J. Peptide Res.

49, 1997, 484-491 Printed in CJK nll rights reserved

~

Copyright 0 Munksgaard 1997 JOURNAL OF PEPTlDE RESEARCH

ISSN 1397-002X

Efficient peptide ladder sequencing by MALDI-TOF mass spectrometry using allyl isothiocyanate
QU-MING GU and GLENN D. PRESTWICH

Department o Chemistry and Department of Biochemistrj, and Cell Biology. University at Stony Brook, Stony Brook, f New York, U S A

Received 2 August. accepted for publication 20 December 1996

A new modification of the peptide ladder sequencing technique is described in which allyl isothiocyanate (AITC) replaces trifluoroethyl isothiocyanate as the volatile amine-modification reagent. AITC is commercially available, readily purified, stable up to 80 =C and reacts cleanly and rapidly with all amino groups of polypeptides. Several model peptides and two side chain-modified peptides were sequentially degraded using AITC and the cleavage reagent heptafluorobutyric acid (HFBA) up to seven amino acids from the N-terminus. Matrix-assisted laser-desorption and ionization coupled with time-of-flight (MALDITOF) mass spectroscopy of the peptide mixture provided a clear ladder-like mass profile with consecutive molecular ions corresponding to each shortened peptide at picomole range. The results indicate the general utility of this analytical protocol by the use of AITC as the amine-coupling reagent. 0 Munksgaard 1997.
Key words: allyl isothiocyanate; MALDI-TOF mass spectrometry; peptide ladder sequencing: post-translational and chemical modification of protein: protein sequencing

The sequence analysis of proteins and peptides by the stepwise removal of amino acids from the aminoterminus by Edman chemistry has been widely used for over 30 years (1, 2). The classical method utilizes phenyl isothiocyanate (PITC) as coupling reagent for N-terminal a-amino group, and generates phenylthiohydantoin (PTH) derivatives of amino acids that are then separated and identified by reversed-phase HPLC. However, most post-translational and chemical modifications of proteins and peptides cannot be identified by automatic Edman degradation (3). A complementary technique, peptide ladder sequencing (4), uses Edman chemistry to sequentially remove amino acids from the N-terminus of peptides. However, instead of analyzing in sequence the cleaved PTH amino acids, the peptide ladder sequencing method analyzes the mixture of shortened peptides by mass spectrometry. The development of matrix-assisted laser-desorption and ionization coupled with time-of-flight (MALDI-TOF) mass spectrometer ( 5 ) has made it possible to analyze rapidly small quantities of mixtures of peptides with ragged N-terminal ends. The original protein ladder sequencing method (4) has been successfully applied to only a few peptides, in part due to the use of organic solvents in an extraction
484

step that can lead to loss of hydrophobic peptides. This inherent disadvantage has limited the applications of this technique. Recently, another ladder sequencing protocol has been developed (6) using the volatile trifluoroethyl isothiocyanate (TFITC) as the N-terminal a-amino group coupling reagent and anhydrous heptafluorobutyric acid (HFBA) for the acid-catalyzed cleavage to release trifluoroethyl ureacoupled amino acids, plus a shortened peptide. This minimized sample loss during work-up and increased the sensitivity of analysis; however, problems remained with generality of the procedure due to unpredictable results with lysine-containing peptides, the need for a separate tagging reaction to improve the mass intensities, and the absence of a commercial source of TFITC. MALDI-TOF mass spectrometry (7) has emerged as a popular choice for analysis of mixtures of peptides and protein for reasons of high sensitivity, speed, accuracy and ease of operation (8-11). Proteolytic maps can be readily obtained without the need for prior HPLC purification; the technique for generating masses for protein identification is called peptide mass fingerprinting (12, 13). The variation of this MALDI-TOF technique has been used for rapid sequence determination of a peptide by analysis

