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Microsequence analysis of peptides and proteins
ANALYTICAL
BIOCHEMISTRY
147, 3 15-330 (1985)
Microsequence
Analysis of Peptides and Proteins
V. Design and Performance of a Novel Gas-Liquid-Solid DAVID
H. HAWKE,
DAVID
C. HARRIS,
Phase Instrument
AND JOHN E. SHIVELY
Division of Immunology, Beckman Research Institute of the City of Hope, Duarte. California 91010 Received October 3 1, I984 We describe the construction and performance of a novel, automated, Edman chemisttybased microsequencer. The reagent and solvent delivery system, the reaction cartridge for coupling and cleavage, and the conversion flask are all constructed from chemically inert perfluoroelastomers. The delivery valves are of a new design incorporating the use of electromagnetically actuated solenoids and zero-dead-volume construction, and may be connected in a modular fashion resulting in multiple inputs with a single output line which can be flushed with inert gas. The bottle closures are of a new design based on an all-Teflon compression fitting. The reaction cartridge and conversion flask are thermostated by solid-state heaters in an aluminum block. The overall size of the instrument is 25 X 34 X 14 in. The chemistry utilizes 2% aqueous triethylamine as the coupling base which is delivered to the reaction cartridge via a stream of nitrogen. The ‘gas-phase” delivery of the coupling base and the cleavage acid (trifluoroacetic acid) is modeled after the method described by R. M. Hewick et al. (J. Biol. Chem. 256, 7990-7997, 1981). The instrument has performed well over a period of 3 years in terms of low background peaks, sensitivity in the picomole range, and reliability of operation. The use of economical components, ease of construction and operation, and sensitive analytical capability make this instrument a useful tool for microsequence analysis of peptides and proteins. Q 1985 Academic Press, IIIC.
Improvements in the techniques and automation of Edman chemistry have led to the routine sequence analysis of subnanomole levels of peptides and proteins. Since the introduction of the first spinning cup sequenator in 1967 (I), improvements in the commercially available instruments (mainly the Beckman 890 series) were described by Wittman-Liebold and co-workers (2,3), Hunkapiller and Hood (4), and us (5). The so-called “modified” spinning cup instruments were able to sequence subnanomole amounts of proteins, but were not generally available. The contribution to their success of other factors was hotly debated, and eventually it was clear that modestly improved spinning cup instruments were also capable of increased sensitivity ((6) the Beckman 890M sequenator). The other factors include automated conversion of anilinothiozolinone 315
(ATZ)’ to PTH derivatives (3) the use of polybrene as a carrier for peptides and small amounts of proteins (7,8), rigorous purification of reagents and solvents (2,4,5,9), and the high sensitivity analysis of PTH amino acid derivatives by reverse-phase HPLC. The development of solid-phase Edman chemistry by Laursen ( 10) involved covalent attachment of peptides or proteins to solid i Abbreviations used: ATZ, anilinothiazolinone; PTH, phenylthiohydantoin; PITC, phenylisothiocyanate; SPITC, 3-sulfophenylisothioyanate; PTC, phenylthiocarbamyl derivative; TEA, triethylamine; DEA, diethylamine; TMA, trimethylamine; DMA, dimethylamine; DPTU, diphenylthiourea; DPU, diphenylurea; PFA, perfluoroalkoxy-Teflon; TFA, trifluoroacetic acid; DTT, dithiothreitol; MeCN, acetonitrile; DTE, dithioerythritol; AUFS, absorbance units full scale; VIA, versatile interface adapter; RAM, random access memory: LED, light emitting diode. 0003-2697/85 $3.00 Copyright 0 1985 by Academic Press. Inc. All rights of reproductmn rn any form resewed.
316
HAWKE,
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supports, but was usually limited to tens of nanomoles of small peptides. Recent improvements have extended the range to OS1.O nmol of small peptides (11). The noncovalent entrapment of peptides and proteins in a Polybrene film (78) is an alternative to covalent attachment and was adapted from the spinning cup instrument to a cartridge type of instrument described by Hewick et al. (12). Other changes in this instrument included delivery of the coupling base and cleavage reagent as gases (or more accurately as vapor swept through by a carrier gas) and miniaturization of valves and the delivery system and therefore a lower consumption of solvents. This new instrument is commercially available and has performed well in a number of laboratories ( 13- 17). The concept of delivering reagents as gases is not new (18-20), but was first successfully adapted to an automated instrument by Hewick et al. (12). In spite of the obvious simplification of instrumentation for a gas-liquid-solid phase instrument compared to a spinning cup instrument, the commercial instrument is costly and utilizes many difficult-to-machine components, including the miniaturized Wittman-Liebold valves, the Wittman-Liebold converter (as originally described), the allglass reaction cartridge, and the custom fraction collector. Our experience with building modified spinning cup instruments prompted us to develop a gas-liquid-solid phase instrument based on the prototype machine of Hewick et al. (12). This work led to the design of new economical zero-dead-volume valves, an all perfluoroelastomer delivery and reaction system, a compression type of closure for reagent and solvent bottles, and computercontrol by an inexpensive Apple II plus computer. Our instrument is less expensive and more compact, utilizes a different coupling base, has a silylated glass disk impregnated with Polybrene for a solid phase, and has a shorter cycle time than the commercially available instrument. This report de-
scribes its construction and the performance of the prototype over the past 3 years. MATERIALS
Instrument
AND METHODS
Design
A schematic of the &rstrument is shown in Fig. 1. The overall schematic differs from that of Hewick et al. (12) in the following respects. The reaction cartridge (Fig. 2) is made of Teflon (turned from 0.50 in bar stock), and utilizes either a Kal-Rez gasket (0.020 in.) or no gasket (the Teflon is selfsealing). The Teflon lines are sealed in the chamber by a “pull-through” fit.2 The glass filter (1 cm diameter) rests on a Teflon screen (mesh opening 0.004 in., Savallex, Minnetonka, Minn.). The reaction chamber is in an aluminum holder thermostated by an Omega CN300 controller (Omega Engineering, Stamford, Conn.). The conversion flask is a 6-ml conical PFA-Teflon vial (Savillex) with a self-sealing closure. The Teflon lines are sealed with “pull-through” fittings. The conversion flask (not shown) is heated by a separate solid-state system. The inert, zerodead-volume valves (Fig. 3) control the flow of reagents and solvents into the cartridge and conversion flask. The output of the cartridge and the conversion flask are controlled by a Teflon three-to-one miniature valve (Model 330TFE, Brunswick Technetics, Cedar Knolls, N. J.) with up to three input lines and a common output line. The reagent bottles (Wheaton, Millville, N. J.) are constructed from 200-ml conical bottles to which are attached 0.7%in. precision-bore glass tubing. The modified swage ’ A “pull through” fit is made by drilling a hole with an i.d. slightly less than the o.d. of the tubing to be drawn through the desired fitting (both fitting and tubing are made of Teflon). The tubing is heated to softness with a heat gun and drawn to a taper in order to insert it into the hole. Once inserted, it is drawn through with a needlenose pliers and trimmed to the appropriate length. None of the heated, tapered line should remain since it may have unwanted properties.
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FIG. 1. Schematic of gas-liquid-solid phase microsequencer. Identification of components is shown to the right of the schematic. The vacuum system is shown in dotted lines because it is optional.
fitting (Fig. 4) can be finger tightened and holds up to 20 psi with no gas or liquid leaks. The tubing is inserted through a “pullthrough” fitting.2 The solvent bottles are drawn from 90-mm Pyrex tubing, made conical at the bottom and fit to 0.75in. tubing at the top, and have a capacity of 700 ml. The reagent and solvent bottles have no vent lines, eliminating the maintenance problems associated with vent solenoids. Bottle pressures are set between 0.5 and 3.0 psi with precision 4-in. regulators (Watts Fluid Air, Inc., Kittery, Mass.). The fraction collector is a Gilson slave unit (Gilson Medical Electronics, Middleton, Wise.), externally triggered by a relay closure ( 100-200 ms). The closure time-out was per-
formed by either a hardware or a software clock. Polypropylene tubes (13 X 100 mm, Sarstedt, Haywood, Calif.) are used in the fraction collector. Smaller conical polypropylene tubes (1.4 ml, Sarstedt) can be used as inserts if smaller volumes are desired. The entire assembly is housed in a Plexiglas housing flushed with argon at a flow rate of 20 ml/min. The prototype was run with a vacuum system until May 1984. The optional vacuum system is similar to that described by Hewick et al. (12). Two bench-top models have been run in this laboratory without a vacuum system. The control system consists of an Apple II plus computer with 48K RAM and a 5.25
318
HAWKE,
HARRIS, AND SHIVELY
Reagents and Solvents The composition and usage per cycle of the reagents and solvents are shown in Table 1. A description of the purification of some of the reagents has been previously published (9). The reagents and solvents are sparged with argon (at least 10 min at a rate of 5 ml/ min) prior to connection to the system (if DTT or DTE is added, sparge before and after the addition; DTT or DTE should be used exclusively to avoid complications inherent in having both present at the same time). The effect of sparging the solvent is to reduce the degree of destruction of PTH amino acids (see Ref. (9)). PITC (Aldrich, Milwaukee, Wise.) is twice distilled in vacua
FIG. 2. Reaction chamber. (A) Knurled aluminum nut for applying pressure to seal Teflon body (B). (C) Interface between two halves of Teflon body (B) may be self-sealing or used with a Kal-Rez gasket. (D) Slot for thermometer. (E) Heater.
in. disk drive. A 32-port parallel, 6522-based VIA (John Bell, San Carlos, Calif.) plugs into slot 7 of the Apple and triggers the appropriate drivers for 24 VDC solenoids. The front panel (Fig. 5) includes switches for manual control and temperature readout. Red-green LEDs indicate whether a given function is manually (red) or automatically (green) actuated. The software is menu oriented and user friendly, and allows for using either stored running programs or creating new running programs. The operating system is a compiled version of BASIC, but can also be edited to add new features. The running programs and operating system occupy only 20K of memory and are stored on a floppy disk for retrieval if necessary. Connection of the computer to a line printer is optional. The 1984 cost of the instrument including hardware, software, and labor (at standard rates) is $15,000.
FIG. 3. Zero-dead-volume valve. The main block is made from Teflon or Kel-F and has two inlet ports and a continuous outlet with two fittings. The inlets and outlets are threaded for standard i-28 flare fittings. The piston and diaphragm are made from one piece of Teflon with the lower portion shown in the closed position and the upper portion shown in the open position. The solenoid coil and housing are shown removed in the upper portion. The flow of liquid into the inlet and out of the outlet is indicated by arrows. It is assumed that the hidden view of the outlet is connected to gas pressure controlled by a two-way valve which when activated expels liquid in the central lumen in the direction of the arrow.
