Effects of Tryptic Peptide Esterification in MALDI Mass Spectrometry

Anal. Chem. 2005, 77, 4185-4193

Effects of Tryptic Peptide Esterification in MALDI Mass Spectrometry Tae-Young Kim,† Yves V. Brun,‡ and James P. Reilly*,†

Departments of Chemistry and Biology, Indiana University, Bloomington, Indiana 47405

The effect of esterification on MALDI ion yield is investigated by using alcohols having different aliphatic chain lengths. For peptides whose ionization yields increase with derivatization, more hydrophobic alcohols tend to yield greater peak enhancements. The completeness of the reaction increases from propanol to methanol. Undesired solvolysis of the amide group in the side chain of Asn or Gln leads to unexpected ester products. Ethanol is suggested as the optimal alcohol for esterification in proteomics experiments since it yields almost complete esterification without substantial solvolysis. