Microsequence analysis of peptides and proteins

ANALYTICAL

BIOCHEMISTRY

192,

Microsequence IX. An improved, Jimmy

Calaycay,

23-31 (19%)

Analysis Compact,

Miro

Received

July

Automated

Rusnak,*

Division of Immunology, Beckman and *Biomedical Instrumentation

of Peptides

Research Seruices,

and John

Instrument E. Shively

Institute of the City City of Hope, Duarte,

Academic

of Hope, California

Duarte,

California

91010;

91010

6, 1990

We describe the construction of an improved, compact protein sequencer with a vertical flow path and continuous flow reactor (CFR). Unique features include a hexagonal valve for six fluid inputs to the CFR, which connects vertically to a transfer valve that allows sample, reagent, and solvent input to a conversion flask (CF). The simplified CF contains only two inputs at the top, one for sample, reagent, and solvent input, and the other a vent. The CF drains from the bottom, connecting to a switching valve which allows either delivery to waste or to an on-line HPLC for the analysis of phenylthiohydantoin amino acid derivatives. Approximately 90% of the sample is analyzed by use of a sonic flow detector. The overall vertical flow path of the sequencer is about 16 cm. The size of the instrument (25 w X 38 h X 44 d cm) is smaller than that of commercially available sequencers or HPLC systems. The performance of the instrument includes reduced background peaks and high-sensitivity sequence analysis at the 5-10 pmol level. The simplified sequencer is more economical and portable than conventional sequencers. 0 1SSl

and Proteins

Press,

Inc.

The sequence analysis of proteins by Edman chemistry has depended heavily on the design and performance of automated instruments. The first automated instrument described by Edman and Begg (1) in 1967 was capable of sequencing proteins at the 50-100 nmol level for 30-60 cycles, and survived well into the mid-1970s. A key feature of this and subsequent instruments was the immobilization of protein samples in a film in a spinning cup. The spinning cup instrument was mechanically improved by Wittman-Liebold (2) and also included automated conversion of ATZ’ to PTH amino 1 Abbreviations hydantoin; PITC,

used: ATZ, anilinothiazolinone; phenylisothiocyanate; PTC,

0003-2697/91$3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form

PTH, phenylthiophenylthiocarbamyl;

acid derivatives (3). These improvements, the use of Polybrene as a carrier for sequencing peptides (4,5), and the use of reversed phase HPLC to identify PTH amino acids (6,7) led to a lOO- to lOOO-fold improvement in the sensitivity of sequence analysis (8,9). The instrumentation was further improved by the introduction by Hewick et al. (10) of the coupling base (TMA) and cleavage acid (TFA) in the vapor phase, and the use of a glass fiber disk as a sample support in place of the spinning cup. The so-called “gas-phase sequencer” has become the most widely used instrument today and has a sequencing sensitivity in the range 20-100 pmol. Various versions of this design have been described (ll-13), including the introduction of a continuous flow reactor (CFR) which simplifies and miniaturizes the sample support and liquid flow pattern (14). An alternative to physically immobilizing samples was the covalent attachment of peptides to silica or polystyrene supports described by Laursen in 1971 (15). This approach was popular for peptides until the introduction of Polybrene as a physical carrier for peptides in 1978 by Tarr and co-workers (4). Since even small hydrophobic peptides could be sequenced in a Polybrene film, there was no need to perform the additional step of covalent attachment. Recently, this approach has received new attention through the introduction of a new commercial solid-phase sequencer in which proteins are covalently attached to PVDF membranes (16). In theory, the solid-phase sequencer should have lower backgrounds because of the use of more polar solvents to wash the sample and should result in better repetitive yields (longer sequencer runs) since the sample cannot

TEA, triethylamine; TMA, trimethylamine; DEA, diethylamine; DMA, dimethylamine; EA, ethylamine; DTE, dithioerythritol; DPTU, diphenylthiourea; TFA, trifluoroacetic acid; PVDF, polyvinylidenedifluoride; CFR, continuous flow reactor; CF, conversion flask; PTFE, pertrifluoroethylene; DMSO, dimethylsulfoxide; LED, light-emitting diode.