A NOVEL
4
%COom
MICROSEQUENCER
+
FIG. 4. Bottle closure for reagents and solvents. (A) A one-piece Teflon shroud machined to fit over the cylindrical neck of the bottle (C). The shroud has two holes drilled for accepting Teflon pressurizing and delivery lines, sealed by pulled-through fittings. Stainless-steel nuts (D) and (E) tighten the compression fitting to the bottle against a standard ferrule (B). (D) and (E) are male and female nuts on a 0.75in-tube cap fitting. The bottle neck (C) is precision 0.75~in. tubing.
and stored at -20°C in sealed glass ampoules (5-10 ml). Heptane (Baker or Burdick and Jackson, HPLC grade) is distilled from CaH2 under N2 and stored in 50-ml sealed-glass ampoules. Triethylamine is either obtained from Pierce Chemical Company (Sequenal grade; Rockford, Ill.), or purchased from Aldrich and distilled at reduced pressure first from CaHz and then from tosyl chloride. Trifluoroacetic acid (Sigma or Aldrich) is distilled first from Cr@ and then from A&O3 under nitrogen. Ethyl acetate (Baker, HPLC grade) is purified as previously described (9) briefly charcoal treated, and distilled first from ninhydrin and then from a mixture of SPITC, DTT, and Quadrol. 1-Propanol (Baker, HPLC grade) is distilled from CaH2. Instrument Operation Sample loading. The cartridge is disassembled and a Whatman GF/C glass-fiber disk (1 cm) which has been trimethylsilylated is
319
dropped onto the Teflon screen. The silylation treatment is as follows: soak disk in HN03 (coned AR) for 3-l 2 h; rinse with water, methanol, and THF; treat with 10% trimethylchlorosilane (Regis, Morton Grove, Ill.) in THF (v/v) for 2-3 h at room temperature; rinse with THF and methanol; and air dry and store in a sealed bottle at 4°C. The disk is prewetted with MeCN or ethanol (5 ~1) to prevent beading, aqueous Polybrene ( 100 mg/ml) is spotted (20 pl), and the disk is cycled for 2-3 program cycles. The sample is repeatedly spotted on the precycled disk (20 ~1 per application) and dried. Drying is accomplished by delivering N2 with the cartridge assembly slightly opened (3-5 min per dry cycle). Closing the cartridge during sample drying may inadvertently lead to loss of sample. Following analysis of a protein it is recommended that the disk be disposed of unless background peaks are at an acceptable level after a run. Following peptide analysis, the disk can be reused. More polybrene is spotted on disks that have undergone 75100 cycles. Glycylglycine is omitted from the polybrene in order to reduce background and to speed precycling of the Polybrene film. If performance is poor, Polybrene can be spotted on the filter with the sample. ln some cases, we have used one disk for 400 cycles, at which point some discoloration is apparent. Program. The general features and timing of the program are described in Table 2. One complete cycle comprises a single coupling of 6 min, a double cleavage of 6 min and 7 min, and conversion of ATZ to PTH derivatives for 22 min. Coupling is performed at 49-52°C and conversion at 52-57°C. The program begins first with a short predelivery of base and then of R 1, followed by a longer delivery of base. Analysis of PTH derivatives. The system in use has been previously described by us (21). Briefly, we use an Altex Ultrasphere C18 column (4.5 X 250 mm), with solvent A composed of 16 mM TFA containing 4.5 mM HAc, titrated to pH 5.8 with 5 N NaOH and adjusted to 5% MeCN (v/v), and solvent
320
HAWKE,
HARRIS,
AND SHIVELY
FIG. 5. Front view of gas-liquid-solid phase microsequencer. The instrument contains the computer and associated electronics on the right half and the plumbing and associated delivery valves on the left half. The delivery valves are located directly behind the reaction chamber (left) and conversion flask (right). The fraction collector (not shown) is housed in an inert atmosphere. The approximate measurements are 25 X 34 X 14 in.
B composed of 15 mM TFA, titrated to pH 3.4 and adjusted to 75% MeCN (v/v); a linear gradient is run from 5% B to 100% B over 10 min with a flow rate of 1.2 ml/min and a temperature of 50°C. A typical separation is shown in Fig. 6. This system gives better baseline, resolution, and peak definition for PTH derivatives than does the CN column with acetate buffer previously described ( 12,13) and performance approximately equal to the chromatogram shown in Fig. 1 of a recent review by Hunkapiller and Hood (22). However, the CN column separation shown in Fig. 2 of Ref. (22) shows a number of poorly resolved pairs (ST; H,D; Y,V; P,M; broad R) of PTH derivatives. It appears that the CN columns are highly variable and must be selected for routine optimal performance. The Altex Ultrasphere C- 18 columns using our solvent system have been more reliable in our hands for routine analysis (i.e., the columns are not hand selected). Peaks are integrated by a Spectra Physics 4000 integra-
tion system and converted to picomoles using an average KF value for PTH amino acids. Samples are delivered to the fraction collector in a volume of about 3 ml, dried at room temperature on a Savant Speed Vat (Hicksville, N. Y.), and redissolved in 40 ~1 of solvent (MeCN/H20/TFA; 75/25/O. 1, v/v) containing the internal standard diethylphthalate (amount depends on sample level). Forty percent of the sample is injected using a Waters 710B Autosampler. Yields are normalized to 100% injection using the internal standard as a volume marker. RESULTS
The separation of the commonly encountered PTH derivatives at the 40 and 10 pmol levels is shown in Fig. 6. At higher sensitivity settings (AUFS = 0.004 or 0.002) as little as 1 pmol of each derivative can be detected at acceptable signal-to-noise ratios (about 10: 1). Representative chromatograms from the
A NOVEL TABLE I COMPOSITION AND USAGE OF REAGENTS AND SOLVENTS~ Reagent/solvent
Usage per cycle
RI 5% PITC in heptane R2 2% TEA in waterb R3 TFA’ R4 25% TFA in water SI S2 Ethyl acetated S3 Butyl chloride S4 Acetonitrite
20 /.d 40 IL1 250 pl 20 /.d 2.5 ml 1.0 ml 2.5 ml
321
MICROSEQUENCER
’ R4, S2, S3, and S4 contain approximately 10 mg/ liter DTT. R2 and R3 are liquids delivered as gases by bubbling Nz through the liquid. b One bottle (150 ml) of R2 is used to 75-100 cycles, at which point 1 ml of TEA is added for the next 75100 cycles. After this, R2 is discarded and a new batch is added. ‘Approximately 50 ml of R3 is consumed per 200 cycles. d S2 contains 0.25% I-propanol.
one run to the next. The yield of PTH-serine at cycle 3 is about 40% of PTH-valine at cycle 1. The combined yields of PTH-serine and its DTT adduct are 72% of PTH-valine at cycle 1. The yield of PTH-histidine at cycle 12 is 80% of the yield of PTH-valine at cycle 10, a result showing significant improvement in the yield of PTH-histidine compared to the past performance of the spinning cup instrument (often 10% of the previous cycle). The carryover of PTH-leucine from cycle 9 to cycle 10, and from cycle 11 to cycle 12, is 2 and 3%, respectively. The apparent increase in yield of PTH-alanine from cycle 15 to cycle 19 is due to an increasing background (5- 10 pmol) over this portion of the analysis. This type of result is peculiar to a given protein. TABLE 2 MICROSEQUENCERPROGRAM SUMMARY a
analysis of 250 pmol of sperm whale apomyoglobin are shown in Fig. 7. The initial yield is 78%, the repetitive yield from cycles 1 to 10 (both valine) is 90%, and the average repetitive yield (Fig. 8) is 94.6%. These results are rather typical for the analysis of myoglobin over the range of 100-400 pmol (Fig. 8) and reflect a dropoff in yield from cycles 1 to 2 or 2 to 3, followed by a leveling off to an average repetitive yield in the range 9395%. The background peaks encountered at cycle 1 (especially near valine) are due to the PTC derivative of diethylamine (DEA, a contaminant of the coupling base TEA) and diphenylthiourea (DPTU). These two background peaks potentially interfere with the PTH derivatives of proline and tryptophan, respectively, but since they rapidly drop to the 5-pmol level or less, they have no effect on a run at this level. A third background peak is encountered between the positions of PTH-arginine and PTH-methionine. Although this artifact peak does not interfere with analysis, it has not been chemically identified and its presence is variable from
Reaction chamber
DV Deliver R2 Deliver RI Deliver R2 (couple) Deliver S2 DV Deliver R3 (cleave) DV Deliver S3 DV DW Deliver R3 (cleave) DW Deliver S3 W
Conversion flask
Duration
Total time
Convert Convert Convert
0.5 1.0 1.0
0.5 1.5 2.5
Dry Dry Deliver S4
9.0 2.5 3.0
11.5 14.0 17.0
Pause Dry DV Deliver R4
6.0 1.5 I.0 2.5 0.5
23.0 24.5 25.5 28.0 28.5
Convert Convert Convert Convert
7.5 2.5 1.0 2.5
36.0 38.5 39.5 42.0
a The delivery steps include a 5-s pressurize, a variable amount of delivery times (for volumes see Table 1). and a blow-out step of 30- 150 s, depending on the reagent or solvent. S2 is delivered in four aliquots of 5, 5, 5, and 15 s. S3 is delivered in two aliquots of 2 s. S4 is delivered to the conversion flask (9 s), blown-out (5 s), agitated with Nz (10 s), and blown over to fraction collector (50 s); this process is then repeated.
322
HAWKE, AUF5
.0064
HARRIS, AND SHIVELY
z 0.000 Std
t 40 moles I
;I
.0056
.0048
.0040 s x % .0032
10 pmoles
.0024
.OOE
.0008 7
A8
L
I
‘1
4
8
12
TIME
(m(n)
FIG. 6. HPLC separation of PTH amino acids. A mixture of 19 PTH amino acids at the 40-pmol (left) and IO-pmol (right) levels were separated on an Altex Ultrasphere C- I8 column using the TFA/acetate/MeCN system described under Materials and Methods. Cys’” (not shown) elutes just before Glu (E). The internal standard is diethylphthalate.