Peptide ladder sequencing using allyl isothiocyanate of the shortened peptide mixtures after degradation either chemically (14, 15) or enzymatically (16, 17) from the N-terminus or C-terminus. We present herein an improvement on the ladder sequencing method using the volatile amine-selective reagent, allyl isothiocyanate (AITC). The principle of peptide ladder sequencing using AITC is demonstrated in Scheme 1. This strategy consists of four steps. First, the peptide is allowed to react with AITC to yield an Na-ally1 thiourea peptide; all free amino groups are modified quantitatively during this process. Second, the AITC-tagged N-terminal amino acid is removed from the peptide using organic acid to produce a shortened peptide. Third, an aliquot of the original peptide is added, and the AITC/HFBA cycle reaction is repeated. Steps 2 and 3 are iterated for i cycles. Fourth, the individual masses of the peptides in the mixture are measured by MALDI-TOF, providing a ladder of peptide masses that contains intact peptide ions and consecutive molecular ions corresponding to loss of 1 to i amino acids. The mass differences between consecutive peaks represent the corresponding amino acid residue lost. RESULTS AND DISCUSSION TFITC was synthesized as described by the authors (6), since a commercial source was unavailable. Use of TFITC with several model peptides revealed that this isothiocyanate reagent could react with a variety of nucleophilic side chain functionalities, such as the hydroxyl groups of serine or threonine, as well as lysine &-aminogroups. In the course of the coupling with a-amino group of peptides during ladder sequencing of peptide standards containing lysine residues, reaction conditions often determine the extent of modification of the lysine residue to a. trifluoroethyl thiourea. Substance-P (RPKPQQFFGLM-NH,) was chosen as a model peptide, since it contains one internal lysine at the 3-position from its N-terminus and two proline residues that create peptide linkages that are intrinsically difficult to hydrolyze. A complete substitution with trifluoroethylthiourea (CF,CH,NHCSNH-) at the side chain &-aminogroup of Lys was observed after 10 min of coupling at 75 "C; 50-60% substitution occurred at 75 "C for 4 to 5 min. The partial substitution reaction at Lys e-amino group produced additional peptide peaks in the mass spectrum and seriously complicated the analysis of the peptide ladder sequencing after more than one cycle. On the other hand, longer reaction time increased side reactions and substantially reduced the repetitive yields. A complication arose during acid cleavage of TFITC-modified substance-P. When HFBA-catalyzed cleavage of thiourea bond released the trifluoroethylthiourea-derivatized N-terminal Arg, the substituted trifluoroethylthiourea at the side chain of Lys was oxidized to the stable trifluoroethylurea group (CF,CH,NHCO-, MW = 125) This again increased the spectral complexity and compromised the generality of the protocol. Using trifluoroacetic acid (TFA) for removal of trifluoroethylthiourea group failed to improve the situation. The mass

