ANALYTICAL

206,344-352

BIOCHEMISTRY

(1992)

Sequencing of Peptides and Proteins from the Carboxy Terminus Victoria

L. Boyd,l

Applied

Biosystems,

Received

April

MeriLisa

Bozzini,

Inc., 700 Lincoln

Gerald

Zon,

Richard

Centre Drive, Foster City, California

Academic

and Robert

J. Mattaliano

94404

24, 1992

A new chemical method for carboxy-terminal (C-terminal) protein sequencing has been developed. This approach has been successfully used to sequence 5 residues of standard proteins and 6 to 10 residues of synthetic peptides at low nanomole levels. The sequencing procedure consists of converting the C-terminal amino acid into a thiohydantoin (TH) derivative, followed by transformation of the TH into a good leaving group by alkylation. Next, the alkylated TH is cleaved mildly and efficiently with { N=C = S}- anion, which simultaneously forms a TH on the newly truncated protein or peptide. Thus, after the initial TH derivatization, there is no return to a free carboxyl group at the C-terminus. An additional benefit of this method is that the alkylating moiety can be chosen with a variety of properties allowing for variation in the detection method. This chemistry has been adapted to automated protein sequencers with a cycle time of about 1 h. 0 1992

L. Noble,

Press,

Inc.

The amino acid sequence of a protein or peptide establishes the identity of the molecule and provides information regarding its structure, function, and homology to related proteins. Chemical microsequencing of proteins and peptides from the amino terminus (N-terminus)2 continues to play a key role in the identification i To whom correspondence should be addressed. 2 Abbreviations used: N-terminus, amino terminus; C-terminus, carboxy terminus; TH, thiohydantoin; ATH, alkylated thiohydantoin; Me,SiNCS, trimethylsilyl isothiocyanate; DMF, dimethylformamide; NMP, N-methylpyrrolidone; CH,CN, acetonitrile; HSCN, thiocyanic acid; TFA, trifluoroacetic acid, WRK, Woodward’s reagent K; DITC, 1,4-phenylene diisothiocyanate; DSS, disuccinimidyl suberate; 1,5-I-AEDANS, 1,5-N-iodoacetyl-N’-(sulfo-l-napthyl)ethylenediamine; E&N, triethylamine; DIEA, diisopropylethylamine; MeOH, methanol; CD,OD, deuterated methanol; HBTU, 2-(lHbenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; PyBroP, bromo-tris(pyrrolidino)phosphonium hexafluorophosphate; &lac, &lactoglobulin; AAA, amino acid analysis.

of rare proteins. Many of these proteins are developmentally regulated or found in small quantities and often are the target of gene cloning efforts. Sequence information from the carboxy terminus (C-terminus) of proteins would facilitate the production of more specific probes for gene cloning. Also, a practical method for C-terminal sequencing would provide an alternative approach for proteins and peptides that are N-terminally blocked and potentially yield information on posttranslational modifications at the C-terminus. The development of an efficient method for C-terminal sequencing that is analogous to Edman degradation (1,2) used for N-terminal sequencing has been a difficult goal. Unlike an amino moiety, selective derivatization of a carboxyl group is difficult to achieve. Chemical methods used to derivatize a carboxyl group can also modify other functional groups within a protein or peptide. Repeated exposure of the sample to reagents, high temperatures, and long reaction times increases the probability of additional unwanted side reactions. The activated terminal carboxy group must then be treated with a nucleophile to suitably functionalize the C-terminal amino acid residue and subsequently provide a detectable derivative, such as a thiohydantoin (TH) (3). After successful derivatization, selective and efficient cleavage of the amino acid derivative is required. In comparison, the selective cleaving of the derivative in N-terminal sequencing is neatly avoided by a facile intramolecular cyclization/cleavage process, which contributes significantly to the overall success of Edman degradation (4). Prior to our work, the most developed approach to C-terminal sequencing was that of Stark (3), which was based upon the earlier method of Schlack and Kumpf (5). Activation with acetic anhydride to form a mixed anhydride at the C-terminus is followed by treatment with thiocyanate {N=C=S}anion, producing a TH derivative. This TH moiety is similar in structure to the phenylthiohydantoins formed in Edman degradation. Several strategies for the subsequent cleavage step have

344 All

Copyright 0 1992 rights of reproduction

0003-269’7192 $5.00 by Academic Press, Inc. in any form reserved.