23 Inc. reserved.

24

CALAYCAY,

RUSNAK,

0 Regulator

@ Rusnak

valves

Swtchlng

valve

e z-way

valve

0 Bottle

waste FIG. 1.

Sequencer on the right.

schematic.

Individual

components

are identified

be washed out by the solvents or reagents employed in Edman chemistry. These potential advantages have not been achieved, with sensitivities and repetitive yields no better than the gas-phase sequencers employing sample immobilization in Polybrene. Common features of commercially available instruments are high cost, large size, and relatively complicated, cumbersome valving and liquid flow systems. In the past 2 years we have experimented with reducing to a minimum the size, cost, and complexity of the automated instrument. This report describes the construction of such an instrument and demonstrates improved performance in terms of the magnitude and number of background peaks. This instrument is capable of routinely sequencing samples in the range 5-20 pmol. MATERIALS

AND

METHODS

Instrument design. A schematic of the instrument is shown in Fig. 1. Solvent and reagent deliveries to the CFR and conversion flask (CF) are achieved by maintaining nominal pressures on each bottle (0.5-1.5 psi) and performing timed deliveries. Argon gas is brought into the instrument gas manifold at 20 psi and distrib-

AND

SHIVELY

uted to five Porter (Model 8311) stainless-steel regulators (letters A-E). Digital pressure readouts (DP-352, Acculex, Tauton, MA) for each of the regulators are indicated on the front panel of the instrument (two readouts can be toggled to read all five regulators). The A regulator is dedicated to the function “bubble CF” which delivers a small stream of argon to the bottom of the CF, allowing a slow drying of the ATZ sample prior to conversion. The B regulator controls pressure to Rl (PITC) which is delivered in extremely small amounts to the CFR. The C regulator supplies pressure both to R2 (TEA/water) and to R3 (TFA), the latter of which is further protected from the system by a second isolation valve. The D regulator supplies pressure to R4, S2, S3, S4, CFR blowout, and the S3 loop via a valve manifold constructed from standard Rusnak valves (11). The S3 loop delivers a measured amount of S3 to the CF via the CFR. Since S3 includes an internal standard, this ensures a constant amount per cycle. The E regulator is dedicated to the S4 loop, which delivers a measured amount of S4 to the CF and is used to empty the CF. Specialized valves are made from Kel-F and Teflon, have zero-dead volumes, and are actuated by 24Vdc solenoids which are dropped to 12 Vdc 100 ms after actuation. A cutaway sketch of the hex-valve, transfer valve, and switching is shown in Fig. 2. The standard Rusnak valve has already been described (11). The hexvalve allows the delivery of up to six solvents or reagents through a central lumen to the CFR. The other end of the central lumen is connected to a two-way valve which allows argon gas to displace liquids in the central lumen (blowout function). The transfer valve allows transfer of liquids from either the CFR or S4 and R4 to the CF. It also allows transfer of solvent washes (S2) from the CFR to waste. This valve greatly simplifies the plumbing associated with the CF and allows a vertical placement of the entire liquid delivery system from the hexvalve to the final switching valve which conveys the PTH sample to the auto-injector. The CF was constructed from +-in. Kel-F rod (Fluorocarbon, Anaheim, CA) and externally machined on one end with $16 threads to accept a Teflon cap machined from $-in. bar stock. The CF has a +-in. o.d., has a +-in. i.d., is 1.0 in. long, and is internally machined at the other end with a 30” conical taper and a 0.06-in. exit hole. The exit hole is slightly under & in. to allow the insertion of &-in.-o.d. Teflon tubing for a “pull through” fitting (11). The interior of the CF was polished to allow proper draining of liquids. The CF is lighted from behind with a high-intensity red LED in order to inspect sample delivery and drying steps. The CF cap was internally machined from t-in. Teflon rod with $16 threads. The cap is + in. long and has two 0.06-in. holes to accept two &-in. Teflon lines, one from the transfer valve, and the other a vent line (see Fig. 1).

