Anal. Chem.

2005, 77, 7594-7604

Enrichment and Identification of Cysteine-Containing Peptides from Tryptic Digests of Performic Oxidized Proteins by Strong Cation Exchange LC and MALDI-TOF/TOF MS
Jingquan Dai, Jinglan Wang, Yangjun Zhang, Zhuang Lu, Bing Yang, Xiaohai Li, Yun Cai, and Xiaohong Qian*

Beijing Institute of Radiation Medicine, 27 Taiping Road, Beijing 100850, China

The extreme complexity of sample and uninformative fragmentation of peptides in MS/MS experiments are two of several real challenges faced by proteomics. In this work, a strategy aimed at tackling these two problems is presented. Briefly, proteins were first oxidized by performic acid to cleave the disulfide bonds and simultaneously convert cysteine residue into its sulfonic form. Then the resultant sulfonic peptides were enriched by SCX chromatography, exploiting the negative solution charge of sulfonic group. The sulfonic peptide could be easily detected by MALDI-MS in negative mode and showed both enhanced fragmentation efficiency and a simplified spectrum in MALDI-MS/MS experiment in positive mode. The strength of the strategy was demonstrated by applying it to bovine serum albumin. Potential use of the strategy in proteomics was also discussed.
The completion of the sequencing of the human genome and genomes of a number of other key species provides an invaluable resource for biologists. However, it has proven to be impossible to approach a complete understanding of cellular processes without focusing experimental effort at the protein level. Thus, proteomics has rapidly grown into one of the most attractive subdisciplines of life sciences in the postgenomic era.1 Although the increasing availability of complete or near-complete genome sequences is expanding the scope of experiments; proteomics has been a technology-driven endeavor in terms of the tremendous complexity of the proteome. Mass spectrometry plays a central role in proteomics research; however, even the most advanced MS instrumentation available today is not able to deal with very complex biological samples such as plasma as a whole, without prior prefractionation. Gel-based strategy and shotgun strategy were commonly used for this purpose. While gel-based strategy still can be considered the most widely used technique in proteomics, shotgun strategy is increasingly gaining popularity.1,2 Shotgun strategy overcomes some disadvantages of gel-based
* Corresponding author. E-mail: qianxh@nic.bmi.ac.cn. Tel: +86-10-66930257. Fax: +86-10-6827-9585. (1) Patterson, S. D.; Aebersold, R. H. Nat. Genet. 2003, 33 (Suppl.), 311-323. (2) McDonald, W. H.; Yates, J. R., III. Curr. Opin. Mol. Ther. 2003, 5(3): 302309.

technique such as limitations in the pI and molecular weight of proteins, difficult automation, and reproducibility. Another advantage of shotgun strategy is its flexibility. Although the last step before mass spectrometry is generally reversed-phase liquid chromatography, various methods such as ion exchange chromatography3 and affinity chromatography could be used as the first-dimension separation mode. By using appreciate combinations, a specific subset of peptides such as phosphopeptides,4 glycopeptides,5 and cysteine-containing peptides6 could be enriched from a proteolyzed lysate. Shotgun proteomics takes advantage of the higher separation efficiency of chromatographic techniques on the peptide rather than the protein level. However, the peptide mixture produced in the initial proteolysis step still overwhelms the analytical capacity of current LC/MS systems, both in number and in dynamic range.7 On the other hand, there is no need bother analyzing each peptide in a tremendously complex mixture if the specific purpose, such as proteomics surveying or specific posttranslational modification, is mainly pursued. The enabling principle for shotgun proteomics is attributed to that the unit of identification is a single peptide rather than a group of peptides.2 In the ideal case, one peptide is enough for both qualitative and quantitative identification of each protein. Thus, in respect of different biological questions being addressed, various selective enrichment methods were developed. Among all these methods, quite a few were directed toward cysteine residues for reducing the complexity of the sample mixture with ICAT as a good case in point.6 The reason is that the sulfhydryl group of cysteine is very reactive and can be specifically modified, for example, by reagents possessing both iodoacetyl and vinyl functionalities, and an affinity tag thus can be selectively enriched on an affinity chromatography. The thiol group of cysteine can also form a disulfide bond with a thiol-affinity resin through a disulfide exchange process. Thus Cys-containing
(3) Link, A. J.; Eng, J.; Schieltz, D. M.; Carmack, E., Mize, G. J.; Morris, D. R.; Garvik, B. M.; Yates, J. R., III. Nat. Biotechnol. 1999, 17, 676-682. (4) Beausoleil, S. A.; Jedrychowski, M.; Schwartz, D.; Elias, J. E.; Villen, J.; Li, J., Cohn, M. A.; Cantley, L. C.; Gygi, S. P. Proc. Natl. Acad. Sci. U.S.A. 2004, 101 (33), 12130-12135. (5) Kaji, H.; Saito, H.; Yamauchi, Y.; Shinkawa, T.; et al. Nat. Biotechnol. 2003, 21, 667-672. (6) Gygi, S. P.; Rist, B.; Gerber, S, A.; Turecek, F.; Gelb, M. B.; Aebersold, R. Nat. Biotechnol. 1999, 17, 994-999. (7) Nilsson, C. L.; Davidsson, P. Mass Spectrom. Rev. 2000, 19, 390-397. 10.1021/ac0506276 CCC: $30.25 2005 American Chemical Society Published on Web 10/26/2005