Chromatogmms from the sequence analysis of 1 nmol of the small peptide NHz-PheAsn-Gly-Leu-Arg-Gly-CONH2 is shown in Fig. 9. This peptide is used in our laboratory to rapidly evaluate the performance of an instrument (requires 4 h for analysis) and to detect the possible N-acyl rearrangement associated with the Asn-Gly peptide bond (a rearrangement resulting in an internally blocked peptide). The initial yield of the analysis is 47% (calculated at cycle 2). In the analysis shown (selected from over 30 analyses), the background from PTC-DEA and DPTU is significantly elevated on cycle 1 and decreases dramatically on cycle 2. This is a common phenomenon in gas-liquidsolid phase sequence analysis and is shown to illustrate the type of background peaks occasionally encountered (contrast Fig. 7). In this example, the Polybrene was precycled with glycylglycine, and a small amount of glycine is found on each cycle. The glycine
background is essentially eliminated if glycylglycine is omitted from the Polybrene. The yield of PTH-asparagine at cycle 2 is greater than that of PTH-phenylalanine at cycle 1, a result consistently observed for this peptide, but for which we have no explanation (it is not due to background at PTH-asparagine). These chromatograms illustrate the ability of the instrument to sequence even a small peptide with reasonable repetitive yields (estimated as 87%) through the penultimate residue. The last residue (glycinamide, cycle 6, not shown) is not usually detected. It is assumed that this last residue is not retained by Polybrene even when sequenced at the lnmol level, thus illustrating the phenomenon of washout, commonly observed for the last one or two residues of peptides ending in neutral or hydrophobic residues (glycinamide is uncharged and represents an unfavorable last residue). The yield of PTH-arginine is approximately twice the equivalent run of this peptide on the spinning cup instrument (data not shown), suggesting that yields of PTH-arginine and PTH-histidine are improved on the gas-phase instrument. The ultimate test for microsequence analysis is a sample which has been isolated at the subnanomole level and for which the sequence is unknown. In this category, 200 pmol of a tryptic peptide from human insulin receptor was isolated by reverse-phase HPLC (for a description of the method see Ref. (23), and of the sample preparation Ref. (24)) and subjected to microsequence analysis on the gas-liquid-solid phase instrument. The chromatograms shown in Fig. 10 give the complete sequence of the 15-residue peptide, a result in agreement with amino acid analysis (Table 3). Since only 40% of each cycle is analyzed, only 35 pmol of PTH-isoleucine is injected at cycle 1, giving an initial yield of 44%, and by cycle 10 the amount injected is less than 1 pmol (detected but not integrated at this level). The interpretation of the chromatograms is unambiguous through cycle 8 (AUFS at 0.004), but the baseline level and sequencer noise become significant compared
A NOVEL
323
MICROSEQUENCER S 14
a
j- /4UFS
AUFS = 0.016
.0120r
‘CYCLE
.0090
.0075
1
1
CYCLE
= 0.008
IO
I
12
GTT .0030
-
6
12
6
TIME
b
AUFS:
.0064
-
.0056
-
.0048
-
CYCLE
, 12
(mln)
0.008 15
CYCLE
16
K Sld 20
CYCLE
18
24
.0040 z :ru .0032 T r
(A),I
I
6
I2
TIME
6
12
(min)
FIG. 7. Chromatograms from microsequence analysis of 250 pmol of myoglobin. Forty percent of each cycle was analyzed on the same system described in Fig. 6. The pmol quantitated for each injection are shown below the peak identified. The carryover peak from the previous cycle is shown in parentheses. (a) Cycles 1, 3, 10, and 12. (b) Cycles 15, 16, 18, and 19.
to signal level for cycles 9-l 6 (AUFS at 0.002). In spite of this the identifications are clear at least through cycle 14, and require some experience to identify PTH-arginine at cycle 15. These results illustrate a reasonable
lower level of analysis for a peptide. If the analysis began with 10 pmol at cycle 1 and gave similar results only 0.1 pmol would be detected by cycle 9, a situation beyond our current level of analysis.
324
HAWKE,
Ave. Rep. Yield
HARRIS, AND SHIVELY
= 94.6%
VLSEGEWPLVLHVWAKVEA+
lo-
2
4
6
CYCLE
8
10
12
14
16
18
20
NUMBER
FIG. 8. Yields of PTH amino acids from microsequence analysis of 250 pmol of myoglobin. The top, middle, and bottom curves show the analysis for 400, 250, and 100 pmol, respectively. A least-squares fit of the yield of PTH amino acid at each cycle was used to calculate the average repetitive yield for each curve.
Efect of Glass Components on Performance
Initially, we constructed the cartridge and conversion flask from glass and noted poor yields of PTH-serine and PTH-threonine. For serine the major peaks observed were dehydroserine and the so-called DTT-serine adduct (4) and for threonine, dehydrothreonine was observed. It had been suggested that the dehydration reaction is facilitated by acylation of the hydroxyl groups during cleavage (25), and that the addition of methanol to the sample would prevent or reverse the reaction. Since it is only convenient to add methanol during the conversion phase of the chemistry, this was tried. Indeed, a dramatic increase in the yields of PTH-serine and PTH-threonine were observed for the model peptide tested (Table 4). In spite of the improvement, the hypothesis was incorrect, since the same measure of protection
was observed by prewetting the conversion flask with acetonitrile, which is unlikely to act as a scavenger or transesterification agent. We reasoned that exposure of PTH-serine and PTH-threonine to dry glass at 55°C resulted in the dehydration reaction, and that the predelivery of solvent in some way (hydrogen bonding) masked the reactive silanol groups. To further explore this possibility, we constructed a conversion flask from PFATeflon (this material is sufficiently translucent for liquid to be easily seen in the converter) and repeated the experiment. The same high yields of PTH-serine and PTH-threonine were observed whether or not the flask was prewetted with solvent. We conclude that the glass surface is primarily responsible for the dehydration reaction. Since it also seemed likely that lower yields of PTH-histidine were possible on glass surfaces (PTH-histidine gives low yields off of poorly “endcapped” reversephase columns), we also constructed the reaction cartridge from Teflon (entry and exit of liquids can be monitored by careful observation of the Teflon delivery lines). One consequence of this change was lower overall background levels for DPTU and PTC-DEA. Although at first we thought this was due to higher retention of these background peaks on glass surfaces3 we now consider it more likely that we are effecting a better seal in the Teflon cartridge compared to the glass cartridge. The observation that the level of DPTU background reflects cartridge seal is easily demonstrated by purposely (or accidentally) compromising the seal in either system. In this respect, we believe that a Teflon flare fit to a glass surface is more prone to leak and cause background accumulation than are the pull-through fittings in our cartridge. 3 It was this idea which prompted us to trimethylsilylate the sample disks. This resulted in shghtty better backgrounds. The suggestion of one reviewer to try to “cover” sites by using more Polybrene actually gave higher backgrounds, presumably by increased retention of byproducts by Polybrene itself.
A NOVEL AUFS
MICROSEQUENCER
T 0.032
,024
1: YCLE
190 Ll
2
CYCLE
4
CYCLE
5
,018
,006
L 6
12
I 6
12
TIME
I
,
1
6
12
6
12
(men)
FIG. 9. Chromatograms from microsequence analysis of a synthetic small peptide. One nanomole of the peptide FNGLRG-amide was sequenced and analyzed as described in Fig. 7. The background peaks near PTH-phe (F) on cycle 1 are identified in cycle 2; DEA refers to the PTCderivative of DEA. The background at PTH-gly (G) is due to precycling Polybrene with glycylglycine. The last amino acid (glycinamide) is not detected.
DISCUSSION
Reagent and Solvent Delivery System Design A critical feature of the instrumentation is the delivery system, since both strong base and strong acid are used in Edman chemistry. In order to avoid cross-contamination, the reagents are delivered through “zero-deadvolume,” chemically inert valves and the lines are flushed with solvents compatible with the reagents. The Wittman-Liebold valve in its original (2) and miniaturized version (12) was an improvement over existing technology because of its low dead volume and the ability to have multiple inputs with a single exit line which is cleared of liquid by a pressurized-gas blow-out line integral to the system. Disadvantages of the valves were the great difficulty in machining them to proper tolerances, the use of a single valve block for multiple inputs (if one malfunctioned or was machined improperly the entire block had to be discarded), the fact that the liquid seal must be maintained over a large
surface area (limiting allowable back-pressures), and the use of positive gas pressure to maintain the closed position (loss of gas pressure causes the valves to leak and crosscontaminate reagents and solvents). The latter problem was partially solved by introducing mechanical solenoids to close the valve diaphragms, but this approach was not totally successful since a “vacuum assist” is also required to actuate the valves. A second problem encountered in the delivery system of some instruments is the bottle seal. The use of conventional O-rings or gaskets is not recommended unless they are truly inert to the reagents and solvents; in this regard, the material Kal-Rez (Du Pont) would be expected to perform well. A third problem is the tendency of the bottle-pressure regulators to drift. Since consistent delivery volumes (for liquids or gases) depend on reproducible bottle pressures, both the bottle seals and the bottle pressures must be reproducible. In consideration of these problems, our instrument has several unique features. The delivery valves (Fig. 3) are zero dead volume,
326
HAWKE,
a
AUFS
.0064
HARRIS, AND SHIVELY
= 0.008 I 35 Sid
.0056 CYCLE
1
1 CYCLE
I .0048-
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3
CYCLE
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IJe---e-&h
b ,;
TIME
b
AUFS
(min)
= 0.004
.0036
.0032
Sld
CYCLE
5
CYCLE
6
;td
CYCLE
7
CYCLE
8
.0028
! DTT
I
I
4
L
8
/
J I
I
12
4
8
/
12 TIME
I
I
4
8
I
I2
(min)
FIG. 10. Chromatograms from microsequence analysis of a tryptic peptide from human insulin receptor. The sample (200 pmol) was analyzed as described in Fig. 7. (a) Cycles 1-4; (b) cycles 5-8; (c) cycles 912; and (d) cycles 13-16.
made of inert materials (Teflon or Kel-F), modular (two inputs per valve, as many valves can be linked as isnecessary), electromechanically actuated (no compressed air or vacuum required), able to withstand high
backpressures (the seal is formed over a small area; backpressures up to 30 psi are tolerated), and easy to machine (the lumen is a straight pass in drilling; the unit cost is about $100). The valves have performed well on both gas-
A NOVEL C
AUFS
327
MICROSEQUENCER
= 0.002
stu
.0016-
Std
CYCLE
CYCLE
10
(ZYCLE
11
12
I T
DT
I
d .0016 .0016-
.0014
r
I
I
I
I
I
I
4
8
12
4
8
12
‘-e-&-e
TIME
(min)
AUFS
Std
CYCLE
I
I
I
4
8
12
CYCLE
16
= 0.002
I I: -
DTT
Std
CYCLE
13
14
CYCLE
.OOlZ -
15
DTT
DTl
OTT
.OOlO f z
.0008i .0006
-
.0004
-
.oooz .0002
t I
1 I 4
/ 8
I 12
I
I
I
4
8
12
II
4
TIME
FIG.