MICROSEQUENCE

PEPTIDE

wnml lumm (to blawut I am of 01~ inputs

Transfer

val

mlltml CFR

lumen

from

.to waata

to

caltral hlmen - CFandthrw inputs hat shown)

Switching

valve

FIG. 2. Specialized valves. The hex-valve has six inputs and a central lumen, the top connected to a two-way valve for blowout, and the bottom connected to the CFR. The transfer valve has one port with two outlets for either delivery to waste or to the CF and three ports for input of R4 and S4 and for overflow of the S4 loop to waste. The switching valve allows one input to be diverted to one of two outputs.

The CF is heated by a split, hinged aluminum block which allows easy access to the CF assembly. Teflon tubing was used for all plumbing except connections to the CFR from the hex and transfer valves, which used &-in.-o.d., 0.4-mm-i.d. Tefzel tubing. All pressurize and delivery lines were $-in.-o.d., 0.5mmi.d. PTFE tubing (Zeus Industrial products, Raritan, NJ), with the exceptions of the R4 delivery line, which was 0.3-mm id., and the Rl delivery line, which was 0.2-mm i.d. Tubing connections to the valves were made via pull through fittings to machined Kel-F inserts placed in standard polypropylene $28 fittings from Rainin (Woburn, MA). Reagent and solvent bottles were amber 2-0~ (50 ml) or 4-0~ (100 ml) bottles obtained from Scientific Products/Baxter (P/N B7465-18 or -19) with Teflon-lined caps. The bottles were inserted into machined Kel-F holders and sealed with either Kal-Rez gaskets or poly-

AND

PROTEIN

ANALYSIS

25

propylene bottle seals (P/N 400612, Applied Biosysterns, Foster City, CA). A Rheodyne (Cotati, CA) Model 7126 injector was used to transfer the PTH sample to the HPLC. Liquid exiting the Teflon sample loop is detected by an ultrasonic detector (Series 900, Introtek, Deer Park, NJ) which transmits a signal to the computer/controller and triggers an injection. In this manner up to 90% of the sample can be injected in a highly reproducible manner. The Rheodyne injector is actuated with pneumatic solenoids connected to the argon gas supply. The instrument is divided vertically by a steel bulkhead into electronics and chemistry sections. The electronics section contains the power supply, system controller, and temperature and pressure readouts. The chemistry side includes the gas regulators, delivery valves, and associated plumbing. Both sides have exhaust fans, of which the chemistry side is vented to the outside of the building. The electronic side of the front panel has digital readouts for the temperature of the CFR and CF, and the pressure on each of the five regulators. Photographs of the instrument are shown in Figs. 3 and 4. Figure 3 shows the overall layout and size of the instrument, and Fig. 4 is a detailed view of the vertical arrangement of the sample delivery valves. An Lshaped translucent door provides access to the delivery valve assembly from the front and side. Access to reagents and solvents is on the side of the instrument by removal of a metal panel. The top of the instrument containing the bottle assembly holders and the hex-valve is accessed through a hinged cover. The overall dimensions are 25 (width) X 38 (height) X 44 (depth) cm. Sequencer and HPLC controllers. The sequencer system computer/controller is an IBM XT-compatible computer (Little Board/PC, Ampro Computers, Mountain View, CA) equipped with a 3.5-in. disk drive and a monochrome/EGA controller. The software (available upon request) is menu driven and allows facile entry and control of sequencer functions. The HPLC is a Beckman System Gold (Model 126 pumps, Model 167 detector) plumbed for narrow-bore HPLC use. HPLC control and data analysis are accomplished through the Beckman System Gold chromatography software and an IBM PS2-70 computer equipped with a 30-MByte hard drive. Programming of the sequencer can be performed on the IBM computer and then downloaded to the internal computer in the sequencer. In theory, a separate window in the HPLC system software can be used to monitor the activities of the sequencer while it is in operation; but in practice, we have dedicated a separate monitor to the sequencer. This situation may be improved as more software updates become available from Beckman and IBM (multitasking/OS2). A typical program for the order and timing of the delivery of reagents

26

CALAYCAY,

FIG.