CARBOXY-TERMINAL

R’

SEQUENCING

OF

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AND

PROTEINS

345

resultant alkylated thiohydantoin (ATH) is a good leaving group in contrast to the parent TH. Subsequent cleavage using the Lewis acid trimethylsilyl isothiocyanate (Me,SiNCS) not only releases the ATH, but also simultaneously converts the freshly exposed C-terminus into a thiohydantoin. This novel approach allows the sequencing of 2 nmol or less of standard proteins for up to 5 cycles and up to 10 cycles on 2 nmol or less of synthetic peptides.

r\

J

base

R-X MATERIALS

has:*

HX

‘N=C=S

i

i-R

SCHEME

1. Two-step cycle for the stepwise degradation of a protein or peptide from the carboxy terminus. A peptidyl thiohydantoin is S-alkylated under basic conditions to form a good leaving group. In the second step, the isothiocyanate anion with acid assistance cleaves the alkylated thiohydantoin (ATH) while simultaneously forming a new thiohydantoin at the truncated C-terminus.

met with varying degrees of success. Acid and base hydrolysis, as well as nucleophilic cleavage under basic conditions followed by hydrolysis, have each been investigated (6-12). Practical application of the Stark approach is nevertheless compromised by a rapid drop in efficiency, presumably due to competing side reactions during activation and cleavage. Even with improved reagents (13,14), reliable sequencing requires several nanomoles of peptides composed of certain amino acids and proceeds for only a few cycles (6,9,11,12,15). There are considerably fewer claims for sequencing proteins or smaller quantities (~1 nmol) of peptides (7,8,10). Here we report a new approach to C-terminal sequencing (Scheme 1) which is based on some properties of the thiohydantoin ring system. As in the Stark approach, the C-terminus is first derivatized to a thiohydantoin. However, unlike all previously reported methods, the C-terminal TH is next S-alkylated. The

AND

METHODS

The following reagents and solvents were purchased from Aldrich (Milwaukee, WI): l-bromomethylnaphthalene (lachrymator, irritant), Me,SiNCS, 1,5-Niodoacetyl-N’-(sulfo-1-naphthyl)ethylenediamine (1,5I-AEDANS), triethylamine (EtaN), pyridine, formic acid, hydrogen peroxide (30% in water), dimethylformamide (DMF), 2-ethyl-5-phenylisoxazolium-3’-sulfonate (Woodward’s reagent K; WRK), and ethoxycarbonyl isothiocyanate. Trifluoroacetic acid (TFA; extremely corrosive), acetonitrile (CH,CN), methanol (MeOH), N-methylpyrrolidone (NMP), 2-(lH-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 50% cross-linked polystyrene beads (16), and t-butoxycarbonyl (t-Boc)-amino acids were supplied by Applied Biosystems, Inc. (ABI, Foster City, CA). Apomyoglobin (horse) attached to glass beads via 1,4-phenylene diisothiocyanate (DITC) and /3-lactoglobulin (P-lac) were obtained from Sigma (St. Louis, MO). Disuccinimidyl suberate (DSS) was purchased from Pierce (Rockford, IL). Bromo-tris(pyrro1idino)phosphonium hexafluorophosphate (PyBroP) was purchased from Novabiochem (San Diego, CA). Methylene chloride (CH,Cl,) was obtained from Burdick and Jackson (Muskegon, MI). Silica gel (60 A, 70-230 mesh) was purchased from Scientific Products (McGaw Park, IL). Initially, all experiments were performed manually in solution using amino acids, dipeptides, and tripeptides. The products and intermediates were purified by column chromatography, when possible, and were characterized by ‘H NMR spectroscopy (300 MHz, Varian). Synthesis of an alkylated TH reference standard. One millimole each of ethoxycarbonyl isothiocyanate (13,14), pyridine, and a t-Boc-amino acid are dissolved in CH,CN (10 ml) and allowed to stir 1-18 h. The resulting N-1-t-Boc-TH is purified by column chromatography (silica gel, 9:l CH,Cl,:MeOH). The purified t-Boc-TH is reacted with 1 M equivalent each of triethylamine and l-bromomethylnaphthalene in a small amount of CH,CN at 60°C for 10 min. The Et,N * HBr salt precipitates at -4°C and is removed by filtration. The t-Boc protecting group is removed by treating the t-Boc-ATH with TFA:CH,CN (25:75) for 30 min at 60°C. The ATH precipitates out of the TFA:CH,CN at -4°C. The ATHs