I
8
I
12
I
I
4
8
12
(min)
IO-Conhued.
liquid-solid phase and spinning cup instruments over a period of 3 years (if a solenoid malfunctions it can be replaced, leaving the valve body still functional). The bottle closure design (.Fig. 4) is based on a compression fitting which allows repeated opening and
resealing, requiring only a finger-tight seal. Only Teflon contacts the liquid or vapor, and the tubing seals are made by “pullthrough” fittings. The bottle-pressure regulators are 4-in. precision regulators and do not drift with time. The vent system, standard
328
HAWKB,
HARRIS, AND SHIVELY
TABLE 3 AMINO
ACID COMIWSITION OF TRYPTIC PEPTIDE OF HUMAN INSULIN RECEPTORS
Amino acid
picomoles
Residue
Cys'" Asx Thr Ser GlX Pro GlY Ala Val Met Be Leu TY~ Phe His Trp LYS AC3
20 93 37 37 180 27 50 50 0 0 15 39 0 0 0 0 0 32
0.6 (1) 2.9 (3) 1.1 (I) 1.1 (0) 5.6 (6) 0.8 (0) 1.6 (0) 1.6 (1) 0 (0) 0 (0) 0.5 (I) 1.2 (1) 0 (0) 0 (0) 0 6-o 0 (0) 0 (0) 1.0 (1)
’ The sample (60 ng, or 40 pmol) was hydrolyzed for 24 h at 110°C in 6 N HCl containing 0.2% 2-mercaptoethanol. Amino acid analysis is described in paper VI of this series (submitted). Since the sample was S carboxymethylated, cysteine was determined as the cys”” derivative. The values are normalized to one arginine per mole (32 pmol). Predicted values from sequence analysis are shown in parentheses. An equivalent acid hydrolysis blank gives IO-20 pmol of Ser, Gly, and Pro; thus, these amino acids are considered to be at background levels.
matic in the TMA-water system. This change in TMA concentration with time is slower in the “blow-across” method than in the “bubble-through” method. The number of cycles which can be run per bottle of TMA (25 or 12.5%) is greater than for 2% TEA (200-300 versus 100) but the overall base consumption is greater in the TMA-based system. Chemistry and Background
Peaks
The biggest problem associated with gasliquid-solid phase chemistry at the beginning ( 198 1- 1982 in our laboratory) was the enormous background due to DPTU (often >500 pmol). This was in contrast to the spinning cup instrument for which the DPTU background can be entirely eliminated. We reasoned that the stronger base in this system (pH > 9) and the increased amounts of PITC (15% v/v in heptane) led to increased hydrolysis of PITC to aniline which in turn reacted with PITC to give DPTU. DPTU is especially stubborn to remove, since it is only marginally soluble in ethyl acetate (S2). Reducing the PITC level to 5% reduced the DPTU peak (actually two peaks are observed, one is DPTU and the other DPU) to about the
TABLE 4
in older design instruments, has been eliminated as an unnecessary variable in the bottlepressure-delivery system. R2 and R3 are delivered by bubbling nitrogen through the liquids to carry vapor over to the cartridge as described by Hewick et al. (12). Currently, the Applied Biosystems instrument uses a “blow-across” method for delivering R2, an approach which probably reduces the amount of water and base vapor delivered, thus lowering base related background. We have seen no difference between the two methods for delivering R2 (TEAwater) in our system, suggesting that the difference is related to the rapid change in partial pressure of base, an effect more dra-
EFFECT OF PREWETTING CONVERSION FLASK (GLASS) WITH S4 (ACETONITRILE) ON YIELDS OF SER AND THR
Prewetted (pmol) Amino acid
Yes
No
Thr Ser Glu LYS Ser Gin
361 (2.7) 385 (1.9) 137 1185 195 277
201 (.58) 74 (.32) 742 1269 71 279
Notes. The approximate ratio of authentic to dehydro derivative is given in parenthesis. The results are the same for methanol or acetonitrile as S4. Approximately 1.5 nmol of the peptide TSEKSQTP was sequenced in duplicate.
A NOVEL
MICROSEQUENCER
lOO-pmol level, a level still not compatible with high-sensitivity analysis. A further improvement was observed in changing R2 from 25% TMA to 2% TEA. The background was highest with freshly made TMA and slowly decreased with use, while the background with 2% TEA was lower and remained steady throughout use. Apparently, the latest solution to this problem with the Applied Biosystems instrument is to reduce PITC to 2.5% and TMA to 12.5%, and to “blow across” rather than “bubble through” R2. A third factor involved was the cartridge seal. This factor became obvious in the glasscartridge system when we noticed that the background changed each time the cartridge was opened and the Zitex seal was disturbed or replaced. We therefore replaced the Zitex seal with a Kal-Rez gasket and used a Teflon screen as a support for the glass-fiber filter. This tactic reduced the DPTU background to 5 pmol or less. A similarly low background was seen for an all-Teflon cartridge, with no difference in background for a Zitex seal, a Kal-Rez seal, or no seal (Teflon is self-sealing). We concluded that an improperly sealed cartridge will accumulate DPTU in the space between the cartridge surfaces, and will slowly leach out during an S3 rinse to the conversion flask, rather than be washed out entirely during the S2 rinse to waste. Furthermore, the situation is not remedied by following the S2 rinse with an S3 rinse prior to cleavage (data not shown). Other background peaks include DTT (or DTE) which is added to preserve the solvents, and the PTC derivative of DEA (or DMA in the case of TMA), a contaminant in TEA. The possible overlap of PTH-Asp (and sometimes PTH-Glu) with the DTT peaks (the reduced and oxidized forms) has prompted Hewick et al. (12) to convert them to their methyl ester derivatives. This is most conveniently performed during the conversion reaction by using methanolic HCI instead of 25% aqueous TFA as R4. Although we have not taken this approach, we see no major disadvantage other than the fact that the
329
reagent (MeOH/HCl) is unstable at room temperature and is known to rapidly convert to a mixture of methanol, water, HCl, and methyl chloride (26). The removal of DEA from TEA or DMA from TMA is achieved by distillation of the base from a substance which reacts irreversibly with primary or secondary amines. For this purpose we have chosen tosyl chloride, and Hewick et al. (12), phthalic anhydride. In either case, the last traces of secondary amine are not removed, as evidenced by the presence of their PTCderivatives in published chromatograms (12, 13,22, this work). More careful distillation and processing of the bases may lead to lower levels of these peaks in chromatograms. The use of aqueous TEA as a coupling base may have a number of advantages over aqueous TMA. We have a cycle time of 42 min compared to over 60 min for the Applied Biosystems instrument. This is due to shorter coupling times, and shorter delivery and blowout times. The latter is achieved by using standard-bore (0.04-in.) Teflon tubing in place of microbore (0.01 in.). As originally described (12) a double-coupling program was advocated together with 15% PITC, probably leading to increased production of DPTU since two deliveries of PITC were necessary. These problems have now been solved by lowering the PITC concentration in Rl and by using a single-coupling program. Conclusion The advent of automated microsequence analysis has helped to revitalize and update the efforts of protein chemists in terms of the types and amounts of samples which can be analyzed. The availability of the instrumentation has substantially improved our knowledge of the Parameters which affect performance and the requirements of the sample preparation for compatibility. Since the field is new the variety in instrumentation is limited and the price high. It is our hope that the description of a new instrument with similar capabilities but different design fea-
330
HAWKE,
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tures will further stimulate the field and suggest commercially attractive alternatives. ACKNOWLEDGMENTS We gratefully acknowledge the advice and encouragement of Dr. Chas. W. Todd. We are pleased to acknowledge the design input and many hours of careful work in construction of these instruments by Clifford Sailor and Lance Richardson at the City of Hope Biomedical Instrumentation Department. The design and construction of the zero-dead-volume valve was by Miro Rusnak from the same department. We thank Dr. Michael Hunkapiller for discussing the early Caltech prototype instrument with us before publication, and Chad BenAvram for a critical appraisal of the manuscript.
REFERENCES 1. Edman, P., and Begg, G. (1967) Eur. J. B&hem. 1, 80-91. 2. Wittman-Liebold, B. (1973) Hoppe-Seyler’s Z. Physiol. Chem. 354, 1415-1431. 3. Wittman-Liebold, B., Graffunder, H., and Kdhls, H. ( 1976) Anal. Biochem. 75, 62 1-633. 4. Hunkapiller, M. M., and Hood, L. E. (1978) Biochemistry 17, 2 124-2 133. 5. Shively, J. E. (1981) in Methods in Enzymology (Pestka, S., ed.), Vol. 79, pp. 31-48, Academic Press, New York. 6. Bhown, A. S., Cornelius, T. W., Mole. J. E., Lynn, J. D., Tidwell, W. A., and Bennett, J. C. (1980) Anal. Biochem. 102, 35-38. 7. Tarr, G. E., Beecher, J. F., Bell, M., and McKean, D. J. (1978) Anal. Biochem. 84,622-627. 8. Klapper, D. G., Wilde, C. E., III, and Capra, J. D. (1978) Anal. Biochem. 85, 126-131. 9. Shively, J. E., Hawke, D., and Jones, B. N. (1982) Anal. Biochem. 120, 312-322. 10. Laursen, R. A. (197 1) Eur. J. Biochem. 20, 89-102. I I. LItalien, J. J., and Strickler, J. E. (1982) Anal. Biochem. 127, 198-212.