1. R-N=C=S 2 CF3CF2CF2C02H

After i cycles

H2N-AA2-AA3--AAi---AAn.1-~"-OH H,N-AA~--AAi---AA".l-~"-OH HzN------+i--AAn.pUn-OH H-AA-- H N - ~O , ~A.A , ,I

One-Step MALDI-TOF Analysis

ir

H~N-AAI-AA~-AA~--AA~----AA,~-AA,-OH

SCHEME 1. General procedure for the peptide ladder sequencing using AITC. 485

Q.M. Gu and G.D. Prestwich

differences after only two cycles illustrate the difficulty. The difference between the original undigested ) substance-P [(M+N' = 1348.6)] and subsequent peptide molecular positive-ion peak [ ( M +H)' = 1317.4)] was 31.2 Da instead of the expected 156.2 (Arg). No positive-ion peak at ( M + H ) += 1192.4 was observed, indicating that Arg was lost but trifluoroethylurea had been gained on the peptide. The peak at m/z 1220.4 corresponded to the loss of Arg and Pro, and the peak at m/z 967.2 corresponded to the loss of Arg, Pro and E-NHCONHCH,-CF,-Lys. After each cycle, the sample residue must be completely dried in vucuo after the acid-catalyzed hydrolysis to remove the last traces of acid. The pH of the reaction solution must then be checked and adjusted to 8.5-9.0 prior to the addition of the next cycle peptide sample and the coupling reagent. The coupling reaction does not proceed quantitatively at a pH lower than 7.0, leading to partial modifications and generation of spurious mass peaks in the peptide ladder mixture. a-amino group of phenylalaninamide demonstrated that the coupling rate at pH8.8 was about 25% of that with TFITC (Table l ) , coupling could be accelerated with excess AITC or by increased reaction temperature. (TFITC was twice as active as PITC in reacting with the or-amino group of phenylalaninamide.) The moderate reactivity of AITC thus makes it more suitable for specific modification of primary amino groups of peptides. Indeed, as demonstrated in Figs. 1-3, AITC gives readily-interpreted, clean MALDI-TOF peptide ladders. The AITC ladder technique can be employed using picomolar amounts of peptide when a-cyano-4-hydroxycinnamic acid (CCA) was used as matrix and the mass analysis was operated by reflector-positive mode. Scheme 2 shows the general working procedure for one cycle of ladder sequencing. The usual reaction time of AITC with a peptide is 10 min at 75 "C. HFBA was employed as acid catalyst for removal of allyl thiourea derivatized-a-amino acid of the peptide and the reaction was normally carried out at 70 "C for 10 min. For both coupling and cleavage reactions, 70-75 "C is the optimal temperature range, and a reaction time of 5-15 min gives reproducible data. It was found that HFBA-catalyzed acid hydrolysis also caused the conversion of allylthiourea at the lysine side chain of substance-P to the corresponding allylurea via oxidation, resulting in a mass decrease

Trifluoroethyl isothiocyanate

Allyl isothiocyanate

In order to minimize the side products of coupling reaction and to develop a general ladder sequencing technique suitable for analysis of unknown affinitylabeled peptides, we investigated alternative volatile amino-coupling reagents. AITC (b.p. = 150 "C) was readily available and appeared more suitable for the peptide ladder sequencing by several criteria. The double bond of the allyl group activates isothiocyanate, making it more reactive towards amino groups than either methyl or ethyl isothiocyanate. AITC is commercially available, inexpensive, and easy to handle. It is quite stable at elevated temperatures (70-80 "C) and reacts cleanly and selectively with amino groups of peptides. Although its coupling with

Cheek pH before next cyde OR Add 3 4 pl of coupling bufferand speed vac again (20min, 24C)

[ Coup'ing I

2 \

pl peptide m coupling buffer 2 p coupling reagent in CHJCN I

10 min at 7~ 5\

SCHEME 2. Diagram of one cycle of peptide ladder sequencing using AITC.

TABLE 1 Comparison of ally1 isothiocyanate wiili trlpuoreliiyl isotiiiocvanate as volatile ladder sequencing reagent Trifluoroethyl isothiocyanate Structure B.P. ("C) MW Commercial availability Reactivity (PITC as 100%1)~ Couping time (min) Speed vacuum (min) Mass spectra
a

Allyl isothiocyanate H, =CH-CH,-N=C=S 150 99 Available, inexpensive 50% 10 20 Clean, reproducible

CF,CH,-N=C=S 92-94 141 Not available" 200"" 10 15 Complex

The precursor for the synthesis of this reagent is commercially available. Relative reactivities of isothiocyanates were determined by reacting isothiocyanates with phenylalaninamide in the coupling buffer at 75 'C for 10 min and quantitation by thin layer chromatographic analysis.

486

Peptide ladder sequencing using ally1 isothiocyanate

a.i.

1200

1000 1047.2
I

800

932 2
I

600

379.1
I

616.6 776.2
1 1

51;
400

200

300

400

so0

600

700

800

900

lo00

1100

1200

m/z

FIGURE 1 Positive-ion MALDI-TOF reflector mode spectra of AITC-ladder sequencing of human angiotensin-I1 (DRVYIHPF) after five cycles. The x-axis (m/z) represents positive-ion at (Mf H)' and y-axis (ail represents the absolute intensity. Degradation of the peptide was performed using 4 pmol for each cycle. The peak at (MfH)' 1047.2 corresponds to the undigested peptide. Peaks at (M+H)+ 932.2, 776.2, 676.6, 513.7 and 400.2 correspond to the digested peptides that have lost Asp, Arg, Val, Tyr and Ile, respectively, from the Nterminus of the peptide. The peak at (M+H)+ 379.1 is the doubly charged ion of CCA matrix.