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peptides were captured by the solid phase and enriched through a covalent chromatography.8 Another challenge encountered by shotgun proteomics is that even if dramatical reduction of the complexity of the sample mixture was achieved, most proteins have to be identified through much less candidate peptides. However, not all peptides could give high-quality MS/MS spectra. In fact, it was reported that more than 80% of tandem MS spectra could not give satisfactory results after a database search.9 To enhance the fragmentation efficiency and simplify the resultant spectra, various derivatization methods were developed.10 No great success was achieved until Keough and co-workers presented a strategy to introduce a negatively charged sulfonic acid moiety on the N-terminal amino group of the tryptic peptide.11 Although the N-terminal sulfonation method showed promise in peptide de novo sequencing, the main disadvantage was a reduction in sensitivity12 by a factor of 10. Other drawbacks including intrinsically incomplete chemistry and multiple steps of operation made its integration into proteomics research difficult. So although a commercially available kit (the CAF MALDI sequencing kit from Amersham Bioscience) could be conveniently obtained, no successful application of the method in shotgun proteomics was reported to our knowledge. Performic oxidation is a well-established method for scission of the disulfide bond.13 During performic oxidation, methionine and cysteine are converted into their stable oxidation states, i.e., methionine sulfone (M(O2)) and cysteinesulfonic acid (C(O3)), respectively. Recently, it was reported that performic oxidation was used as an alternative to the more popular reduction/ alkylation method to cleave the disulfide bond before hydrolysis with a protease in proteomics research.14,15 Due to its efficiency and many other advantages, performic oxidation deserves more attention now and some methodology research have already been carried out.16 Here we report a novel strategy coupling performic oxidation with subsequent strong cation exchange HPLC separation and matrix-assisted laser desorption/ionization tandem mass spectrometry for selective enrichment and identification of cysteine-containing peptides. The strength of the strategy was demonstrated with bovine serum albumin (BSA) as a model protein. Possible use in proteomics research was also discussed. EXPERIMENTAL SECTION Chemicals and Materials. Model peptide NVQCRPTQVQLRPVQVR (PBP2) was purified from a tryptic digest of a recombinant platelet-derived growth factor isoform (PDGF-BB, obtained from Jin-Sai-Shi Biotech Inc., Beijing, China) by reversed-phase high-performance liquid chromatography as described before.16 BSA was purchased from Sigma (St. Louis, MO) and used without
(8) Liu, T.; Qian, W. J.; Strittmatter, E. F.; Camp, D. G.; Anderson, G. A.; Thrall, B. D.; Smith, R. D. Anal. Chem. 2004, 76, 5345-5353. (9) Sadygov, R. G.; Liu, H.; Yates, J. R., III. Anal. Chem. 2004, 76,1664-1671. (10) Roth, K. D. W.; Huang, Z. H.; Sadagopan, N.; Watson, J. T. Mass Spectrom. Rev. 1998, 17, 255-274. (11) Keough, T.; Youngquist, R. S.; Lacey, M. P. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 7131-7136. (12) Standing, K. G. Curr. Opin. Struct. Biol. 2003, 13, 595-601. (13) Hirs, C. H. W. Methods Enzymol. 1967, 11, 197-199. (14) Oda, Y.; Nagasu, T.; Chait, B. T. Nat. Biotechnol. 2001, 19, 379-382. (15) Knight, Z. A.; Schilling, B.; Row: R. H.; Kenski, D. M.; Gibson, B. W.; Shokat, K. M. Nat. Biotechnol. 2003, 21, 1047-1054. (16) Dai, J.; Zhang, Y.; Wang, J.; Li, X.; Lu, Z.; Cai, Y.; Qian, X. Rapid Commun. Mass Spectrom. 2005, 19, 1-10