12. Hewick, R. M., Hunkapiller, M. W., Hood, L. E., and Dryer, W. J. (1981) J. Biol. Chem. 256, 7990-7997. 13. Esch, F. S. (1984) Anal. Biuchem. 136, 39-47. 14. Currie, M. G., Geller, D. M., Cole, B. R., Siegel, N. R., Fok, K. F., Adams, S. P., Eubanks, S. R., Galluppi, G. P., and Needleman, P. (I 984) Science (Washington, D. C.) 221, 67-69. 15. Roberts, A. B., Anzano, M. A., Meyers, C. A., Wideman, J., Blather, R., Pan, Y.-C. E., Stein, S., Lehrman, S. R., Smith. J. M., Lamb, L. C., and Spom, N. B. (1983) Biochemistry 22, 56925698. 16. Downward, J., Yarden, Y., Mayes, E., Scrace, G., Totty, N., Stockwell, P., Ulhich, A., Schlessinger, J., and Wateriield, M. D. (1984) Nature (London) 307, 52 l-527. 17. Ezra, E.. Blather, R., and Udenfriend, S. (1983) Biochem. Biophys. Res. Commun. 116, 10761083. 18. Fraenkel-Conrat, H. (1954) J. Amer. Chem. SK 76, 3606-3607. 19. Schroeder, W. A. ( 1967) in Methods in Enzymology (Hits, C. H. W., ed.), Vol. 11, pp. 445-461, Academic Press, New York. 20. Waterheld, M. D., Lovins, R. E., Richards, F. F., Salomone, R., Smith, G. P., and Haber, E. (1968) Fed. Proc. 27, 455. 2 I. Hawke, D., Yuan, P.-M., and Shively, J. E. (1982) Anal. Biochem. 120, 302-3 1I. 22. Hunkapiller, M. W., and Hood, L. E. (1984) in Methods in Enzymology (Hirs, C. H. W., and Timasheff, S. N., eds.), Vol. 91, pp. 486-493, Academic Press, New York. 23. Yuan, P.-M., Pande, H., Clark, B. R., and Shively, J. E. (1982) Anal. Biochem. 120, 289-301. 24. Fujita-Yamaguchi, Y. (1984) J. Biol. Chem. 259, 1206-1211. 25. Margolies, M. N., Brauer, A., Oman, C., Klapper, D. G., and Horn, M. J. (1982) in Methods in Protein Sequence Analysis (Elzinga, M., ed.), pp. 189-203, Humana Press, Clifton, N. J. 26. Pritchard, D. G., and Todd, C. W. (1977) J. Chromatogr. 133, 133-139.
BIOCHEMISTRY
147, 3 15-330 (1985)
Microsequence
Analysis of Peptides and Proteins
V. Design and Performance of a Novel Gas-Liquid-Solid DAVID
H. HAWKE,
DAVID
C. HARRIS,
Phase Instrument
AND JOHN E. SHIVELY
Division of Immunology, Beckman Research Institute of the City of Hope, Duarte. California 91010 Received October 3 1, I984 We describe the construction and performance of a novel, automated, Edman chemisttybased microsequencer. The reagent and solvent delivery system, the reaction cartridge for coupling and cleavage, and the conversion flask are all constructed from chemically inert perfluoroelastomers. The delivery valves are of a new design incorporating the use of electromagnetically actuated solenoids and zero-dead-volume construction, and may be connected in a modular fashion resulting in multiple inputs with a single output line which can be flushed with inert gas. The bottle closures are of a new design based on an all-Teflon compression fitting. The reaction cartridge and conversion flask are thermostated by solid-state heaters in an aluminum block. The overall size of the instrument is 25 X 34 X 14 in. The chemistry utilizes 2% aqueous triethylamine as the coupling base which is delivered to the reaction cartridge via a stream of nitrogen. The ‘gas-phase” delivery of the coupling base and the cleavage acid (trifluoroacetic acid) is modeled after the method described by R. M. Hewick et al. (J. Biol. Chem. 256, 7990-7997, 1981). The instrument has performed well over a period of 3 years in terms of low background peaks, sensitivity in the picomole range, and reliability of operation. The use of economical components, ease of construction and operation, and sensitive analytical capability make this instrument a useful tool for microsequence analysis of peptides and proteins. Q 1985 Academic Press, IIIC.
Improvements in the techniques and automation of Edman chemistry have led to the routine sequence analysis of subnanomole levels of peptides and proteins. Since the introduction of the first spinning cup sequenator in 1967 (I), improvements in the commercially available instruments (mainly the Beckman 890 series) were described by Wittman-Liebold and co-workers (2,3), Hunkapiller and Hood (4), and us (5). The so-called “modified” spinning cup instruments were able to sequence subnanomole amounts of proteins, but were not generally available. The contribution to their success of other factors was hotly debated, and eventually it was clear that modestly improved spinning cup instruments were also capable of increased sensitivity ((6) the Beckman 890M sequenator). The other factors include automated conversion of anilinothiozolinone 315
(ATZ)’ to PTH derivatives (3) the use of polybrene as a carrier for peptides and small amounts of proteins (7,8), rigorous purification of reagents and solvents (2,4,5,9), and the high sensitivity analysis of PTH amino acid derivatives by reverse-phase HPLC. The development of solid-phase Edman chemistry by Laursen ( 10) involved covalent attachment of peptides or proteins to solid i Abbreviations used: ATZ, anilinothiazolinone; PTH, phenylthiohydantoin; PITC, phenylisothiocyanate; SPITC, 3-sulfophenylisothioyanate; PTC, phenylthiocarbamyl derivative; TEA, triethylamine; DEA, diethylamine; TMA, trimethylamine; DMA, dimethylamine; DPTU, diphenylthiourea; DPU, diphenylurea; PFA, perfluoroalkoxy-Teflon; TFA, trifluoroacetic acid; DTT, dithiothreitol; MeCN, acetonitrile; DTE, dithioerythritol; AUFS, absorbance units full scale; VIA, versatile interface adapter; RAM, random access memory: LED, light emitting diode. 0003-2697/85 $3.00 Copyright 0 1985 by Academic Press. Inc. All rights of reproductmn rn any form resewed.
316
HAWKE,
HARRIS, AND SHIVELY
supports, but was usually limited to tens of nanomoles of small peptides. Recent improvements have extended the range to OS1.O nmol of small peptides (11). The noncovalent entrapment of peptides and proteins in a Polybrene film (78) is an alternative to covalent attachment and was adapted from the spinning cup instrument to a cartridge type of instrument described by Hewick et al. (12). Other changes in this instrument included delivery of the coupling base and cleavage reagent as gases (or more accurately as vapor swept through by a carrier gas) and miniaturization of valves and the delivery system and therefore a lower consumption of solvents. This new instrument is commercially available and has performed well in a number of laboratories ( 13- 17). The concept of delivering reagents as gases is not new (18-20), but was first successfully adapted to an automated instrument by Hewick et al. (12). In spite of the obvious simplification of instrumentation for a gas-liquid-solid phase instrument compared to a spinning cup instrument, the commercial instrument is costly and utilizes many difficult-to-machine components, including the miniaturized Wittman-Liebold valves, the Wittman-Liebold converter (as originally described), the allglass reaction cartridge, and the custom fraction collector. Our experience with building modified spinning cup instruments prompted us to develop a gas-liquid-solid phase instrument based on the prototype machine of Hewick et al. (12). This work led to the design of new economical zero-dead-volume valves, an all perfluoroelastomer delivery and reaction system, a compression type of closure for reagent and solvent bottles, and computercontrol by an inexpensive Apple II plus computer. Our instrument is less expensive and more compact, utilizes a different coupling base, has a silylated glass disk impregnated with Polybrene for a solid phase, and has a shorter cycle time than the commercially available instrument. This report de-
scribes its construction and the performance of the prototype over the past 3 years. MATERIALS
Instrument
AND METHODS
Design
A schematic of the &rstrument is shown in Fig. 1. The overall schematic differs from that of Hewick et al. (12) in the following respects. The reaction cartridge (Fig. 2) is made of Teflon (turned from 0.50 in bar stock), and utilizes either a Kal-Rez gasket (0.020 in.) or no gasket (the Teflon is selfsealing). The Teflon lines are sealed in the chamber by a “pull-through” fit.2 The glass filter (1 cm diameter) rests on a Teflon screen (mesh opening 0.004 in., Savallex, Minnetonka, Minn.). The reaction chamber is in an aluminum holder thermostated by an Omega CN300 controller (Omega Engineering, Stamford, Conn.). The conversion flask is a 6-ml conical PFA-Teflon vial (Savillex) with a self-sealing closure. The Teflon lines are sealed with “pull-through” fittings. The conversion flask (not shown) is heated by a separate solid-state system. The inert, zerodead-volume valves (Fig. 3) control the flow of reagents and solvents into the cartridge and conversion flask. The output of the cartridge and the conversion flask are controlled by a Teflon three-to-one miniature valve (Model 330TFE, Brunswick Technetics, Cedar Knolls, N. J.) with up to three input lines and a common output line. The reagent bottles (Wheaton, Millville, N. J.) are constructed from 200-ml conical bottles to which are attached 0.7%in. precision-bore glass tubing. The modified swage ’ A “pull through” fit is made by drilling a hole with an i.d. slightly less than the o.d. of the tubing to be drawn through the desired fitting (both fitting and tubing are made of Teflon). The tubing is heated to softness with a heat gun and drawn to a taper in order to insert it into the hole. Once inserted, it is drawn through with a needlenose pliers and trimmed to the appropriate length. None of the heated, tapered line should remain since it may have unwanted properties.