346

BOYD

for the amino acids derived from Ala, Leu, Phe, Tyr, Gly, Gln, and Glu have been obtained in this manner. The NMR spectra are consistent with the presence of a single monoalkylated product, and the HPLC retention times are identical to those of the ATHs obtained from the sequencing runs of synthetic peptides and proteins. The ATH from glutamic acid retains an underivatized side-chain carboxyl group from this synthetic procedure. At this time, the molecular weights of independently synthesized ATH-amino acid derived from Leu and Ala have been verified using the ABI BioIon mass analyzer. Preparation of modelpeptides. Peptides were synthesized using an ABI Model 431A peptide synthesizer with the standard ABI Fastmoc reaction cycles on a 0.25mmol scale. Deprotection and cleavage were by standard methods. Products were characterized by HPLC and amino acid analysis (AAA) using an ABI Model 420H hydrolyzer amino acid analyzer. Covalent attachment of peptides and proteins to an amino-functionalized support. Attachment to 50% cross-linked aminomethyl polystyrene beads (26 pmol amine/g) was achieved using DSS. DSS (15 mg) was dissolved in 760 ~1 NMP containing 10% (v/v) pyridine and diisopropylethylamine (DIEA). The polystyrene beads (100 mg) were shaken with the DSS solution for 1 h at room temperature and then washed with NMP. TFA-treated /3-lac (17) or a model peptide (lo-15 mg, -10 pmol) was dissolved in NMP/lO% pyridine (360 pl), added to the polystyrene beads, and shaken overnight at room temperature. The beads were sequentially washed with NMP and CH,CN and then dried under vacuum. &Lac was oxidized to convert Cys to cysteic acid using performic acid (18) at 0°C for 1 h. The performic acid was removed under vacuum. Formation of a C-terminal TH on a peptide or protein using Woodward’s reagent K (19). The C-terminus was activated with 500 ~1 of a 0.12 M ketenimine solution formed from WRK (62 mg) and DIEA (50 ~1) in 2 ml CH,CN. After 4 h at room temperature the ketenimine solution was removed, and the beads were washed with CH,CN twice and then shaken overnight in 500 ~1 of a 10% solution of Me,SiNCS in CH,CN. After washing with CH,CN and H,O, the beads were dried and used for sequencing. Automated C-terminal sequencing. Sequencing was performed on an ABI Model 477A protein sequencer, without any hardware modifications, using the solvents, reagents, and representative reaction and conversion cycles indicated in Table 1. The HPLC conditions are provided in Table 2. The reaction temperature was 55°C and the conversion flask was 4O’C. Automated preparation of a TH at the C-terminus. The C-terminus was converted to a thiohydantoin in a

ET

AL. TABLE

1

Representative Reaction and Conversion an ABI Model 477A Protein Sequencer for mated Carboxy-Terminal Sequencing” A. Reaction

cycle

Step

Function

8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

Prep Sl Deliver Sl Pause Argon dry Prep R3 Deliver R3 Argon dry Pause Argon dry Prep R3 Deliver R3 Argon dry Prep Sl Deliver Sl Argon dry Pause Argon dry Deliver S2 Argon dry Deliver S2 Argon dry Prep Rl Deliver Rl Pause Argon dry Pause Prep R2 Deliver R2 Pause Argon dry Prep transfer Transfer w/S3 Pause Transfer w/argon Transfer w/S3 Pause Transfer w/argon End transfer Deliver S2 Argon dry

Cycles Performing

B. Conversion Time(s) 6 15 10 6 6 2 8 700 10 6 1 8 6 2 6 600 10 60 20 60 120 6 5 10 5 60 6 20 600 300 15 14 20 10 14 20 10

Step

8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Used on Auto-

cycle

Function Clear Deliver Argon Empty Prep Deliver Argon Pause Clear Empty Ready Argon Pause Load Argon Load Argon Pause Argon Pause Load Inject Pause Prep Deliver Argon Empty Load Argon Empty Argon

inj to waste S4 dry R5 R5 dry inj to waste to receive dry S4 dry S4 dry dry injector

R4 R4 dry S4 dry dry

Time(s) 120 10 4 30 6 5 4 5 200 20 90 120 5 8 5 8 160 30 30 15 24 1 120 6 12 10 30 6 4 60 120

