in various thicknesses, from 
Millipore Corporation, Bedford, Mass. under the trademark "Immobilon PVDF 
transfer membrane"..sup.2/ PVDF membranes have a high surface area and a 
controlled open porous structure which provides strong retention of 
proteins adsorbed via hydrophobic interactions. The chemical structure of 
PVDF membranes provides stability and resistance to sequencer chemistry. 
FNT 2/ See "Immobilon.TM. PVDF Transfer Membrane: A New Membrane Substrate for 
Western Blotting or Proteins", Pluskal et al. (1986) Biotechniques 4:272. 
The invention also includes a membrane formed in part from a Polymeric 
quaternary ammonium salt such as Polybrene and in part from PVDF. A 
PVDF-Polybrene composition containing from about 25% to about 75%, 
preferably about 50% by weight of Polybrene or the like is appropriate for 
the formation of such membranes. 
In a preferred practice of a method of this invention protein or peptide 
samples are electrotransferred from SDS gels, e.g., 8% or 12% gel to PVDF 
membranes. Conventional electrotransfer techniques are useful. Transfer 
yields on the order of 30-50% at 50 V (500 mA) have been observed even at 
short transfer times of 10 minutes. Transfer yields may be improved by 
utilizing voltage settings of 25-30 V (300 mA), adding a plurality of 
layers of membranes, for example, two layers and prewetting the membranes 
with methanol or acrylontrile. 
Preferred procedure entails transfer to a Polybrene coated PVDF membrane or 
to a membrane of combined Polybrene-PVDF. Over a range of 25-100 pmol 
samples on 12% SDS gels optimum transfer time for lactoglobulin, ovalbumin 
and bovine serum albumin is about 100 minutes. Transfer efficiency of 
about 80% to about 95% is achieved. The actual value is a function of the 
sample. Longer transfer times have been observed to decrease recovery. 
Samples can be stained with Coomassie Blue directly on the membranes. In 
the absence of Polybrene no background staining is observed. In the 
presence of Polybrene a uniform background is observed, but does not 
interfere with the detection of the samples. 
Samples transferred to PVDF membranes, preferably after staining are cut 
into strips from about 0.05 to 2 mm in width and from about 1-3 
centimeters in length for convenient insertion into a cylindrical reactor. 
EXAMPLE 1 
A .beta.-lactoglobulin standard obtained from Sigma Chemical Company and 
radioiodinated with I.sup.125. The radiolabelled protein was separated 
from free iodine by centrifuged gel permeation chromatography. The 
specific activity of the iodinated protein was in the range of 0.02-0.03 
.mu.Ci/.mu.g. The labelled sample was mixed with an unlabelled sample to 
give 100,000 Cpm per 100 pmol of protein. 
PVDF membranes were treated with Polybrene (10 mg/ml in methanol/water 1/1) 
for 1-2 seconds, air dried and rinsed with methanol. Two layers of 
Polybrene treated PVDF membranes, wet with methanol, were applied to the 
electrotransfer apparatus for transfer of the sample from 12% SDS gel. The 
transfer was accomplished in about 100 minutes at a voltage setting of 
25-30 V at 300 mA. Transfer efficiency was 90-95%. 50 pmol, 20 pmol and 10 
pmol samples were prepared. 
The PVDF bearing the sample was stained with Coomassie Blue and cut into 
multiple 1 to 1.5 mm strips 46 which were inserted into the reactor tube 
14 of a continuous flow reactor as shown by FIG. 10. Sequencing was 
carried out generally in the manner described with reference to FIG. 5. 
The initial yields were 50%-60% and repetitive yields in the range of 
91-94%. Little or no background peaks were observed from the stained PVDF 
strips. 
EXAMPLES 2 AND 3 
Example 1 was repeated using ovalbumin and bovine serum albumin instead of 
.beta. lactoglobulin as the sample. Similar results were observed. The 
efficiency of electrotransfer was 90-95% for ovalbumin and 80-90% for 
bovine serum albumin. 
For bovine serum albumin, the initial yield from a 50 pmol sample was about 
20% and the repetitive yield ranged from about 94% to about 98%. 
The continuous flow reactors of this invention have important advantages. 
These reactors are: 
(1) inexpensive, disposable, simple to construct, and easy to install into 
and remove from a sequenator. These features permit a series of reactors 
to be precharged and subsequently inserted into the sequenator as needed; 
(2) have little unswept volume, i.e., volumes not flushed with fluids, 
where materials can accumulate. Substantially, leakproof seals to vapors 
and fluids are provided throughout the length of the reactor. These 
features minimize the accumulation of by-products or successive amino 
acids isolated from the sequenced peptide and, hence, the background which 
may interfere with the chromatogram identification of successive amino 
acids from the peptides; 
(3) have few, if any, surfaces at which amino acids or by-products can be 
entrapped and accumulated which would later leach back into the flow 
stream and generate background signals in successive isolations of amino 
acids derivatives from a peptide; 
(4) the discrete objects 20 disposed in the reaction tubes, e.g. tube 14 
can be used as a sample concentrator in a method similar to that used in 
reverse phase high performance liquid chromatography technology. The 
continuous flow reactors, e.g. the reactor 10 is compatible with high 
performance liquid chromatography technology and Edman and Stark chemistry 
technology. The discrete objects 20 in the continuous flow reactor 10 
provide for good mass transfer characteristics which are clearly superior 
to the characteristics in existing cartridge technology. 
The above discussion and related illustrations of the present invention are 
directed primarily to preferred embodiments and practices of the 
invention. However, it is believed that numerous changes and modifications 
in the actual implementation of the concepts described herein will be 
apparent to those skilled in the art, and it is contemplated that such 
changes and modifications may be made without departing from the scope of 
the invention as defined by the following claims.