of 16 Da. Partial oxidation of the methyl sulfide group of methionine was also common, affording a 16 Da mass increase. Following both coupling and cleaving procedures, excess reagents must be completely removed under vacuum prior to performing next step reaction; 20min is the minimum recommended Speed-Vac time. Results obtained on human angiotensin-I1 (DRVYIHPF) using AITC are shown in Fig. 1. The ladder sequences of six residues (k 0.3 Da) were successfully generated after five cycles. For the MALDI-TOF MS analysis, a-cyano-4-hydroxycinnamic acid was used as matrix. The clean mass spectral profile clearly indicated the general usefulness of AITC-ladder sequencing of the standard model peptide. Previous ladder sequencing protocols have focused on methodology, and consequently synthetic model peptides or structurally simple peptides were employed. However, to be useful to the practicing protein chemist and mass spectroscopist, the technique must be general, since the aim is to analyze unknown peptides. In particular, a general and efficient ladder sequencing technique would be ideal for phosphorylated, sulfated, glycosylated and other post-translationally modified peptides, as well as for

chemically modified peptides arising from affinity labeling experiments. Thus, we illustrate below two examples of chemically modified peptides that possess reactive and labile functionalities. First, a photoaffinity analog of substance-P modified at the Lys amino group was prepared [RPK(NBZDC)PQQFFGLM-NH,]. The benzoyldihydrocinnamoyl (BZDC) group is a useful benzophenone photophore that has been widely used as a photoaffinity ligand (18, 19). Four cycles of ladder sequencing produced consecutive positive-ion mass peaks appearing at ( M + H ) + 1584.5, 1428.4, 1331.4, 967.4 and 870.1 in the MALDI-TOF spectrum (Fig. 2), and the differences between adjacent ion peaks, 156.1, 97.0, 364.0 and 97.3, correspond to Arg, Pro, NBZDC-Lys and Pro, respectively. The masses were obtained with errors of 0.2 Da. Minor peaks appearing in the spectrum correspond to Na+ , K + and NH4+ salts of the degraded peptide fragments. Oxidation of Met was again observed during the hydrolytic reaction. The N-BZDC-labeled lysine was lost intact as the modified amino acid, indicating that the amide bond between BZDC and lysine was stable under the conditions of acidic cleavage of the peptide linkage. Next, a 12-amino acid phosphopeptide
487

Q.M. Gu and G.D. Prestwich

a.1.

900

800

700

I 5 Ell

600

500

400

300

200

100
700
900

1100

1300

1500

1700

FlGURE 2 Positive-ion MALDI-TOF reflector mode spectra of AITC-ladder sequencing of [N-BZDC-Lys"1substance-P [RPK(NBZDC)PQQFFGLM-NH,] after four cycles. Degradation of the peptide was performed using 2 pmol for each cycle. The peak at ( M f H)' 1584.5 corresponds to the undigested BZDC-modified peptide. Peaks at ( M + H ) ' 1428.3, 1331.4, 967.4 and 870.1 correspond to the loss of amino acids of Arg. Pro, N-BZDC-Lys and Pro from the N-terminal of the peptide during successive cycles of digestion. The labeled peak at 989.6 represents the Na' salt of the peak of 967.4.
a.i.
2500

1485.5
I

2000

1500

1000 1 I 2,8

4 802 2
500

0 2,8 7
I I

931
I

600

800

1000

1200

1400

1600

mlz

FIGURE 3 Negative ion MALDI-TOF reflector mode spectra of AITC-ladder sequencing of phosphopeptide [ FLPVPEY (0-PO,H,)INQSV] after seven cycles. Degradation of the peptide was performed using 5 pmol for each cycle. The pcak at (M-H).. 1485.5 corresponds to the undigested phosphopeptide. Peaks at ( M - H ) - 1338.3, 1225.1. 1128.3. 1028.7, 931.6, 802.2 and 558.4 correspond to the loss of amino acids of Phe, Leu. Pro, Val. Pro, Glu and Phospho-Tyr during successivc digestion cycles. The peaks in the 500-600 range are mostly from CCA matrix.