100 120

D Reagents and solvents: Rl, 10% Me,SiNCS in CH,CN; R2, TFA; R3, 2.5% bromomethylnapthalene in CH,CN; R4, MeOH; R5, CH,CN; Sl, 10% DIEA in CH,CN; S2, CH,CN; S3, MeOH; S4, 7% CH,CN in H,O.

modified first cycle using chlorouronium chloride activation (20). A 10% solution of 1,1,3,3-tetramethyl-chlorouronium chloride in CH,CN is delivered from the Xl bottle position. After deliveries of Sl, Xl, and Rl, there is a 5-min pause, a second delivery of Rl, and then a 100-s pause. The sequencing is continued as described in Table 1.

CARBOXY-TERMINAL TABLE

SEQUENCING

OF

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2

HPLC Conditions for the Analysis of the Cleaved Amino Acid Derivatives (ATHs) from Carboxy-Terminal Sequencing” Run time Maximum pressure Minimum pressure Target pressure Target time Equilibration time Time (min)

Concentration (%70)

20.0 25.0 35.0 35.1

s

35.1 min psi 0 psi 2000 psi 0.1 min 12.0 min

Flow (jdlmin)

20 57 75

0.0 10.0

0

5000

B

300 300 300 300 300

100 100

347

PROTEINS

2

20

It

Event 34 4 4 4 4 0

D An ABI Model 120 HPLC was equipped with an ABI PTC, C-18, 5-pm column; oven temperature, 38°C; solvent A, 50 mM sodium acetate buffer, pH 5.4; solvent B, 70% CH,CN/32 mM sodium acetate, pH 6.2.

RESULTS AND DISCUSSION Once formed at the C-terminus, an amino acid-TH is difficult to cleave from the parent peptide or protein because the required hydrolysis or nucleophilic displacement involves the scission of a rather stable bond. TH release strategies as previously described are nevertheless conceptually attractive because these five-membered heterocycles are structurally analogous to PTHamino acids. PTH-amino acids are the more stable rearrangement product of the anilinothiazolinones produced during Edman degradation (21) (Fig. 1). On the other hand, the chemical reactivity and stability of a TH are very different from those of a PTH-amino acid. A resonance stabilized anion of the TH can be formed by deprotonation at the N-3 position, the position occupied by the phenyl substituent in a PTH. Therefore, a TH or peptidyl-TH has a relatively low pK,, value of -7 (3~0) (Fig. 2) and is deprotonated readily, thus offering the prospect of rapid alkylation under basic conditions.

FIG.

2.

Resonance

structures

for the ionized

peptidyl-TH.

An investigation of the nucleophilic reactivity of the peptidyl-TH anion revealed that alkylation of N-l-acylTH in fact occurs rapidly and results predominantly in the S-alkylated product as expected. Experimentally, this regioselective alkylation is supported by basic methanolysis of an N-1-t-Boc-amino acid-TH that has been alkylated with bromoethylacetate. If alkylation is occurring at the sulfur atom in the TH moiety, then the sulfhydryl (2-mercaptoacetate) should be obtained. This

H

R

/O +f HNvN 4-R' OH

S-R'

S-R'

R

OH

NYNH PTH FIG. 1. toin (TH), peptidyl-TH,

TH

ATH

Structures of a phenylthiohydantoin (PTH), a thiohydanand an alkylated thiohydantoin (ATH). In a proteinylor the site of attachment is through the N-l position.

FIG. 3. Tautomerism uct of the sequencing R is the amino acid alkylating reagent.

of an alkylated-TH (ATH), the cleaved prodapproach presented in Scheme 1. In this figure, side chain, and R’ is the alkyl group from the

348

BOYD

MfLL Cycle 1 0.024A”FS

W

Cycle 2

II

ET

AL.

pounds S-alkylate a tripeptide-TH model system within 10 min at 60°C. Benzyl bromide analogs in particular are advantageous because of their high reactivity, inherent ability to introduce a uv-detectable moiety onto the alkylated product, and relative stability in solution. Also, numerous benzyl bromide analogs possess the added benefit of high volatility, rendering them good candidates for vapor-phase delivery on automated instrumentation. Alkylation with benzyl bromide analogs increases the hydrophobicity of the resultant ATHs, which significantly increases their retention on reversed-phase HPLC columns relative to the nonalkylated THs or

Cycle3 Ii

us

Phe

Cycle 1 0.05AUFS

-_.,II

Cycle 4

.

. .

..I.

,_.,I, Cycle 2

HIS

Cycle 3

&i&!-L

FIG. 4.

C-terminal sequence data of 1.6 nmol of sequence-grade apomyoglobin (Sigma) covalently attached to DITC glass beads. The C-terminus is converted to a thiohydantoin in a modified first cycle using chlorouronium chloride activation as described under Materials and Methods. The HPLC analysis for the sequence run did not resolve ATH-Phe from the artifact peak due to the thiocyanate derivative of l-bromomethylnaphthalene in cycle 3. The uv was monitored at 254 nm and the absorbance scale is 24 mAUFS for each cycle shown.

sulfhydryl compound was in fact isolated and then verified by comparison to authentic material utilizing TLC and NMR spectroscopy. Additionally, the S-alkylation of cyclic thioureas, including 2-thiohydantoins, is supported by literature reports (22-25). Alkylating reagents of the type X-CH,-aryl and XCH,C(O)Y, where X = halogen, triflate, etc., and Y = alkyl, aryl, OR, NR,, etc., are known to be useful for protein modification (26). In the course of the presently described investigations, we have found that these com-

Cycle 4

Gill

...

Cycle 5

(‘3~)

FIG. 6.

C-terminal sequence data on 1.3 nmol of @-lac using the cycles and HPLC conditions described in Tables 1 and 2. Attachment to 50% cross-linked aminomethyl polystyrene heads and preparation of the proteinyl-TH using WRK were achieved as described under Materials and Methods. The uv was monitored at 254 nm. The ahsorbance scale is 54 mAUFS for each cycle shown.

CARBOXY-TERMINAL

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349

PROTEINS

II-

Cycle 6

GIY

lJi

Cycle 7

Cycle 2 Phe

t

I Gln

Cycle 6

1

Cycle 9

Cycle 4

il

I

Cycle 10

Cycle 5

I

Time (min.)

Leu

16.0

20.0

cJ!A __

22.0

A 12.0

14.0

16.0

16.0

20.0

22.0

FIG. 6. An example of C-terminal sequencing data on 2.8 nmol of a synthetic peptide attached to polystyrene beads; the peptidyl-TH formed using WRK activation and the sequencing protocol is described in Table 1. Sequence calling was unambiguous for nine cycles. was monitored at 254 nm and the absorbance scale is 40 mAUFS for each cycle shown.

was The uv

350

BOYD ET AL.

those aikylated with charged reagents like 1,5-I-AEDANS. Their increased retention facilitates the separation of the ATH sequencing products from the hydrophilic by-products formed during this sequencing chemistry. Several other late-eluting compounds are usually seen. A major by-product has been identified by independent synthesis as the thiocyanate derivative of the alkylating reagent. Residual alkylating reagent and the hydroxyl version of the alkylating reagent are also seen. With respect to sequencing efficiency, the best results so far have been obtained using l-bromomethylnaphthalene as the alkylating reagent. It is not known if the low melting point (45’C) of this reagent or its high vapor pressure at the temperature (55°C) used to develop the automated chemical approach contributes to the success of this particular bromide as a sequencing reagent. Both DMF, a solvent of choice for alkylations (27), and CH,CN are used for the presently reported chemistry. Additionally, alkylation with l-bromomethylnaphthalene affords corresponding ATH reference standards readily isolated by precipitation. Alkylation of the THs with other alkylating agents results in products that appear to be less stable than those alkylated with benzyl bromide analogs. Such instability is possibly due to desulfurization of the TH to hydantoin (28) or to hydrolytic dealkylation. Cleavage of the N-l acyl bond that joins the ATH to the parent peptide/protein frees the lone-pair electrons of nitrogen that, prior to cleavage, are resonance delocalized toward the exocyclic carbonyl carbon. These lone-pair electrons, in combination with enolization of the ATH carbonyl at C-4, constitute a 4n + 2 electron orbital system. We believe that the energy barrier to the transition state for the formation of this cleavage product, relative to that for the unalkylated TH, is lower and therefore facilitates the cleavage reaction. The ‘H and 13CNMR spectra of two model ATH compounds support that enolization occurs (Fig. 3). In CD,OD, no carbonyl carbon resonance signal is seen for the ATH of Leu in the 13CNMR spectrum, and a vinylic doublet for the methylene attached at the C-5 position is seen in the ‘H NMR spectra. Similarly, the methyl group of the ATH of Ala is seen predominantly as a vinylic singlet at -2 ppm. No evidence of enolization is seen in the ‘H or 13CNMR spectra of the corresponding (nonalkylated) THs ( 19). The cleavage of the ATH is an acid-catalyzed reaction. Me,SiNCS is successful as a cleavage reagent presumably because the Me,Si group serves as a proton mimic. Under the reaction conditions, Me,SiNCS could hydrolyze to thiocyanic acid (HSCN); however, a role for HSCN, if it has any, has not been established. On the other hand, when a strong acid such as TFA is added after Me,SiNCS, more facile cleavage/derivatization occurs, compared to that with Me,SiNCS alone. A pre-