3.

Photograph

of instrument.

Not

shown

RUSNAK,

AND

are the monitor,

and solvents is shown in Table 1. The overall time is 50 min and was made to coincide with the time taken to analyze the PTH amino acid derivatives and recycle the HPLC column to initial conditions. PTH amino acid derivatives are separated on either a Beckman Ultrasphere 5-pm ODS column (250 X 2.0 mm i.d.) or an Applied Biosystems 5-pm PTH-222 Cl8 column (220 X 2.1 mm i.d.). Two HPLC solvent systems were evaluated. The first employs ammonium acetate and a methanol/acetonitrile gradient and has been previously described by us (14). The second has been described by Fischer and Myerson (17) and employs TEA/acetate and an isopropanol/acetonitrile solvent system. PTH amino acids are detected at 269 nm on a Beckman 167 detector. PTH amino acid standards were obtained from Pierce (Rockford, IL). Reagents and soluents. The composition and usage per cycle of the reagents and solvents are shown in Ta-

SHIVELY

on-line

HPLC,

and computer

for data

analysis.

ble 2. PITC, heptane, and TEA were sequencer grade from Pierce (Rockford, IL). Trifluoroacetic acid (sequencer grade) was obtained from Aldrich (Milwaukee, WI). Acetonitrile and methanol (Omnisolv grade) were obtained from EM Science (Gibbstown, NJ). Ethyl acetate and butyl chloride were obtained from Burdick and Jackson (Muskegon, MI). The ethyl acetate was redistilled and purified as previously described (11). Water was purified on a Mill;& system. Reagents and solvents were sparged with argon (5 ml/min) for 10 min before connection to the instrument. R2 is changed after every 100 cycles or recharged by adding 2 ml of neat TEA to 100 ml of spent reagent. Sample loading. The hex-valve is mounted on a hinged steel plate which, when lifted, pulls the Tefzel tubing away from the CFR, allowing access to the PVDF strip containing the sample. A diagram with the dimensions of the CFR is shown in Fig. 5. PVDF (Immobilon

MICROSEQUENCE

PEPTIDE

AND

PROTEIN

27

ANALYSIS

ferred (3.0-mm o.d., 1.5-mm i.d., P/N 6406-42, Cole Parmer, Chicago, IL.). The PFA tubing should be slightly under &-in. (1.59-mm) i.d. to ensure a tight seal to the Tefzel tubing. Tefzel tubing is preferred because of its stiffness. The CFR is lighted from the back with a high-intensity red LED to facilitate timing of reagent and solvent deliveries. The CFR is heated by a split, hinged aluminum block similar to the CF assembly. The heater assembly snaps closed to tightly enclose the CFR during operation. RESULTS The instrument design (Fig. 1) incorporates a short, vertical flow path for the introduction of reagents and solvents via a hexagonal valve (Fig. 2) to a continuous

TABLE

1

Sequencer Program Summary Flow reactor

Conversion flask

Dry

FIG. 4. Detail of instrument. Shown are the CFR and CF and their heaters, the transfer valve connecting the two, and the switching valve at the bottom which transfers the sample to the on-line HPLC.