further purification. Formic acid (98%), trifluoroacetic acid (99%), and R-cyano-4-hydroxycinnamic acid were purchased from Sigma. Hydrogen peroxide (30%) was purchased from China National Medicine Group Shanghai Chemical Reagent Co. (Shanghai, China). HPLC grade acetonitrile was acquired from J. T. Baker. Potassium chloride was from Beijing Chemical Reagent Co. (Beijing, China). Deionized water was produced by a Milli-Q A10 system from Millipore (Bedford, MA). Performic Oxidation and Tryptic Digestion. Performic oxidation of BSA and PBP2 was performed as described previously.17 Briefly, protein/peptide was dissolved in formic acid to 1 mg/mL, and incubated on ice for 2.5 h with 2-3 volumes of performic acid; the latter was prepared by mixing 1 part hydrogen peroxide with 19 parts formic acid and incubating at room temperature for 2 h. Then 5 volumes of cold Milli-Q water were added, and the resulting solution was lyophilized. Oxidized BSA (Ox-BSA) was dissolved in 50 mM NH4HCO3 to 1 mg/mL. Then TPCK-modified porcine trypsin (Promega, Madison, WI) was added to a final protease/protein ratio of 1:25 (w/w), and the mixture was incubated at 37 C overnight. The resultant digest was desalted on a C18 column (250 4.6 mm) purchased from Elite (Dalian, China) and then completely dried by a Speed Vac (ThermoSavant, NY). Strong Cation Exchange (SCX) Chromatography. SCX chromatography was performed on an Elite P230 HPLC and UV detector (Elite). Solvent A (5 mM KH2PO4/30% acetonitrile, pH 3.0) and solvent B (solvent A with 500 mM KCl) were used to develop a salt gradient. Dried tryptic peptides from Ox-BSA were dissolved in 500 L of SCX solvent A immediately before analysis. Tryptic peptides were separated at pH 3.0 by SCX chromatography by using a strong cation exchange column (150 4.6 mm) from Hypersil and a flow rate of 0.75 mL/min. UV detection was at 214 nm. A gradient was developed consisting of 10 min at 100% solvent A, 1-min gradient to 15% solvent B, 20 min at 15% solvent B, 1-min gradient to 100% solvent B, 20 min at 100% solvent B, 1-min gradient to 100% solvent A, and then 20 min at 100% solvent A. Fractions were collected every minute during the analysis. MALDI-TOF/TOF Analysis. All mass spectra were acquired on an Applied Biosystems 4700 TOF/TOF Proteomics Analyzer instrument (Framing, MA) unless mentioned otherwise. All HPLC fractions from the SCX column were directly mixed 1:1 with saturated R-cyano-4-hydroxycinnamic acid in 50% acetonitrile containing 0.1% trifluoroacetic acid, and 0.5 L was applied to the MALDI target plate. Before the spots were completely (1 min later) dried, the remaining liquid was absorbed by a piece of absorbent paper to desalt.18 Prior to analysis, the mass spectrometer was externally calibrated with a mixture of angiotensin I, Glu-fibrinopeptide B, ACTH(1-17), ACTH (18-39), ACTH(7-38), and des-Agr-bradykinin. For MS/MS experiments, the instrument was externally calibrated with fragments of Glu-fibrinopeptide. MS/MS experiments were all performed under metastable suppressor off conditions in positive reflectron mode. All MS experiments were
(17) Marshak, D. R.; Kadonaga, J. T.; Burgess, R. R.; Knuth, M. W.; Brennan, W.A., Jr.; Lin, S. H. In Strategies for protein purification and characterization: a laboratory course manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 1996; Addenda 6. (18) Gobom, J.; Schuerenberg, M.; Mueller, M.; Theiss, D.; Lehrach, H.; Nordhoff, E. Anal. Chem. 2001, 73, 434-438.

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Figure 1. Scheme for Cys-containing peptide enrichment by SCX chromatography. (a) At pH 3.0, most peptides produced by trypsin proteolysis have a solution charge state of 2+, whereas after conversion of sulfydryl group to sulfonic group by performic oxidation Cys-containing peptides have a charge state of only 1+ or lower. (b) Solution charge-state distribution of tryptic peptides (5-35 aa) produced by a theoretical digestion of the human protein database with cysteines in their sulfonic forms and (c) native or carbamidomethylation forms (n ) 543 4252 peptides). (d) Experimental scheme summarizing the method for enrichment of the sulfonic peptides and subsequent identification by database searching or de novo sequencing with resulted high-quality MALDI-MS/MS data.