mixed solution of Me,SiNCS and TFA is unstable and rapidly turns red, most likely due to polymerization of HSCN that forms under the strongly acidic conditions (29). Although TFA itself rapidly cleaves the ATH-peptidy1 bond to give the same ATH that forms from the cleavage reaction with Me,SiNCS, this leaves the truncated peptide with a free carboxyl group that requires subsequent reactivation. As previously stated, the C-terminal amino acid must be derivatized to a TH to begin sequential C-terminal degradation of the peptide or protein. Initial TH formation can be achieved using one of several approaches. Candidate chemical methods include the Stark conditions (3,5-12), acylisothiocyanate/pyridine (13,14), and WRK (19). Although both the Stark reagents and an acylisothiocyanate reagent have been used for sequencing on automated instrumentation (6-9,13,14), we have found that their use is limited by competing side reactions and by-products that interfere with subsequent product identification by HPLC. In contrast, a sample can be cleanly prepared for sequencing by forming the first peptidyl C-terminal TH in solution employing WRK. However, WRK chemistry requires an overnight reaction with Me,SiNCS under mild conditions, rendering this procedure less suitable for rapid, automated sequencing. The potential benefits of an alternative activation chemistry prompted an investigation of the uronium salts of various N-hydroxy compounds, a new class of reagents for activation of carboxyl groups (20,30). A reactive l-hydroxybenzotriazolyl ester derived from HBTU does not react efficiently with thiocyanate anion to form a TH. 1,1,3,3-Tetramethyl-chlorouronium chloride, a more reactive halogenated precursor of HBTU, is not favored in peptide synthesis as it promotes racemization, but is suitable for our purposes. The combination of chlorouronium chloride and a tertiary amine (or a sterically hindered secondary amine, such as diisopropylamine) plus Me,SiNCS rapidly converts the C-terminus of peptides and proteins to a TH. The sequencing data for apomyoglobin shown in Fig. 4 was obtained using a chlorouronium reagent as part of a modified firstreaction cycle, followed by repeated application of the reaction cycle described in Table 1. The commercially available reagent, PyBroP (31), was also used in the automated formation of the peptidyl-TH, but with limited success. A reliable, routine, and sensitive method of assaying the free carboxyl content of proteins analogous to the ninhydrin assay for amines (32) is unavailable, to our knowledge. To estimate the efficiency of the activation of the carboxyl group during peptidyl-TH synthesis, the uv absorbance of the ATH formed and cleaved in the first cycle of sequencing can be compared chromatographically to that of an independently synthesized ATH standard of known concentration. The amount of