P, 20-pm thickness, pore size 0.45 pm) was obtained from Millipore (Bedford, MA). A fresh PVDF strip (10 X 1 mm, folded once to a total length of 5 mm) is spotted with 2-4 ~1 of 50 mg/ml of Polybrene (Aldrich) in water/ methanol (50/50, v/v) and allowed to air-dry (1-2 min), followed by a similar application procedure for the sample. Large pieces of PVDF can be batch treated with Polybrene if desired. For sample application, the PVDF strip can be held with a hemostat and the spotting process repeated as necessary, depending on the sample volume. Preferred sample application solvents are water or aqueous TFA/acetonitrile, methanol, or isopropanol, or mixtures of these with DMSO or hexafluoroacetone trihydrate. Once a sample is spotted it can be stored indefinitely before sequence analysis (store in a polypropylene tube at 0 or -20°C). The PVDF strip is inserted into the CFR tubing with forceps. The CFR is inserted onto the Tefzel tubing connected to the transfer valve, and the hex-valve-Tefzel tubing assembly lowered onto the top of the CFR to complete the seal to the CFR. In order to monitor liquid deliveries to the CFR, transparent FEP tubing is pre-

Deliver Deliver Deliver Deliver Deliver Deliver Pause Deliver Pause Deliver Pause Deliver Pause Pause Pause Pause DV Pause Deliver Deliver Deliver Deliver Deliver Deliver Pause Deliver DW Pause Deliver Pause Deliver Pause Pause Pause Deliver Deliver Deliver a The blowout.

R2 Rl R2 Rl R2 S2 S2 S2 S3

R3 R3 R3 R3 R3 R3 R3

S3 S3

R3 S2 S3

Duration”

Convert Convert Convert Convert Convert Dry Dry Dry Dry Dry Dry Dry Dry Dry Deliver S4 Bubble CF Transfer to injector Pause Clear S4 line Deliver S4 Bubble CF Blow out S4 to waste Deliver R4 Bubble CF Blow out R4 to waste Pause Pause Pause Deliver R4 Pause Dry CF Pause Dry CF Deliver R4 Bubble CF Convert Convert Convert

first time All times

is pressurize, in seconds.

Total

175 240 281 591 632 962 1009 1029 1076 1096 1143 1163 1213 1233 1256 1266 1366 1426 1466 1479 1489 1529 1544 1554 1704 1734 2009 2099 2111 2171 2271 2331 2431 2443 2446 2643 2725 2665

0, 1750 5, 30, 30 5, 1, 35 5, 225, 80 5, 1, 35 5,225, 100 5, 2, 40 0, 20, 0 5, 2, 40 0, 20,o 5, 2, 40 0, 20, 0 5, 5, 40 0, 20, 0 5, 8, 10 0, 0, 10 0, 0, 100 0, 0, 60 0, 0, 40 59% 0 0, 10, 0 0, 40, 0 595, 5 0, 0, 10 0, 150, 0 0,30, 0 0, 275, 0 0, 90, 0 5, 2, 5 5, 5, 50 0, 100, 0 5, 5, 50 0, 100, 0 5, 2, 5 60, 5 5, 100, 90 5, 2, 75 5, 5, 150

the second

delivery,

time

and

the third

28

CALAYCAY, TABLE Composition

AND

SHIVELY 100

2

-

and Usage of Reagents and Solvents” Reagent/solvent

Rl R2 R3 R4 Sl s2 s3 s4

RUSNAK,

Usage

per cycle

2d

5% PITC in heptane 2% TEA in water TFA TFA/water/MeOH (1.5/28.5/70) (Spare, not used) MeCN/Ethyl acetate (15/85) MeCN/Butyl chloride (15185) Water/MeCN (10190)

gas gas 60 /.d 4 x 45 crl 4 x 60 /d 140 al l~,.,.,.,.,.,.,.,...,.,.,.,.~...~.,

a R3, S2, S3, and S4 contain a spare.