A NOVEL
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FIG. 1. Schematic of gas-liquid-solid phase microsequencer. Identification of components is shown to the right of the schematic. The vacuum system is shown in dotted lines because it is optional.
fitting (Fig. 4) can be finger tightened and holds up to 20 psi with no gas or liquid leaks. The tubing is inserted through a “pullthrough” fitting.2 The solvent bottles are drawn from 90-mm Pyrex tubing, made conical at the bottom and fit to 0.75in. tubing at the top, and have a capacity of 700 ml. The reagent and solvent bottles have no vent lines, eliminating the maintenance problems associated with vent solenoids. Bottle pressures are set between 0.5 and 3.0 psi with precision 4-in. regulators (Watts Fluid Air, Inc., Kittery, Mass.). The fraction collector is a Gilson slave unit (Gilson Medical Electronics, Middleton, Wise.), externally triggered by a relay closure ( 100-200 ms). The closure time-out was per-
formed by either a hardware or a software clock. Polypropylene tubes (13 X 100 mm, Sarstedt, Haywood, Calif.) are used in the fraction collector. Smaller conical polypropylene tubes (1.4 ml, Sarstedt) can be used as inserts if smaller volumes are desired. The entire assembly is housed in a Plexiglas housing flushed with argon at a flow rate of 20 ml/min. The prototype was run with a vacuum system until May 1984. The optional vacuum system is similar to that described by Hewick et al. (12). Two bench-top models have been run in this laboratory without a vacuum system. The control system consists of an Apple II plus computer with 48K RAM and a 5.25
318
HAWKE,
HARRIS, AND SHIVELY
Reagents and Solvents The composition and usage per cycle of the reagents and solvents are shown in Table 1. A description of the purification of some of the reagents has been previously published (9). The reagents and solvents are sparged with argon (at least 10 min at a rate of 5 ml/ min) prior to connection to the system (if DTT or DTE is added, sparge before and after the addition; DTT or DTE should be used exclusively to avoid complications inherent in having both present at the same time). The effect of sparging the solvent is to reduce the degree of destruction of PTH amino acids (see Ref. (9)). PITC (Aldrich, Milwaukee, Wise.) is twice distilled in vacua
FIG. 2. Reaction chamber. (A) Knurled aluminum nut for applying pressure to seal Teflon body (B). (C) Interface between two halves of Teflon body (B) may be self-sealing or used with a Kal-Rez gasket. (D) Slot for thermometer. (E) Heater.
in. disk drive. A 32-port parallel, 6522-based VIA (John Bell, San Carlos, Calif.) plugs into slot 7 of the Apple and triggers the appropriate drivers for 24 VDC solenoids. The front panel (Fig. 5) includes switches for manual control and temperature readout. Red-green LEDs indicate whether a given function is manually (red) or automatically (green) actuated. The software is menu oriented and user friendly, and allows for using either stored running programs or creating new running programs. The operating system is a compiled version of BASIC, but can also be edited to add new features. The running programs and operating system occupy only 20K of memory and are stored on a floppy disk for retrieval if necessary. Connection of the computer to a line printer is optional. The 1984 cost of the instrument including hardware, software, and labor (at standard rates) is $15,000.
FIG. 3. Zero-dead-volume valve. The main block is made from Teflon or Kel-F and has two inlet ports and a continuous outlet with two fittings. The inlets and outlets are threaded for standard i-28 flare fittings. The piston and diaphragm are made from one piece of Teflon with the lower portion shown in the closed position and the upper portion shown in the open position. The solenoid coil and housing are shown removed in the upper portion. The flow of liquid into the inlet and out of the outlet is indicated by arrows. It is assumed that the hidden view of the outlet is connected to gas pressure controlled by a two-way valve which when activated expels liquid in the central lumen in the direction of the arrow.
A NOVEL
4
%COom
MICROSEQUENCER
+
FIG. 4. Bottle closure for reagents and solvents. (A) A one-piece Teflon shroud machined to fit over the cylindrical neck of the bottle (C). The shroud has two holes drilled for accepting Teflon pressurizing and delivery lines, sealed by pulled-through fittings. Stainless-steel nuts (D) and (E) tighten the compression fitting to the bottle against a standard ferrule (B). (D) and (E) are male and female nuts on a 0.75in-tube cap fitting. The bottle neck (C) is precision 0.75~in. tubing.
and stored at -20°C in sealed glass ampoules (5-10 ml). Heptane (Baker or Burdick and Jackson, HPLC grade) is distilled from CaH2 under N2 and stored in 50-ml sealed-glass ampoules. Triethylamine is either obtained from Pierce Chemical Company (Sequenal grade; Rockford, Ill.), or purchased from Aldrich and distilled at reduced pressure first from CaHz and then from tosyl chloride. Trifluoroacetic acid (Sigma or Aldrich) is distilled first from Cr@ and then from A&O3 under nitrogen. Ethyl acetate (Baker, HPLC grade) is purified as previously described (9) briefly charcoal treated, and distilled first from ninhydrin and then from a mixture of SPITC, DTT, and Quadrol. 1-Propanol (Baker, HPLC grade) is distilled from CaH2. Instrument Operation Sample loading. The cartridge is disassembled and a Whatman GF/C glass-fiber disk (1 cm) which has been trimethylsilylated is
319
dropped onto the Teflon screen. The silylation treatment is as follows: soak disk in HN03 (coned AR) for 3-l 2 h; rinse with water, methanol, and THF; treat with 10% trimethylchlorosilane (Regis, Morton Grove, Ill.) in THF (v/v) for 2-3 h at room temperature; rinse with THF and methanol; and air dry and store in a sealed bottle at 4°C. The disk is prewetted with MeCN or ethanol (5 ~1) to prevent beading, aqueous Polybrene ( 100 mg/ml) is spotted (20 pl), and the disk is cycled for 2-3 program cycles. The sample is repeatedly spotted on the precycled disk (20 ~1 per application) and dried. Drying is accomplished by delivering N2 with the cartridge assembly slightly opened (3-5 min per dry cycle). Closing the cartridge during sample drying may inadvertently lead to loss of sample. Following analysis of a protein it is recommended that the disk be disposed of unless background peaks are at an acceptable level after a run. Following peptide analysis, the disk can be reused. More polybrene is spotted on disks that have undergone 75100 cycles. Glycylglycine is omitted from the polybrene in order to reduce background and to speed precycling of the Polybrene film. If performance is poor, Polybrene can be spotted on the filter with the sample. ln some cases, we have used one disk for 400 cycles, at which point some discoloration is apparent. Program. The general features and timing of the program are described in Table 2. One complete cycle comprises a single coupling of 6 min, a double cleavage of 6 min and 7 min, and conversion of ATZ to PTH derivatives for 22 min. Coupling is performed at 49-52°C and conversion at 52-57°C. The program begins first with a short predelivery of base and then of R 1, followed by a longer delivery of base. Analysis of PTH derivatives. The system in use has been previously described by us (21). Briefly, we use an Altex Ultrasphere C18 column (4.5 X 250 mm), with solvent A composed of 16 mM TFA containing 4.5 mM HAc, titrated to pH 5.8 with 5 N NaOH and adjusted to 5% MeCN (v/v), and solvent
320
HAWKE,
HARRIS,
AND SHIVELY
FIG. 5. Front view of gas-liquid-solid phase microsequencer. The instrument contains the computer and associated electronics on the right half and the plumbing and associated delivery valves on the left half. The delivery valves are located directly behind the reaction chamber (left) and conversion flask (right). The fraction collector (not shown) is housed in an inert atmosphere. The approximate measurements are 25 X 34 X 14 in.
B composed of 15 mM TFA, titrated to pH 3.4 and adjusted to 75% MeCN (v/v); a linear gradient is run from 5% B to 100% B over 10 min with a flow rate of 1.2 ml/min and a temperature of 50°C. A typical separation is shown in Fig. 6. This system gives better baseline, resolution, and peak definition for PTH derivatives than does the CN column with acetate buffer previously described ( 12,13) and performance approximately equal to the chromatogram shown in Fig. 1 of a recent review by Hunkapiller and Hood (22). However, the CN column separation shown in Fig. 2 of Ref. (22) shows a number of poorly resolved pairs (ST; H,D; Y,V; P,M; broad R) of PTH derivatives. It appears that the CN columns are highly variable and must be selected for routine optimal performance. The Altex Ultrasphere C- 18 columns using our solvent system have been more reliable in our hands for routine analysis (i.e., the columns are not hand selected). Peaks are integrated by a Spectra Physics 4000 integra-
tion system and converted to picomoles using an average KF value for PTH amino acids. Samples are delivered to the fraction collector in a volume of about 3 ml, dried at room temperature on a Savant Speed Vat (Hicksville, N. Y.), and redissolved in 40 ~1 of solvent (MeCN/H20/TFA; 75/25/O. 1, v/v) containing the internal standard diethylphthalate (amount depends on sample level). Forty percent of the sample is injected using a Waters 710B Autosampler. Yields are normalized to 100% injection using the internal standard as a volume marker. RESULTS
The separation of the commonly encountered PTH derivatives at the 40 and 10 pmol levels is shown in Fig. 6. At higher sensitivity settings (AUFS = 0.004 or 0.002) as little as 1 pmol of each derivative can be detected at acceptable signal-to-noise ratios (about 10: 1). Representative chromatograms from the
A NOVEL TABLE I COMPOSITION AND USAGE OF REAGENTS AND SOLVENTS~ Reagent/solvent
Usage per cycle
RI 5% PITC in heptane R2 2% TEA in waterb R3 TFA’ R4 25% TFA in water SI S2 Ethyl acetated S3 Butyl chloride S4 Acetonitrite
20 /.d 40 IL1 250 pl 20 /.d 2.5 ml 1.0 ml 2.5 ml
321
MICROSEQUENCER
’ R4, S2, S3, and S4 contain approximately 10 mg/ liter DTT. R2 and R3 are liquids delivered as gases by bubbling Nz through the liquid. b One bottle (150 ml) of R2 is used to 75-100 cycles, at which point 1 ml of TEA is added for the next 75100 cycles. After this, R2 is discarded and a new batch is added. ‘Approximately 50 ml of R3 is consumed per 200 cycles. d S2 contains 0.25% I-propanol.
one run to the next. The yield of PTH-serine at cycle 3 is about 40% of PTH-valine at cycle 1. The combined yields of PTH-serine and its DTT adduct are 72% of PTH-valine at cycle 1. The yield of PTH-histidine at cycle 12 is 80% of the yield of PTH-valine at cycle 10, a result showing significant improvement in the yield of PTH-histidine compared to the past performance of the spinning cup instrument (often 10% of the previous cycle). The carryover of PTH-leucine from cycle 9 to cycle 10, and from cycle 11 to cycle 12, is 2 and 3%, respectively. The apparent increase in yield of PTH-alanine from cycle 15 to cycle 19 is due to an increasing background (5- 10 pmol) over this portion of the analysis. This type of result is peculiar to a given protein. TABLE 2 MICROSEQUENCERPROGRAM SUMMARY a
analysis of 250 pmol of sperm whale apomyoglobin are shown in Fig. 7. The initial yield is 78%, the repetitive yield from cycles 1 to 10 (both valine) is 90%, and the average repetitive yield (Fig. 8) is 94.6%. These results are rather typical for the analysis of myoglobin over the range of 100-400 pmol (Fig. 8) and reflect a dropoff in yield from cycles 1 to 2 or 2 to 3, followed by a leveling off to an average repetitive yield in the range 9395%. The background peaks encountered at cycle 1 (especially near valine) are due to the PTC derivative of diethylamine (DEA, a contaminant of the coupling base TEA) and diphenylthiourea (DPTU). These two background peaks potentially interfere with the PTH derivatives of proline and tryptophan, respectively, but since they rapidly drop to the 5-pmol level or less, they have no effect on a run at this level. A third background peak is encountered between the positions of PTH-arginine and PTH-methionine. Although this artifact peak does not interfere with analysis, it has not been chemically identified and its presence is variable from
Reaction chamber
DV Deliver R2 Deliver RI Deliver R2 (couple) Deliver S2 DV Deliver R3 (cleave) DV Deliver S3 DV DW Deliver R3 (cleave) DW Deliver S3 W
Conversion flask
Duration
Total time
Convert Convert Convert
0.5 1.0 1.0
0.5 1.5 2.5
Dry Dry Deliver S4
9.0 2.5 3.0
11.5 14.0 17.0
Pause Dry DV Deliver R4
6.0 1.5 I.0 2.5 0.5
23.0 24.5 25.5 28.0 28.5
Convert Convert Convert Convert
7.5 2.5 1.0 2.5
36.0 38.5 39.5 42.0
a The delivery steps include a 5-s pressurize, a variable amount of delivery times (for volumes see Table 1). and a blow-out step of 30- 150 s, depending on the reagent or solvent. S2 is delivered in four aliquots of 5, 5, 5, and 15 s. S3 is delivered in two aliquots of 2 s. S4 is delivered to the conversion flask (9 s), blown-out (5 s), agitated with Nz (10 s), and blown over to fraction collector (50 s); this process is then repeated.