carried out in both positive and negative reflectron mode. Precursor ions for MS/MS were selected manually on the basis of the MS spectra to ensure better results. All MS data were analyzed using the Findmond tool (http://us.expasy.org/ tools/findmod/) to match with BSA (UniProt entry P02769 [ALBU_BOVIN]). All MS/MS data were analyzed manually and by GPS Explorer (the analysis software installed on the instrument by the manufacturer). All m/z values quoted in this study are monoisotopic. ESI-Q-TOF Analysis. For comparison purposes, native and oxidized PBP2 (Ox-PBP2) were also analyzed on an ESI-Q-TOF mass spectrometer (Micromass) immediately after separation on the coupled CapLC system (Waters). The instrument was operated in the positive survey mode and externally calibrated with fragments of Glu-fibrinopeptide. The mass spectrometer conditions were as follows: electrospray voltage 3.3 kV, sample cone voltage 45 V, and source temperature 80 C. The flow from pump C was used to load the sample and wash the sample on a 300 m i.d. 5 mm precolumn cartridge (LC Packings) for 3 min with a 0.1% formic acid aqueous solution. Separations were performed with a 75 m i.d. 15 cm PepMap C18 column (LC Packings) using a gradient solvent system (A 0.1%, v/v, formic acid in water/ acetonitrile 95/5, v/v; B 0.1%, v/v, formic acid in water/acetonitrile
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15/85, v/v). The flow from pumps A and B was 2.5 L/min, and this was split to reduce the flow rate through the column to 0.3 L/min. Gradient elution was as follows: 4.5 min 96% buffer A, a 35-min gradient from 4 to 50% buffer B, 5 min 100% buffer B, a 4-min gradient from 100% buffer B to 96% buffer A, and buffer A was thus maintained at 96% for 7 min before column reequilibration. RESULTS AND DISCUSSION Principle of the Performic Oxidation Strategy. The strategy used here exploited the differential net solution charge state of Cys-containing peptides and non-Cys-containing peptides after performic oxidation. Briefly, most tryptic peptides carry a solution net charge state of 2+ under commonly used SCX conditions because only Lys, Arg, His, and the amino terminus of the peptides are charged.4 After performic oxidation, cysteine was converted into C(O3), which maintains a negative charge at acidic pH values, so the net charge state of such a sulfonic peptide is generally 1+ or lower depending on the number of Cys in the sequence (Figure 1a). An in silico tryptic digest of the human protein database from National Center for Biotechnology Information (March 4, 2005) was performed on the assumption that all cysteines were converted into their sulfonic acid forms. It was shown that 3.5% of all

Figure 2. SCX chromatography separation at pH 3.0 of tryptic digestion of BSA after performic oxidation (a) or reduction/carbamidomethylation (b). The dashed line indicates the salt gradient. Peaks corresponding to different solution net charge states of peptides are labeled. Table 1. Peptides Identified from Flow-Through (Fraction 2-7) and Eluent of 15% Phase B (Fraction 21-34)a fraction number 2 3 3 4 4 4 4 4 5 5 6 6 6 7 21 23 24 24 25 25 26 26b 26 33c 34c observed mass (M-H)1710.632 1710.608 1040.408 1040.399 1898.664 1720.557 1118.441 1710.608 1118.451 1482.509 1482.537 1710.557 1727.619 1727.625 1443.486 1057.417 1057.388 1432.568 2129.688 1096.476 2129.672 747.382 1096.47 3009 3009 calculated mass (M-H)1710.5904 1710.5904 1040.3636 1040.3636 1898.6723 1720.5365 1118.41 1710.5904 1118.41 1482.5258 1482.5258 1710.5904 1727.617 1727.617 1443.5009 1057.3902 1057.3902 1432.5907 2129.7215 1096.4626 2129.7215 747.3716 1096.4626 3009.1228 3009.1228 mass (Da) -0.043 -0.017 -0.044 -0.035 0.008 -0.021 -0.03 -0.017 -0.04 0.017 -0.01 0.034 -0.001 -0.007 0.015 -0.026 0.002 0.023 0.033 -0.012 0.05 -0.009 -0.006 potential mod S-S-cyc S-S-cyc S-cyc S-cyc S-S-S S-S-S S-S S-S-cyc S-S S-S S-S S-S-cyc S-S S-S S-S S S S S-S-S S S-S-S S S S-S-S S-S-S no. MC 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 1 1 net charge -1 -1 0 0 0 0 0 -1 0 0 0 -1 0 0 1 1 1 1 1 1 1 1 1 1 1

peptide YNGVGQECCQAEDK YNGVGQECCQAEDK QNCDQFEK QNCDQFEK CCAADDKEACFAVEGPK ECCHGDLLECADDR CCTESLVNR YNGVFQECCQAEDK CCTESLVNR EYEATLEECCAK EYEATLEECCAK YNGVFQECCQAEDK YNGVFQECCQAEDK YNGVFQECCQAEDK TCVADESHAGCEK QNCDQFEK QNCDQFEK YICDNQDTISSK ETYGDMADCCEKQEPER EACFAVEGPK ETYGDMADCCEKQEPER GACLLPK EACFAVEGPKEYEATLEECCAKDDPHAC YSTVFDKEYEATLEECCAKDDPHAC YSTVFDK

position 184-197 184-197 413-420 413-420 581-597 267-280 499-507 184-197 499-507 375-386 375-386 184-197 184-197 184-197 76-88 413-420 413-420 286-297 106-122 106-122 198-204 588-597 375-399 375-399

a S, sulfonic acid; S, Met in its sulfone form; cyc, N-terminal cyclization. b Not confirmed by MS/MS. c Accurate m/z values of [M - H]- could not be obtained due to poor resolution.

the peptides had a net charge lower than or equal to 0 and 13% had a net charge at 1+ (Figure 1b); however, these small quantities of peptides were derived from 37.9 and 81.4% of the whole proteome, respectively. As a whole, peptides having a net charge equal to or lower than 1+ covered 85.8% of the whole proteome. So separation of these peptides through SCX chromatography from the bulk could dramatically reduce the complexity of the sample system and still be able to characterize the proteome with relatively little loss of information (Figure 1d).

Theoretical calculation also showed that most of these peptides were sulfonic peptides (all the 3.5% with net charge of e0 and 11.8% among the 13% with net charge of 1+), which represent 80.3% of the whole proteome. Sulfonic tryptic peptide was known to show enhanced fragmentation efficiency under CID or PSD conditions.19 So it is expected that proteins could be efficiently identified through these relatively small quantity of sulfonic peptides.
(19) Burlet, O.; Yang, C. Y.; Gaskell, S. J. J. Am. Soc. Mass Spectrom. 1992, 3, 337-344

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Figure 3. Positive MS/MS spectra of sulfonic peptides in their N-terminal cyclized and native forms. (a) N-Terminal cyclized T413-420 (solution net charge 0) in the flow-through. (b) Native T413-420 (solution net charge 1) in the eluent of 15% B. (c) N-Terminal cyclized T184-197 (solution net charge 1-) and (d) native T184-197 (solution net charge 0) both in the flow-through. All cysteines are in their sulfonic acid forms. Potential N-terminal cyclized peptides are labeled cyc. For clarity, not all product ions are labeled.

In addition, after performic oxidation the charge-state distribution of the resultant peptides was more evenly distributed (Figure 1b) than before (Figure 1c), wherein cysteines are in their native or carbamidomethylation forms; therefore, SCX chromatography would not necessarily be overwhelmed by those peptides with a charge state of 2+; thus peak capacity of SCX chromatography was more fully exploited. Enrichment and Identification of Cysteine-Containing Peptides. The SCX separation of a complex peptide mixture derived from 250 g of performic oxidized BSA at pH 3.0 generated by trypsin proteolysis is shown in Figure 2a. Peptides identified from flow-through and eluent of 15% B were listed in Table 1. As expected, peptides with solution net charge at 0 or lower were not retained on the SCX column and were identified in the flow-through (fraction 2-7). Those with a net charge at 1+ were eluted in 15% B (fraction 21-34). And those with a net charge at 2+ or higher were eluted in 100% B (fraction 40-61). The three groups of peptides were completely separated under the conditions used here. It was noticeable that all peptides that eluted earlier with solution net charge at or lower than 1+ were Cys-containing
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peptides (Table 1). For comparison purposes, the same amount of tryptic peptides of BSA after reduction/carbamidomethylation were separated using the same conditions (Figure. 2b). No peptides were identified in fractions until phase B was enhanced to 100%. Thus, it was demonstrated that it was feasible to use the strategy proposed for selective enrichment and identification of Cys-containing peptides from a complex digestion. In fact, four of six predicted tryptic peptides (no missed cleavage sites, g500 Da) of Ox-BSA with solution net charge at 0 were found in flowthrough. The two not found were T581-587 CCAADDK and T106-117 ETYGDMADCCEK. However, corresponding peptides T581-597 with net charge at 0 and T106-122 with net charge at 1+, which both had a missed cleavage site in the sequence, were eluted in flow-through and 15% B, respectively (Table 1). Peptides T413-420 and T184-197, indicating N-terminal minus 17 in mass spectra (Table 1 and Figure 3a, c), were also identified from the flow-through. It was reported that N-terminal glutamine could undergo cyclization to form pyroglutamic acid in the basic environment of tryptic digestion,20,21 causing loss of 17 Da from peptide and loss of a positive charge under SCX conditions. Thus,

Figure 4. Effect of performic oxidation on the fragmentation efficiency of Cys-containing tryptic peptide. (a) Positive MS spectrum of T469482 with Cys in the carbamidomethylated form at m/z 1724.975. (b) Positive MS/MS spectrum of T469-482 with Cys in the carbamidomethylated form. (c) Positive MS spectrum of T469-482 with Cys in the sulfonic form at m/z 1747.832. (d) Positive MS/MS spectrum of T469-482 with Cys in the sulfonic form. When cysteine was in its sulfonic form, methionine was in its sulfone form.