250 pg DTE/lOO

ml. The

Sl position

LI”TOTMKGLD,OK”AG

is

Cycle

FIG. 6.

flow reactor (Fig. 5). ATZ amino acid derivatives formed in the CFR are delivered to a conversion flask via a transfer valve which also allows the input of several reagents and solvents to the CF. The position of the transfer valve and its connection to the CF allow a vertical flow configuration and simplify the design of the CF. The CF has a single input line from the transfer valve, a vent line to release pressure and allow drying steps, and a drain line at the bottom. The drain line is also used to dry the CF with a stream of argon. The drain/dry line connects vertically to a switching valve which transfers the PTH amino acid derivatives to an autoinjector connected on-line to an HPLC. The use of a liquid flow detector allows the injection of 90% of the sample to the HPLC. Precise deliveries of S3 (butyl chloride containing the ATZ derivative) from the CFR to the CF are controlled by the use of a loop on a switching valve (Fig. 1). The loop is overfilled flowing to waste (Fig. 1, S3 overflow),

/l/16

FIG. 5. Continuous shown to the right.

flow

reactor.

in

The

OD

Teflon

dimensions

Tubing

of the CFR

are

Sequence analysis of fl-lactoglobulin. 90, 37, and 6 pmol of b-lactoglobulin analyzed.

Yields

are shown

for

and then diverted to the CFR. A similar strategy is employed for the precise delivery of S4 (methanol/water containing the PTH derivative) to the CF. This strategy greatly simplifies the setting of times and pressures for critical deliveries, and helps to prevent cycle-to-cycle variation in performance. Background noise is lowered by deliveries of extremely small amounts of PITC (2 pl), minimization of tubing and valve runs, and a vertical flow arrangement. The low delivery of PITC is achieved by pressurizing the Rl bottle to 0.2 psi and delivery of PITC for 1 s without further pressurization. The Rl delivery line from the bottle to the hex-valve is restricted by the use of 0.2mm-i.d. Teflon tubing. Blowout of Rl to the CFR results in a small drop of Rl touching the PVDF strip. After a few seconds the drop wets the entire PVDF strip and the heptane evaporates, leaving a thin film of PITC coating the sample. The delivery of 2 ~1 of 5% PITC (419 mM) corresponds to 837 nmol, a vast excess over samples usually present in picomole amounts. The performance of the sequencer was evaluated by the sequence analysis of several proteins and peptides. Sequence analysis of the protein standard P-lactoglobulin A at the 90,37, 10, and 6 pmol level was performed. The initial yields varied from 41 to 50%, and the repetitive yields from 97 to 98%. Plots of the yields of the expected amino acids at each cycle are shown for the 90, 37, and 6 pmol runs in Fig. 6. Traces of the original chromatograms (cycles l-18) are shown for the 10 pmol run in Fig. 7. At this level the PTH-Thr peaks are barely detectable at cycle 18, and the PTH-Trp at cycle 19 and PTH-Ser at cycle 21 (not shown) were not detected. These cycles were positively identified at the 37 and 90 pmol level. Two major background peaks are observed: DTE eluting early in the chromatogram, and DPTU eluting between PTH-Val and PTH-Trp. The size of the DTE peak depends on the amounts added to the

MICROSEQUENCE

Time

PEPTIDE

AND

PROTEIN

29

ANALYSIS

(min)

FIG. 7. Chromatograms for the sequence lin. Ten picomoler of sample was analyzed. shown.

analysis of P-lactoblobuThe first 18 cycles are

solvents and varies from 5 to 20 pmol/cycle. The size of the DPTU peak is lo-20 pmol on cycle 1 and decreases to about 5 pmol by cycle 5. Neither DTE nor DPTU coelutes with authentic PTH amino acids. Minor background peaks observed were an unidentified peak (0.51.0 pmol/cycle) coeluting with PTH-Gly, the PTC derivative of ethylamine (0.5-1.0 pmol/cycle, except cycle 1 where it is 2-3 pmol) coeluting with PTH-Ala, the PTC derivative of diethylamine (0.5-1.0 pmol/cycle), and diphenylurea (1.0-2.0 pmol/cycle). The latter two peaks do not coelute with authentic PTH amino acids. All other background peaks are below the 0.5 pmol level. Sequence results for a second protein sequencing standard, horse apomyoglobin at the 20 pmol level, are shown in Figs. 8 and 9. The initial and repetitive yields were 60 and 92%, respectively. PTH-Ser at cycle 3, and PTH-Trp at cycles 7 and 14 were easily identified. A different chromatography solvent system was used in