[FLPVPEY(O-PO,H,)INQSV],a fragment of epidermal growth factor (20), was subjected to seven cycles of AITC-ladder sequencing. The phosphotyrosine appeared to be stable during both the AITC
488

coupling and HFBA cleavage reactions, and the hydrophilic phosphate showed little interference with the ladder sequence protocols. Figure 3 shows that a clean MALDI-TOF MS ladder sequence was

Peptide ladder sequencing using allyl isothiocyanate obtained for this peptide, despite very low yields after two cycles. It appears that the Pro-3 and Pro-5 at the 3 and 5 positions reduce the efficiency of HFBA cleavage. Ser-11 might also be modified after the multiple HFBA treatments. MALDI analysis in the negative-ion mode showed higher peak intensities and better peak resolution for these phosphopeptides than the positive-ion mode. The spectrum showed negative-ion mass peak (M-H)- appearing at 1485.5, 1338.3, 1225.1, 1128.3, 1028.7,931.6, 802.2 and 558.4, corresponding to the degraded peptide fragments after release of amino acid residues, Phe, Leu, Pro, Val, Pro, Glu and the intact 0-phospho-Tyr, respectively. Longer polypeptides were also problematic with earlier protocols. Thus, AITC-ladder sequencing was applied in three cycles of degradation with a synthetic C-terminally amidated 33-mer, pheromone biosynthesis activating neuropeptide (PBAN) (21) (Table 2). This peptide contains one internal lysine residue and has an overall hydrophobic character. Positive-ion masses at ( M + H)' 3904.6, 3891.4, 3804.3 and 3689.2 correspond to the undigested PBAN peptide, one Met-oxidized PBAN with a loss of one Leu and a gain of an allylurea (CH, =CHCH,NHCO-, MW = 83), one Metoxidized PBAN with a loss of Leu, Ser and a gain of an allylurea, and one Met-oxidized PBAN with a loss of Leu, Ser, Asp and a gain of an allylurea, respectively. In contrast, neither ladder sequencing using the reported organic solvent extraction method (4) nor the TFITC method evidenced a single degradation cycle by MALDI-TOF MS. The milder AITC appeared superior in this direct comparison, In conclusion, we have developed a general, efficient and inexpensive approach to peptide ladder sequencing using AITC in conjunction with MALDITOF MS. However, this method is not without caveats. Coupling, acidolysis, and solvent removal require optimization for new 'unknown' peptides. Side-chain-specific reactions may interfere with analysis of methionine- or lysine-rich peptides. Polypeptides with over 30 residues will require proteolysis to obtain useful data from multiple degradation cycles. Nevertheless, the AITC-ladder sequencing method represents a significant improvement, and provides analytical biochemistry with a useful tool for the identification of the post-translationally modified and chemically modified peptides. EXPERIMENTAL PROCEDURES Materials AITC, HFBA, acetonitrile, CCA and sinapinic acid (SA) were obtained from Aldrich Chemical Co. Substance-P, human angiotensin I1 and bovine insulin

TABLE 2 Ladder sequencing of peptides using allyl isofhiocyanates by M A L D P Peptides Cycles of sequencing 5 Matrices Mode Ladder masses observed'

Angiotensin (DRVYIHPF) Substance-Pb (RPKPQQFFGLM-NH,) Synthetic peptide (DRHIYVPF) BZDC-Substance-P [RPK( BZDC)PQQFFGLM-NH,] PBAN (LSDDMPA.'.PRL-NH,) Phosphopeptide from EGFR [ FLPVPEY (PO,H,)INQSV]

CCA

Positive

1047.2, 932.2, 176.2. 676.6, 513.7, 400.1 1348.6, 1317.4, 1220.4, 967.2d

CCA

Positive

CCA

Positive

1047.6, 932.3, 775.6, 638.5. 525.5 1584.5, 1428.4, 1331.4, 967.4, 870.1 3904.6, 3891.4, 3804.3, 3689.2" 1485.5, 1338.3, 1225.1, 1128.3, 1028.7, 931.6, 802.2. 558.4

CCA

Positive

SA

Positive

CCA

Negative

Unless otherwise stated, coupling reactions were performed using AITC at 75 "C for 10 min and cleavage reactions were carried out at 70 "C for 10 min as described in the text. The ladder sequence data were obtained using TFITC as coupling reagent. This column lists the average masses appearing in the profile of ladder sequencing MALDI-TOF spectra. Trifluoroethylurea (CF,CHZNHCO, MW = 125) coupled to the side chain of internal Lys. Allylurea (CH, = CHCH2NHC0, MW = 83), coupled to the side chain of the internal Lys.