the cyclized peptide T413-420 would hold a charge of 0 instead of 1 and was eluted in the flow-through instead of in the eluent of 15% B as the native one (Table 1 and Figure 3b). A similar explanation could be applied to T184-197, and as its cyclized form and native form had a net charge of 1- and 0, respectively, both forms of the peptide were identified in the flow-through (Table 1 and Figure 3c, d). However, no cyclization of N-terminal tyrosine was reported, to our knowledge. It was noted there was no peptide with net charge higher than 0 in flow-through and no other peptide coeluted with peptides with net charge at 1+. However, there were three Cys-containing peptides, i.e., T569-587 (TVMENFVAFVDKCCAADDK), T569597 (TVMENFVAFVDKCCAADDKEACFAVEG PK), and T469482 (MPCTEDYLSLILNR), which had a net charge of 1+, eluted by 100% B. Peptide T469-482 was eluted at last and further bled through four fractions (fraction 58-61). To solve the confusion, the peptide mixture was separated by reversed-phase highperformance liquid chromatography. All three peptides showed

a more hydrophobic characteristic, and T469-482 was the most hydrophobic one (data not shown). Therefore, it indicated that although separation on a SCX column was dominated by electrostatic strength it was also affected by the hydrophobic property of the analyte. Effect on the Fragmentation and Detection. To explore the effect of performic oxidation on the fragmentation behavior of Cyscontaining tryptic peptides, oxidized and carbamidomethylated forms of peptide T469-482 were selected to perform MS/MS experiment, respectively (Figure 4). Although the oxidized form detected at much lower intensity in positive MS (Figure 4c) and accumulated much less shots in positive MS/MS (Figure 4d) than the carbamidomethylated one (Figure 4a, b), it showed a dramatically enhanced fragmentation efficiency and only contiguous y-series ions were produced, which greatly simplified the spectrum (Figure 4d). This was consistent with the results of Burlet and co-workers.19
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Figure 5. Effect of performic oxidation on the fragmentation behavior of Cys-containing tryptic peptide. All cysteines in their sulfonic acid forms and Met in their sulfone forms. All spectra were acquired in positive mode. For clarity, not all product ions are labeled.

A recent achievement in modification for easier peptide sequencing was N-terminal sulfonation.22 After such a modification, only y-series product ions were detected for monoprotonated precursor; thus, the interpretation of the spectrum was greatly simplified. The explanation was that the negative sulfonic group at the N-terminal neutralized the positive charge required for b-series ions and thereby only y-series ions were produced. According to this explanation, those product ions containing sulfonic group should be suppressed in positive MS/MS experiments. However, it was noted in the present study that even the cysteic acid residue was adjacent to the C-terminal (Figure 5a) or up to two cysteic acid residues were involved (Figure 5b, c) and y-series ions containing sulfonic groups were still dominantly produced. In fact, all tryptic peptides with variable numbers of sulfonic groups distributing randomly in the sequences showed enhanced fragmentation efficiency with cysteic acid residues in the product ions (Figure 5d). Spectra shown in Figure 5 or in
(20) Sanger, F.; Thompson, E. O. P.; Kitai, R. Biochem. J. 1955, 59, 509-518. (21) Khandke, K. M.; Fairwell, T.; Chait, B. T.; Manjula, B. N. Int. J. Pept. Protein Res. 1989, 34, 118-123. (22) Keough, T.; Youngquist, R. S.; Lacey, M. P. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 7131-7136.

other figures in the present paper were just a few examples among many such cases we encountered during our study (data not shown). So it was indicated that it was the position of basic amino residue such as arginine that dominated which types of product ions were preferred to others. In fact, it was reported that only b-series ions were observed when arginine located at N-terminal.23 It was reported that adduction of the negative sulfonic group to peptide led to a decreased detection in positive MS mode.12 Similar results were also observed in the present study (Figure 6). When tryptic digestion of Ox-BSA was analyzed in positive MS mode, only eight sulfonic peptides were detected and coverage of 47.85% was obtained (Figure 6a). However, when analyzed in negative MS mode, 28 sulfonic peptides were detected and coverage of 79.07% was obtained (Figure 6b). In fact, only one sulfonic tryptic peptide, i.e., T76-88, with two cysteic acids in its sequence was not observed. However, T76-88 was detected in fraction 21 of the SCX chromatography by negative MS and then confidently identified by positive MS/MS (Figure 7, Table 1). So it was demonstrated that sulfonic peptide could be detected efficiently in negative mode. This could be further verified as
(23) Men, L.; Wang, Y. Rapid Commun. Mass Spectom. 2005, 19, 23-30.