II

FIG. 9. Chromatograms for the sequence analysis myoglobin. Twenty picomoles of sample was analyzed. cycles are shown.

of horse apoThe first 16

this run, resulting in the closer elution of PTH-Val to DPTU, however, in this run the size of the DPTU peak is about l-3 pmol. Sequence results for a small synthetic peptide (16 residues long) at the 10 pmol level are shown in Figs. 10 and 11. This peptide was designed to include all of the difficult amino acids (Ser, His, Arg, and Trp), and terminates in a Lys to simulate a typical tryptic peptide. At this level, only 300-500 fmol of PTH-His, -Arg, and -Trp are recovered. The repetitive yield was 87% with an initial yield of 50% for PTH-Ala (corrected for the PTC-EA background at cycle 1) and the final yield was 10% for PTH-Lys at cycle 16. The overall backgrounds for a small peptide are better than those observed for proteins (see Figs. 7 and 9). A second small peptide with the sequence NH,-Phe-Asn-Gly-Leu-Arg-Gly-CONH, was sequenced at the 5 pmol level with an initial yield of 60% and with identification of each residue through the

1 !,.,.,...,...........,.,.,.,.,..I

GLSDGEWOOVLNVWGK"EAD,A

ALFHGRVSWAMFPNGK

Cycle

FIG. 8. Sequence analysis of horse shown for 20 pmol of horse apomyoglobin

Cycle

apomyoglobin. analyzed.

Yields

are

FIG. 10. Sequence analysis moles of sample was analyzed.

of a small

synthetic

peptide.

Ten pico-

30

FIG. 11.

Chromatograms thetic peptide. Ten picomoles 16 amino acids long.

CALAYCAY,

for the sequence analysis of sample was analyzed.

RUSNAK,

of a small synThe peptide is

fifth cycle (data not shown). The C-terminal CONH, residue was not identified.

Gly-

DISCUSSION

The current generation of gas-phase protein sequencers are large and expensive and have not been significantly changed from the instrument introduced by Hewick et al. in 1981 (10). In this report we describe a more compact, economical sequencer with a new liquid delivery system comprising valves, a continuous flow reactor, and a conversion flask arranged in a vertical flow path of about 16 cm. The sequencer has exceptionally low levels of background peaks, including DPTU, and can routinely sequence samples at the 5-20 pmol level. Several aspects of the simplified design are discussed here. The hex-valve allows the introduction of up to six reagents and solvents into a central lumen directly connected to the continuous flow reactor. The distance from the bottom of the hex-valve to the center of the CFR is about 1 cm. This design feature reduces the amount of liquids needed in sequencing and the potential for crosscontamination to a minimum. Similarly, the liquid flow from the CFR to the conversion flask is reduced to a minimum. The distance from the center of the CFR to the top of the CF is only 10 cm, 5 cm of which comprise the transfer valve which allows the introduction of solvents and reagents into the CF. The novel design of these valves includes construction from inert Kel-F and Teflon, zero-dead volume, and a vertical flow arrangement, allowing liquids to drain by gravity. The transfer valve has permitted the construction of a simplified conversion flask with only two inlets at the top (liquids in and vent) and a drain in the bottom which allows drying of the sample from the bottom (during conversion) and delivery to the autoinjector of the