CARBOXY-TERMINAL

SEQUENCING

protein or peptide attached to a solid support was quantified by AAA. For the sequencing of l-2 nmol of protein or peptide, the initial yield of ATH is 2050%. Comparable initial yields are obtained with either WRK or chlorouronium chloride activation. Results obtained when sequencing 1.6 nmol apomyoglobin attached to DITC glass, and 1.3 nmol of /3-lactoglobulin covalently attached to 50% cross-linked aminomethyl polystyrene, are shown in Figs. 4 and 5. Correct identification of the C-terminal sequence for these proteins proceeded for five and four cycles. The retention times assigned to ATH products were verified by analysis of the independently synthesized ATH reference standards or the sequencing of synthetic peptides containing the same amino acid residues. Sequencing of l-2 nmol of the model synthetic peptides, up to 14 amino acid residues in length, also covalently attached to polystyrene, for 6 to 8 cycles is accomplished routinely. Correct sequence identification for up to 10 cycles with a repetitive yield of -67% is possible in some cases. Figure 6 provides an example of unambiguous sequencing for 9 cycles. While these representative examples with proteins and peptides indicate the current scope and soundness of this new sequencing method, several limitations are evident. Perhaps the most serious shortcoming common to all C-terminal sequencing methods is the obstacle to sequencing through proline. The formation of proline-TH would require quaternization of the nitrogen atom involved in the peptide bond. To form a TH, the proline-peptidyl bond must be cleaved simultaneously. Using the dipeptide model Ala-Pro, prolineTH is formed if water is present for the hydrolysis of the peptide bond during TH synthesis. The possibility of sequencing through proline under conditions compatible with the method outlined in Scheme 1 has not yet been fully explored. Peptides whose composition includes the 13 amino acids Ala, Asn, Arg, Gly, Ile, Leu, Gln, Lys, Phe, Tyr, Trp, Val, and Met have been successfully sequenced and their ATHs uniquely identified by reverse-phase HPLC. Synthetic peptides with these residues have been sequenced at the 1-nmol level for up to eight cycles. Samples containing Asp, Glu, Cys, or His have been sequenced successfully through to the next residues, although their unique ATH derivatives have not consistently been detected by HPLC. Cysteine is identifiable as cysteic acid when the protein or peptide is oxidized prior to sequencing (Fig. 5). Aspartic and glutamic acids do not stop the sequencing, as they are presumably derivatized during the first TH synthesis, which requires carboxyl group activation (chlorouronium chloride or WRK activation). In Fig. 5, a new HPLC peak in the second cycle is seen for the ATH of His; its appearance, although reproducible for this protein, was not seen

OF

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351

PROTEINS

when a synthetic peptide containing His was sequenced. A notable decrease in repetitive yield is often observed after sequencing through one of these four amino acids, except when Cys is oxidized to cysteic acid. Serine and threonine are currently problematic residues. Sequencing fails at the C-terminal residue prececling these amino acids. We assume that the hydroxyl group displaces the alkylated sulfur of the preceding TH ring concurrent with its formation, resulting in a bicyclic structure. Experimentally, sequencing through serine and threonine is successful on peptides synthesized with permanent protecting groups on the hydroxyl moiety. We are currently investigating methods of chemically modifying these amino acid residues. CONCLUSIONS

The unique alkylation and cleavage steps of this presented method may be largely responsible for its utility in sequencing peptides and proteins from the C-terminus. The alkylation step serves the dual purpose of improving the leaving group ability of the parent amino acid-TH, possibly via aromatic stabilization, and offering the added benefit of introducing a uv-absorbing or fluorescent moiety to provide enhanced detection of the cleaved product. The cleavage step is relatively selective and efficient. Further, since the next-in thiohydantoin is formed during cleavage, the activation of the carboxyl group at the beginning of each cycle is obviated. The ease and simplicity of this two-step process, which combines alkylation under basic conditions with cleavage/ derivatization under acidic conditions, has improved the ability for successful and reliable C-terminal sequencing. In summary, we find the presented novel chemical approach an improvement over previously reported chemical methods for sequencing proteins from the carboxy terminus. We recognize a number of challenging problems that must be addressed to improve the capabilities of this method to the level of robustness that has been attained over the years with N-terminal chemistry introduced by Edman. ACKNOWLEDGMENTS We thank Drs. Steven Menchen and Jack Richards for both their guidance and many useful suggestions. We also express appreciation to Sylvia Yuen for technical assistance, Christie McCollum for the aminomethyl polystyrene beads, Ling Chen for the BioIon data, and Tracy Rivas and Karen Felker for the preparation of the scheme and figures.

REFERENCES 1. Edman, 2. Edman,

P., and Begg, G. (1967) Eur. P. (1956) Acta Chem. Stand.

J. Biochem. 1,80-91. 10, 761-768.

3. Stark, G. R. (1968) Biochemistry 7,1796-1807. 4. Edman, P. (1950) Actu Chem. Scar& 4, 283-293.

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5. Schlack, P., and Kumpf, W. 170. 6. Bailey, J. M., Shenoy, N. R., Protein Sci. 1, 68-80. 7. Bailey, J. M., and Shively, J. Chemistry (Villafranca, J. J., Press, San Diego, CA.

(1926)

2. Physiol.

Ronk,

M.,

E. (1991) Ed.), Vol.

154,125-

Chem.

and Shively,

J. E. (1992)

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