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Figure 6. Effect of performic oxidation on the detection of Cys-containing tryptic peptides. (a) Positive and (b) negative MS spectra of tryptic digest of Ox-BSA. Detected Cys-containing peptides are labeled by asterisks.

Figure 7. Sulfonic peptide T76-88 detected in fraction 21 of the SCX chromatography in both positive (a) and negative MS mode (b) and identified by positive MS/MS experiment (c). All cysteines are in their sulfonic acid forms.

shown in Figure 8. PBP2 was hardly detected in its native form in negative mode (Figure 8b) despite its strong intensity in positive mode (Figure 8a). However, PBP2 was easily detected in negative mode (Figure 8c) after performic oxidation although the intensity was still more intense in positive mode (Figure 8d). From our experience and reported data from other researchers,24 the sequence coverage rarely exceeded 60% when BSA was

treated by a reduction/alkylation method before tryptic hydrolysis. The higher coverage obtained here (Figure 6b) also indicated that performic oxidation could efficiently cleave disulfide bonds, thus facilitating subsequent tryptic hydrolysis. The conversion of thiol group into the more hydrophilic sulfonic group might also contribute to a more efficient tryptic hydrolysis.
(24) Brian, T. C.; Sechi, S. Anal. Chem. 1998, 70, 5150-5158.

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Figure 8. Effect of performic oxidation on the detection of Cys-containing tryptic peptides. (a) Positive MS spectra of PBP2. (b) Negative MS spectra of PBP2. (c) Negative MS spectrum of Ox-PBP2. (d) Positive MS spectrum of Ox-PBP2.

It was also noted that peaks corresponding to adduction of potassium were detected when fractions from SCX chromatography were directly analyzed using the method described in Experimental Section (Figure 7a, b; Figure 9a, b). These peaks could be efficiently eliminated when fractions from SCX chromatography were further separated on reversed-phase HPLC as commonly used in proteomics research. Although sulfonic peptide showed decreased sensitivity in positive mode, it did not pose a serious problem because of the dramatically enhanced fragmentation efficiency in the positive MS/MS experiment. As shown in Figure 9, although sulfonic peptides T581-597 and T267-280 were hardly observed in the positive MS spectrum (Figure 9a), both of them gave high-quality spectra in positive MS/MS experiments (Figure 9c, d). Thus, when performic oxidized sample was analyzed, a negative MS scan could be first performed (Figure 9b) and then positive MS/MS experiments carried out according to the results of negative MS by simply adding two protons to the corresponding m/z value to form a precursor. Should such a function integrate into current operating software. the strategy presented here could be carried out in a high-throughput way.
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MALDI-MS/MS versus ESI-MS/MS. It was noted that almost all successful cases of N-terminal sulfonation modification for peptide sequencing were carried out using positive monoprotonated ions as precursors. In fact, it was reported that when multiprotonated ions were used a complex MS/MS spectrum with both b- and y-series ions resulted.25,26 On the other hand, the adduction of the sulfonic group to the N-terminal of a tryptic peptide also led to a serious in-source fragmentation when an ESI source was used.26 As monoprotonated tryptic peptides with internal sulfonic group showed similar fragmentation behavior as monoprotonated N-terminal sulfonated ones, it was expected that similar in-source fragmentation be shown. In fact, serious in-source fragmentation of Ox-PBP2 did occur (Figure 10a) in contrast to the native peptides (Figure 10b) even under the same operating conditions when an ESI-based method was used. Similar results were noted in another report.27 Such a serious in-source fragmentation largely excluded ESI-MS/MS as a method to analyze
(25) Lee, Y. H.; Han, H.; Chang, S.-B.; Lee, S.-W. Rapid Commun. Mass Spectom. 2004, 18, 3019-3027. (26) Bauer, M. D.; Sun, Y.; Keough, T.; Lacey, M. P. Rapid Commun. Mass Spectom. 2000, 14, 924-929.

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Figure 9. Sulfonic peptides efficiently detected in negative mode MALDI-MS and subsequently identified easily in positive mode MALDI-MS/ MS despite their poor detection in positive mode MALDI-MS. (a) Positive MS spectrum of fraction 4 from SCX chromatography of tryptic digest of Ox-BSA. (b) Negative MS spectrum of fraction 4 from SCX chromatography of tryptic digest of Ox-BSA. (c) Positive MS/MS spectrum of T581-597. (d) Positive MS/MS spectrum of T267-280. All cysteines are in their sulfonic acid forms.