489

Q.M. Gu and G.D. Prestwich were obtained from Sigma Chemical Co. [N-BZDCLys3] substance-P [RPK(N-BZDC)PQQFFGLMNH,] was synthesized by reacting substance-P with one molar equivalent of BZDC-NHS at pH 11.5 in DMF-water followed by HPLC purification (19). TFITC (1.8 g) was obtained by reacting 6.3 g of trifluoroethylamine with 4.7 mL of thiophosgene (6), and was purified by distillation (twice) to give a 22% overall yield. PBAN (LSDDMPATPADQEMTRQDPEQIDSRTKYFSPRL-NH,) (21 ) and DRHIYVPF were synthetic peptides. Phosphopeptide [FLPVPEY(O-PO,H,)INQSV] was provided by Dr. W.T. Miller (University at Stony Brook) (20). All other chemicals were from commercial sources and were of analytical grade.
General procedure for peptide ladder sequencing Peptides were dissolved in a mixture of trimethylammonium bicarbonate buffer ( 12.5%, pH 8 . 5 ) / 1 ,I ,1,3,3,3-hexafluoro-2-propanol/water :4 5 ) at a (1 concentration of 2 pmol/pL. To the peptide solution (2 pL) in a Hewlett-Packard 50-pL glass mini-vial was added 2 pL of 10% TFITC or AITC in acetonitrile (2 pL). The sealed vial was heated at 75 "C for 10 min, and the resulting solution was diluted with 4 pL of H,O. Solvent and excess amount of reagents were evaporated using a speed-vacuum at 24 "C for 20min. To the dried residue in the mini-vial was added 2 pL of HFBA, and then heated to 70 "C. After 10 min HFBA was removed in vacuu using a Speed-Vac@ (Savant Instrument Co., Farmingdale, N Y ) at 24 "C for 20 min. Coupling buffer (2 pL) was added to the residue and again removed in vacuo at 24 "C for 15 min. For the next cycle reaction, another 2 p L of peptide solution was added to the dried residue in the mini-vial. The pH of the reaction solution was monitored to be 8.5-9.0 prior to addition of another 2 p L of coupling reagent in acetonitrile. The coupling and degradation reactions were carried out using the same conditions as described as above. This procedure was repeated for i times for the desired i cycle sequencing. The final residue was dissolved in 2 p L of water and used for the sample preparation for mass analysis.

peptides is ca. 1 :900 with a mass accuracy of O.I-0.15%. Steel targets were cleaned by sonication in a 30% aqueous acetonitrile solution containing 0.1% TFA for IOmin, followed by washing with water and acetone, and then air-drying. The saturated CCA matrix solution was freshly prepared by dissolving 10-20 mg CCA in 0.5 mL of 0.1% TFA-30% aqueous acetonitrile solution, followed by vortexing and centrifugation; the supernatant was used for sample preparation. Similar procedure was used for preparation of the matrix solution of saturated SA. Samples ( 1 pL) were mixed with 7-9 pL of the saturated matrix solution and spotted on the target using a clean pipette tip, then air-dried at room temperature for 15 min. All mass data obtained were calibrated using an external calibration standard mixture of human angiotensin-I1 [(M+H ) + = 1047.2)] or bovine insulin [(M+H)' = 5734.56)] with SA [(M+H ) + = 225.08)j. ACKNOWLEDGEMENTS
We thank the NIH (Grant NS 29632 to G.D.P.) and the Center for Biotechnology, a New York State Center of Advanced Technology, for financial support. The Protein TOF instrument was purchased with funding from the NSF (Grant BIR-9419980 to G.D.P.) and the Center for Biotechnology, and with support from Bruker, Inc. We thank Mr. T. Fischer (Center for Analysis and Synthesis of Macromolecules, University at Stony Brook, Stony Brook, NY) for assistance in instrumental operation and peptide synthesis.