AND

SHIVELY

HPLC. Injection of 90% of the sample to the HPLC is achieved with a bubble/liquid sonic detector. The program incorporates a wait step for the signal from the sonic detector. In the case of a fault (no signal), the program can either continue or halt. The chemistry and the program have been optimized for sequencing on PVDF supports in a CFR. In this respect, several differences from the Applied Biosystems Instrument are evident. Our coupling base is aqueous TEA versus TMA used in the ABI instrument. TEA is a stronger base and is less soluble in water than TMA. Further advantages of TEA include its less objectionable odor (it is practically odorless in water) and the need for smaller amounts in sequencing (2% solution versus 12.5% for TMA). Both bases break down, giving peaks corresponding to the PTC derivatives of DEA and DMA, but these peaks do not usually interfere in the identification of the PTH amino acid derivatives. A second chemistry change is the addition of 15% acetonitrile in the ethyl acetate of S2 and the butyl chloride of S3 (Table 2). In both cases the addition of acetonitrile to the solvents improves extraction of impurities or ATZ derivatives from the hydrophobic PVDF membrane. This is especially important for the extraction of the ATZ derivative of aspartic acid, which is otherwise obtained in very low yields from PVDF membranes. The use of a CFR for sequence analysis has a number of advantages. Solvent and reagent flow continues directly through the Teflon tubing with no obstructions, interruptions, or expansions. The entire CFR is flooded during rinses and requires no special gaskets or sealants. The seals are made by inserting one perfluoroelastamer tubing into another. The amount of substrate (in this case PVDF) is reduced to an absolute minimum, thus minimizing background peaks caused by retained chemicals. The CFR is ideal for sequencing on small l-mm-wide PVDF strips, which are preferred in electrotransfer experiments (18). Small PVDF strips are also ideal for experiments involving small sample loads (l-4 ~1). In the near future we predict that samples will be loaded directly from micro-LC runs (l-5 ~1) or capillary electrophoresis runs (lo-50 nl). The sequencing levels achieved in this work depended heavily on the performance of the narrow-bore (2.1-mm-i.d. column) HPLC system. While the background levels and repetitive yields observed encouraged us to sequence less than 10 pmol of sample, it must be admitted that several of the PTH amino acids (Ser, His, Arg, Trp, and sometimes Thr) cannot be detected well beyond 10 cycles. It is clear that further improvements in the detection levels of all the PTH amino acids are in order before femtomole level sequencing can be achieved. Several solutions are possible, with the use of fluorescent derivatives possibly the most attractive (19). Perhaps the most important conclusion from this

MICROSEQUENCE

PEPTIDE

work is that sequencers need not be large, complex, expensive instruments. The currently prohibitive cost of microsequencers has usually limited these instruments to Core labs, thus discouraging investigators from further improvements in Edman chemistry or experiments with new approaches for sequence analysis. It is our belief that the cost of these instruments can be maintained in the same range as HPLC units, thus making them available to the everyday protein chemistry lab. ACKNOWLEDGMENTS This research was supported by the Beckman Research and partially by a grant from the TOSOH Corp. We thank Paxton and Michael Ronk for their helpful suggestions.

Institute Dr. Ray

REFERENCES 1. Edman,

PROTEIN

G. (1967)

Eur.

J. Biochem.

1,80-91.

D. G., Wilde, 85, 126-131.

C. E., III,

and Capra,

J. D. (1978)

31

ANALYSIS

6. Zimmerman,

C. L., Apella, 77,569-573.

them.

E., and Pisano,

J. J. (1977)

And.

Bio-

7. Hawke, D., Yuan, P.-M., and Shively, J. E. (1982) Anal. Biochem. 120,302-311. 8. Hunkapiller, M. W., and Hood, L. E. (1978) Biochemistry 17, 2124-2133.

9. Shively, Vol.

J. E. (1981) in Methods in Enzymology 79, pp. 31-38, Academic Press, San Diego,

(Pestka, CA.

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