322
HAWKE, AUF5
.0064
HARRIS, AND SHIVELY
z 0.000 Std
t 40 moles I
;I
.0056
.0048
.0040 s x % .0032
10 pmoles
.0024
.OOE
.0008 7
A8
L
I
‘1
4
8
12
TIME
(m(n)
FIG. 6. HPLC separation of PTH amino acids. A mixture of 19 PTH amino acids at the 40-pmol (left) and IO-pmol (right) levels were separated on an Altex Ultrasphere C- I8 column using the TFA/acetate/MeCN system described under Materials and Methods. Cys’” (not shown) elutes just before Glu (E). The internal standard is diethylphthalate.
Chromatogmms from the sequence analysis of 1 nmol of the small peptide NHz-PheAsn-Gly-Leu-Arg-Gly-CONH2 is shown in Fig. 9. This peptide is used in our laboratory to rapidly evaluate the performance of an instrument (requires 4 h for analysis) and to detect the possible N-acyl rearrangement associated with the Asn-Gly peptide bond (a rearrangement resulting in an internally blocked peptide). The initial yield of the analysis is 47% (calculated at cycle 2). In the analysis shown (selected from over 30 analyses), the background from PTC-DEA and DPTU is significantly elevated on cycle 1 and decreases dramatically on cycle 2. This is a common phenomenon in gas-liquidsolid phase sequence analysis and is shown to illustrate the type of background peaks occasionally encountered (contrast Fig. 7). In this example, the Polybrene was precycled with glycylglycine, and a small amount of glycine is found on each cycle. The glycine
background is essentially eliminated if glycylglycine is omitted from the Polybrene. The yield of PTH-asparagine at cycle 2 is greater than that of PTH-phenylalanine at cycle 1, a result consistently observed for this peptide, but for which we have no explanation (it is not due to background at PTH-asparagine). These chromatograms illustrate the ability of the instrument to sequence even a small peptide with reasonable repetitive yields (estimated as 87%) through the penultimate residue. The last residue (glycinamide, cycle 6, not shown) is not usually detected. It is assumed that this last residue is not retained by Polybrene even when sequenced at the lnmol level, thus illustrating the phenomenon of washout, commonly observed for the last one or two residues of peptides ending in neutral or hydrophobic residues (glycinamide is uncharged and represents an unfavorable last residue). The yield of PTH-arginine is approximately twice the equivalent run of this peptide on the spinning cup instrument (data not shown), suggesting that yields of PTH-arginine and PTH-histidine are improved on the gas-phase instrument. The ultimate test for microsequence analysis is a sample which has been isolated at the subnanomole level and for which the sequence is unknown. In this category, 200 pmol of a tryptic peptide from human insulin receptor was isolated by reverse-phase HPLC (for a description of the method see Ref. (23), and of the sample preparation Ref. (24)) and subjected to microsequence analysis on the gas-liquid-solid phase instrument. The chromatograms shown in Fig. 10 give the complete sequence of the 15-residue peptide, a result in agreement with amino acid analysis (Table 3). Since only 40% of each cycle is analyzed, only 35 pmol of PTH-isoleucine is injected at cycle 1, giving an initial yield of 44%, and by cycle 10 the amount injected is less than 1 pmol (detected but not integrated at this level). The interpretation of the chromatograms is unambiguous through cycle 8 (AUFS at 0.004), but the baseline level and sequencer noise become significant compared
A NOVEL
323
MICROSEQUENCER S 14
a
j- /4UFS
AUFS = 0.016
.0120r
‘CYCLE
.0090
.0075
1
1
CYCLE
= 0.008
IO
I
12
GTT .0030
-
6
12
6
TIME
b
AUFS:
.0064
-
.0056
-
.0048
-
CYCLE
, 12
(mln)
0.008 15
CYCLE
16
K Sld 20
CYCLE
18
24
.0040 z :ru .0032 T r
(A),I
I
6
I2
TIME
6
12
(min)
FIG. 7. Chromatograms from microsequence analysis of 250 pmol of myoglobin. Forty percent of each cycle was analyzed on the same system described in Fig. 6. The pmol quantitated for each injection are shown below the peak identified. The carryover peak from the previous cycle is shown in parentheses. (a) Cycles 1, 3, 10, and 12. (b) Cycles 15, 16, 18, and 19.
to signal level for cycles 9-l 6 (AUFS at 0.002). In spite of this the identifications are clear at least through cycle 14, and require some experience to identify PTH-arginine at cycle 15. These results illustrate a reasonable
lower level of analysis for a peptide. If the analysis began with 10 pmol at cycle 1 and gave similar results only 0.1 pmol would be detected by cycle 9, a situation beyond our current level of analysis.
324
HAWKE,
Ave. Rep. Yield
HARRIS, AND SHIVELY
= 94.6%
VLSEGEWPLVLHVWAKVEA+
lo-
2
4
6
CYCLE
8
10
12
14
16
18
20
NUMBER
FIG. 8. Yields of PTH amino acids from microsequence analysis of 250 pmol of myoglobin. The top, middle, and bottom curves show the analysis for 400, 250, and 100 pmol, respectively. A least-squares fit of the yield of PTH amino acid at each cycle was used to calculate the average repetitive yield for each curve.
Efect of Glass Components on Performance
Initially, we constructed the cartridge and conversion flask from glass and noted poor yields of PTH-serine and PTH-threonine. For serine the major peaks observed were dehydroserine and the so-called DTT-serine adduct (4) and for threonine, dehydrothreonine was observed. It had been suggested that the dehydration reaction is facilitated by acylation of the hydroxyl groups during cleavage (25), and that the addition of methanol to the sample would prevent or reverse the reaction. Since it is only convenient to add methanol during the conversion phase of the chemistry, this was tried. Indeed, a dramatic increase in the yields of PTH-serine and PTH-threonine were observed for the model peptide tested (Table 4). In spite of the improvement, the hypothesis was incorrect, since the same measure of protection
was observed by prewetting the conversion flask with acetonitrile, which is unlikely to act as a scavenger or transesterification agent. We reasoned that exposure of PTH-serine and PTH-threonine to dry glass at 55°C resulted in the dehydration reaction, and that the predelivery of solvent in some way (hydrogen bonding) masked the reactive silanol groups. To further explore this possibility, we constructed a conversion flask from PFATeflon (this material is sufficiently translucent for liquid to be easily seen in the converter) and repeated the experiment. The same high yields of PTH-serine and PTH-threonine were observed whether or not the flask was prewetted with solvent. We conclude that the glass surface is primarily responsible for the dehydration reaction. Since it also seemed likely that lower yields of PTH-histidine were possible on glass surfaces (PTH-histidine gives low yields off of poorly “endcapped” reversephase columns), we also constructed the reaction cartridge from Teflon (entry and exit of liquids can be monitored by careful observation of the Teflon delivery lines). One consequence of this change was lower overall background levels for DPTU and PTC-DEA. Although at first we thought this was due to higher retention of these background peaks on glass surfaces3 we now consider it more likely that we are effecting a better seal in the Teflon cartridge compared to the glass cartridge. The observation that the level of DPTU background reflects cartridge seal is easily demonstrated by purposely (or accidentally) compromising the seal in either system. In this respect, we believe that a Teflon flare fit to a glass surface is more prone to leak and cause background accumulation than are the pull-through fittings in our cartridge. 3 It was this idea which prompted us to trimethylsilylate the sample disks. This resulted in shghtty better backgrounds. The suggestion of one reviewer to try to “cover” sites by using more Polybrene actually gave higher backgrounds, presumably by increased retention of byproducts by Polybrene itself.
A NOVEL AUFS
MICROSEQUENCER
T 0.032
,024
1: YCLE
190 Ll
2
CYCLE
4
CYCLE
5
,018
,006
L 6
12
I 6
12
TIME
I
,
1
6
12
6
12
(men)
FIG. 9. Chromatograms from microsequence analysis of a synthetic small peptide. One nanomole of the peptide FNGLRG-amide was sequenced and analyzed as described in Fig. 7. The background peaks near PTH-phe (F) on cycle 1 are identified in cycle 2; DEA refers to the PTCderivative of DEA. The background at PTH-gly (G) is due to precycling Polybrene with glycylglycine. The last amino acid (glycinamide) is not detected.