REFERENCES
1. Edman, P. & Gegg, G. (1967) Eur. J. Biochem. 1, 80-91 2. Hewick, R.M., Hunkapillar, M.W., Hood, L.E. & Dreyer, W.J. (1981) J. Biol. Chem. 256, 7990-1997 3. Yan, S.C.B., Grinnell, B.W. &Wold, F. (1989) Trends Biochem. Sci. 14. 264266 4. Chait, B.T., Wang, R., Beavis, R.C. & Kent, S.B.H. (1993) Science 262, 89-92 5. Karas, M. & Hillenkamp, F . (1988) Anal. Chem. 60,2299-2301 6. Bartlet-Jones, M., Jeffery, W.A., Hansen, H.F. & Pappin, D.J.C. (1994) Rapid Commun. Mass Spectrom. 8, 731-742 7. Stults, J.T. (1995) Curr. Opin. Struct. Biol. 5, 691-698 8. Burlingame, A.L., Boyd, R.K. & Gaskell, S.J. (1994) Anal. Chem. 66, 634R-683R 9. Hillenkamp, F., Karas, M., Beavis, R.C. & Chait, B.T. (1991) Anal. Chem. 63, 1193A-1203A 10. Wang, R. & Chait, B.T. (1994) Curr. Opin. Biotechnol. 5,77784 11. Sutton, C.W., Pemberton, K.S., Cottrell, J.S., Corbett, J.M., Wheeler, C.H. & Dunn, M.J. (1995) Elecfrophoresis16, 308316 12. Mann, M. (1994) Sequence Database Searching by Mass Spectrometric Data (VCH publisher Inc., Weinheim) 13. Cottrell, J.S. (1994) Peptide Res. 7, 115-124 14. Vorm, 0. & Roepstorff, P. (1994) Biol. Mass Spectrom. 23, 734-740 15. Kaufmann, R., Kirsch, D. & Spengler, B. (1994) Znt. J. Mass Spectrom. Ion Processes 131, 25538

MALDI-TOF mass spectrometric analysis Mass analysis of the peptide mixture was carried out on a MALDI-TOF mass spectrometer using a Bruker (Billerica, MA) ProteinTOF@ instrument equipped with a reflector. The instrument was operated in reflector model with 17.5-kV ion acceleration and 20-kV reflectron voltages. Ionization is accomplished with a 337-nm beam from a nitrogen laser with a repetition rate of 3 Hz. The signal from the detector was digitized using a transient recorder. The instrument control and data processing were accomplished with software supplied by Bruker using a Sun IPX workstation. The resolution in the reflector mode for
490

Peptide ladder sequencing using allyl isothiocyanate

16. Patterson, D.H., Tarr, G.E., Regnier, F.E. & Martin, S.A. (1995) Anal. Chem. 61, 3971-3978 17. Thiede, B., Wittmann-Liebold, B., Bienert, M. & Krause, E. (1995) FEBS Lett. 351, 65-69 18. Dorman, G. & Prestwich, G.D. (1994) Biochemistry 33, 5661-5673 19. Olszewski, J.D., Dorman, G., Elliott, J.T., Hong, Y., Ahern, D.G. & G.D. Prestwich (1995) Bioconj. Chem. 6, 395-400 20. Gergel, J.R., McNamara, D.J., Dobrusin, E.M., Zhu, G., Saltiel, A.R. & Miller, W.T. (1994) Biochemistry 33, 1467114678 21. Clark, B.A. & Prestwich, G.D. (1996) Znt. J. Peptide Protein Res. 41, 361-368 Address: Professor Glenn D. Prestwich Chair, Department of Medicinal Chemistry 308 Skaggs Hall The University of Utah Salt Lake City, Utah 841 12 USA Tel: 801 581-7063 Fax: 801 581-7087 e-mail: gprestwich@deans.pharm.utah.edu

49 1