sulfonic peptides in proteomics research. Although enhanced insource fragmentation was also observed in a MALDI-TOF/TOF mass spectrometer in our previous study,16 it was not such a serious problem as for an ESI ion source, for example, no in-sourced fragmentation of Ox-PBP2 was observed in Figure 8d. The difference observed between the two types of ion sources might be attributed to the ability of an ESI source to produce multiprotonated ions, which made the sulfonic peptide even more unstable according to the mobile proton model.28 Furthermore, both b- and y-series ions were observed in either single or double protonated forms when double or higher protonated precursor was selected (data not shown). Similar results were also observed in other reports.23 Therefore, a MALDI ion source based mass spectrometer was preferred because of its ability to produce single protonated ions and negligible in-source fragmentation.
(27) Li, W.; Boykins, R. A.; Backlund, P. S.; Wang, G.; Chen, H.-C. Anal. Chem. 2002, 74, 5701-5710. (28) Wysocki, V. H.; Tsaprailis, G.; Smith, L. L.; Breci, L. A. J. Mass Spectrom. 2000, 35, 1399.

CONCLUSION It should be noted that performic oxidation is the critical step in the strategy presented here. Performic oxidation is an efficient method for disulfide scission and had been extensively used for various purposes from its introduction by Sanger.29 The method has several merits in terms of its possible use in proteomics research. First, Cys and Met were both turned into their stable oxidized forms and thus eliminated the possibility of their incomplete oxidation during sample preparation. It was known that such incomplete oxidation could complicate the sample and result in confusion in subsequent mass spectrometric analysis.30,31 Second, performic acid is a strong oxidant and the chemistry was expected to be complete. In contrast, reduction/alkylation for opening the disulfide bond could hardly be complete,32 which
(29) Sanger, F. Biochem. J. 1945, 39, 507-515. (30) Steen, H.; Mann, M. J. Am. Soc. Mass Spectrom. 2000, 12, 228. (31) Mo, W.; Ma, Y.; Takao, T.; Neubert, T. A. Rapid Commun. Mass Spectom. 2000, 14, 2080-2081. (32) Hamdan, M.; Righetti, P. G. Mass Spectrom. Rev. 2002, 21, 287.

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Figure 10. Positive mass spectra of the PBP2: NVQCRPTQVQLRPVQVR obtained on a nanoLC-ESI-Q-TOF instrument for a comparison of the in-source fragmentation after (a) and before (b) performic oxidation. A few fragments observed in (b) should be derived from trace oxidized PBP2 produced during sample preparation or ionization.

might cause inefficient hydrolysis and lead to a more complex sample. Third, a recent study demonstrated that statistical confidence for protein identification could be enhanced due to the incorporation of additional oxygen atoms through performic oxidation.33 Fourth, formic acid is an excellent solvent for proteins and is commonly used for dissolving hydrophobic proteins or peptides.34 In addition, during performic oxidation, cysteine was converted into cysteic acid and simultaneously disulfide bonds were opened, which was also expected to improve the solubility of proteins. Thus, performic oxidation is an attractive pretreatment method for membrane protein that has been a real challenge for proteomics to date. Furthermore, if stable isotope reagents, i.e., 18O and 16O reagents, were available, each cysteine residue means a mass difference of 6 Da for quantification35 could be achieved after oxidation with different stable isotope reagents. Other merits include the volatile regents and the simple operation. In this study, performic oxidation was integrated into a single proteomics strategy and full use of all its merits was made. We showed that sulfonic peptides resulting from performic oxidation could be selectively enriched through SCX chromatography,
(33) Matthiesen, R.; Bauw, G.; Welinder, K. G. Anal. Chem. 2004, 23, 68486852. (34) Glenner, G. G.; Wong, C. W.; Biochem. Biophys. Res. Commun. 1984, 120, 885-890. (35) Lill J. Mass Spectrom Rev. 2003, 22, 182-194.

efficiently detected in negative MALDI-MS mode, and subsequently easily identified by positive MALDI-MS/MS experiments. The strategy presented here has its promise in facilitating enzymatic digestion, selective enrichment of Cys-containing peptides, reduction of the complexity of sample, enhancing fragmentation efficiency/simplifying MS/MS spectrum, and enhancing statistical confidence for protein identification through database searching, i.e., the whole work flow of a proteomics research. It should be noted that although we did not couple SCX separation to reversed-phase HPLC in the present study it could be conveniently carried out during practical use. It is believed that the strategy will see increasing use in proteomics research and a related study in our group is underway. ACKNOWLEDGMENT The authors received financial support for the work from China Technology R&D Project (2002BA711A11, 2004BA711A18), NationalKeyProgramforBasicResearch(2001CB510201,2004CB520802), National Natural Science Foundation (20275044, 20275046, 30321003), and Beijing Municipal Program for Science & Technology (H030230280190). Received for review April 12, 2005. Accepted July 26, 2005.
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