DISCUSSION
Reagent and Solvent Delivery System Design A critical feature of the instrumentation is the delivery system, since both strong base and strong acid are used in Edman chemistry. In order to avoid cross-contamination, the reagents are delivered through “zero-deadvolume,” chemically inert valves and the lines are flushed with solvents compatible with the reagents. The Wittman-Liebold valve in its original (2) and miniaturized version (12) was an improvement over existing technology because of its low dead volume and the ability to have multiple inputs with a single exit line which is cleared of liquid by a pressurized-gas blow-out line integral to the system. Disadvantages of the valves were the great difficulty in machining them to proper tolerances, the use of a single valve block for multiple inputs (if one malfunctioned or was machined improperly the entire block had to be discarded), the fact that the liquid seal must be maintained over a large
surface area (limiting allowable back-pressures), and the use of positive gas pressure to maintain the closed position (loss of gas pressure causes the valves to leak and crosscontaminate reagents and solvents). The latter problem was partially solved by introducing mechanical solenoids to close the valve diaphragms, but this approach was not totally successful since a “vacuum assist” is also required to actuate the valves. A second problem encountered in the delivery system of some instruments is the bottle seal. The use of conventional O-rings or gaskets is not recommended unless they are truly inert to the reagents and solvents; in this regard, the material Kal-Rez (Du Pont) would be expected to perform well. A third problem is the tendency of the bottle-pressure regulators to drift. Since consistent delivery volumes (for liquids or gases) depend on reproducible bottle pressures, both the bottle seals and the bottle pressures must be reproducible. In consideration of these problems, our instrument has several unique features. The delivery valves (Fig. 3) are zero dead volume,
326
HAWKE,
a
AUFS
.0064
HARRIS, AND SHIVELY
= 0.008 I 35 Sid
.0056 CYCLE
1
1 CYCLE
I .0048-
2
CYCLE
3
CYCLE
4
.0040F 5
.0032-
OTT
[
v
7t-s-
IJe---e-&h
b ,;
TIME
b
AUFS
(min)
= 0.004
.0036
.0032
Sld
CYCLE
5
CYCLE
6
;td
CYCLE
7
CYCLE
8
.0028
! DTT
I
I
4
L
8
/
J I
I
12
4
8
/
12 TIME
I
I
4
8
I
I2
(min)
FIG. 10. Chromatograms from microsequence analysis of a tryptic peptide from human insulin receptor. The sample (200 pmol) was analyzed as described in Fig. 7. (a) Cycles 1-4; (b) cycles 5-8; (c) cycles 912; and (d) cycles 13-16.
made of inert materials (Teflon or Kel-F), modular (two inputs per valve, as many valves can be linked as isnecessary), electromechanically actuated (no compressed air or vacuum required), able to withstand high
backpressures (the seal is formed over a small area; backpressures up to 30 psi are tolerated), and easy to machine (the lumen is a straight pass in drilling; the unit cost is about $100). The valves have performed well on both gas-
A NOVEL C
AUFS
327
MICROSEQUENCER
= 0.002
stu
.0016-
Std
CYCLE
CYCLE
10
(ZYCLE
11
12
I T
DT
I
d .0016 .0016-
.0014
r
I
I
I
I
I
I
4
8
12
4
8
12
‘-e-&-e
TIME
(min)
AUFS
Std
CYCLE
I
I
I
4
8
12
CYCLE
16
= 0.002
I I: -
DTT
Std
CYCLE
13
14
CYCLE
.OOlZ -
15
DTT
DTl
OTT
.OOlO f z
.0008i .0006
-
.0004
-
.oooz .0002
t I
1 I 4
/ 8
I 12
I
I
I
4
8
12
II
4
TIME
FIG.
I
8
I
12
I
I
4
8
12
(min)
IO-Conhued.
liquid-solid phase and spinning cup instruments over a period of 3 years (if a solenoid malfunctions it can be replaced, leaving the valve body still functional). The bottle closure design (.Fig. 4) is based on a compression fitting which allows repeated opening and
resealing, requiring only a finger-tight seal. Only Teflon contacts the liquid or vapor, and the tubing seals are made by “pullthrough” fittings. The bottle-pressure regulators are 4-in. precision regulators and do not drift with time. The vent system, standard
328
HAWKB,
HARRIS, AND SHIVELY
TABLE 3 AMINO
ACID COMIWSITION OF TRYPTIC PEPTIDE OF HUMAN INSULIN RECEPTORS
Amino acid
picomoles
Residue
Cys'" Asx Thr Ser GlX Pro GlY Ala Val Met Be Leu TY~ Phe His Trp LYS AC3
20 93 37 37 180 27 50 50 0 0 15 39 0 0 0 0 0 32
0.6 (1) 2.9 (3) 1.1 (I) 1.1 (0) 5.6 (6) 0.8 (0) 1.6 (0) 1.6 (1) 0 (0) 0 (0) 0.5 (I) 1.2 (1) 0 (0) 0 (0) 0 6-o 0 (0) 0 (0) 1.0 (1)
’ The sample (60 ng, or 40 pmol) was hydrolyzed for 24 h at 110°C in 6 N HCl containing 0.2% 2-mercaptoethanol. Amino acid analysis is described in paper VI of this series (submitted). Since the sample was S carboxymethylated, cysteine was determined as the cys”” derivative. The values are normalized to one arginine per mole (32 pmol). Predicted values from sequence analysis are shown in parentheses. An equivalent acid hydrolysis blank gives IO-20 pmol of Ser, Gly, and Pro; thus, these amino acids are considered to be at background levels.
matic in the TMA-water system. This change in TMA concentration with time is slower in the “blow-across” method than in the “bubble-through” method. The number of cycles which can be run per bottle of TMA (25 or 12.5%) is greater than for 2% TEA (200-300 versus 100) but the overall base consumption is greater in the TMA-based system. Chemistry and Background
Peaks
The biggest problem associated with gasliquid-solid phase chemistry at the beginning ( 198 1- 1982 in our laboratory) was the enormous background due to DPTU (often >500 pmol). This was in contrast to the spinning cup instrument for which the DPTU background can be entirely eliminated. We reasoned that the stronger base in this system (pH > 9) and the increased amounts of PITC (15% v/v in heptane) led to increased hydrolysis of PITC to aniline which in turn reacted with PITC to give DPTU. DPTU is especially stubborn to remove, since it is only marginally soluble in ethyl acetate (S2). Reducing the PITC level to 5% reduced the DPTU peak (actually two peaks are observed, one is DPTU and the other DPU) to about the
TABLE 4
in older design instruments, has been eliminated as an unnecessary variable in the bottlepressure-delivery system. R2 and R3 are delivered by bubbling nitrogen through the liquids to carry vapor over to the cartridge as described by Hewick et al. (12). Currently, the Applied Biosystems instrument uses a “blow-across” method for delivering R2, an approach which probably reduces the amount of water and base vapor delivered, thus lowering base related background. We have seen no difference between the two methods for delivering R2 (TEAwater) in our system, suggesting that the difference is related to the rapid change in partial pressure of base, an effect more dra-
EFFECT OF PREWETTING CONVERSION FLASK (GLASS) WITH S4 (ACETONITRILE) ON YIELDS OF SER AND THR
Prewetted (pmol) Amino acid
Yes
No
Thr Ser Glu LYS Ser Gin
361 (2.7) 385 (1.9) 137 1185 195 277
201 (.58) 74 (.32) 742 1269 71 279
Notes. The approximate ratio of authentic to dehydro derivative is given in parenthesis. The results are the same for methanol or acetonitrile as S4. Approximately 1.5 nmol of the peptide TSEKSQTP was sequenced in duplicate.
A NOVEL
MICROSEQUENCER
lOO-pmol level, a level still not compatible with high-sensitivity analysis. A further improvement was observed in changing R2 from 25% TMA to 2% TEA. The background was highest with freshly made TMA and slowly decreased with use, while the background with 2% TEA was lower and remained steady throughout use. Apparently, the latest solution to this problem with the Applied Biosystems instrument is to reduce PITC to 2.5% and TMA to 12.5%, and to “blow across” rather than “bubble through” R2. A third factor involved was the cartridge seal. This factor became obvious in the glasscartridge system when we noticed that the background changed each time the cartridge was opened and the Zitex seal was disturbed or replaced. We therefore replaced the Zitex seal with a Kal-Rez gasket and used a Teflon screen as a support for the glass-fiber filter. This tactic reduced the DPTU background to 5 pmol or less. A similarly low background was seen for an all-Teflon cartridge, with no difference in background for a Zitex seal, a Kal-Rez seal, or no seal (Teflon is self-sealing). We concluded that an improperly sealed cartridge will accumulate DPTU in the space between the cartridge surfaces, and will slowly leach out during an S3 rinse to the conversion flask, rather than be washed out entirely during the S2 rinse to waste. Furthermore, the situation is not remedied by following the S2 rinse with an S3 rinse prior to cleavage (data not shown). Other background peaks include DTT (or DTE) which is added to preserve the solvents, and the PTC derivative of DEA (or DMA in the case of TMA), a contaminant in TEA. The possible overlap of PTH-Asp (and sometimes PTH-Glu) with the DTT peaks (the reduced and oxidized forms) has prompted Hewick et al. (12) to convert them to their methyl ester derivatives. This is most conveniently performed during the conversion reaction by using methanolic HCI instead of 25% aqueous TFA as R4. Although we have not taken this approach, we see no major disadvantage other than the fact that the
329
reagent (MeOH/HCl) is unstable at room temperature and is known to rapidly convert to a mixture of methanol, water, HCl, and methyl chloride (26). The removal of DEA from TEA or DMA from TMA is achieved by distillation of the base from a substance which reacts irreversibly with primary or secondary amines. For this purpose we have chosen tosyl chloride, and Hewick et al. (12), phthalic anhydride. In either case, the last traces of secondary amine are not removed, as evidenced by the presence of their PTCderivatives in published chromatograms (12, 13,22, this work). More careful distillation and processing of the bases may lead to lower levels of these peaks in chromatograms. The use of aqueous TEA as a coupling base may have a number of advantages over aqueous TMA. We have a cycle time of 42 min compared to over 60 min for the Applied Biosystems instrument. This is due to shorter coupling times, and shorter delivery and blowout times. The latter is achieved by using standard-bore (0.04-in.) Teflon tubing in place of microbore (0.01 in.). As originally described (12) a double-coupling program was advocated together with 15% PITC, probably leading to increased production of DPTU since two deliveries of PITC were necessary. These problems have now been solved by lowering the PITC concentration in Rl and by using a single-coupling program. Conclusion The advent of automated microsequence analysis has helped to revitalize and update the efforts of protein chemists in terms of the types and amounts of samples which can be analyzed. The availability of the instrumentation has substantially improved our knowledge of the Parameters which affect performance and the requirements of the sample preparation for compatibility. Since the field is new the variety in instrumentation is limited and the price high. It is our hope that the description of a new instrument with similar capabilities but different design fea-
330
HAWKE,
HARRIS, AND SHIVELY
tures will further stimulate the field and suggest commercially attractive alternatives. ACKNOWLEDGMENTS We gratefully acknowledge the advice and encouragement of Dr. Chas. W. Todd. We are pleased to acknowledge the design input and many hours of careful work in construction of these instruments by Clifford Sailor and Lance Richardson at the City of Hope Biomedical Instrumentation Department. The design and construction of the zero-dead-volume valve was by Miro Rusnak from the same department. We thank Dr. Michael Hunkapiller for discussing the early Caltech prototype instrument with us before publication, and Chad BenAvram for a critical appraisal of the manuscript.
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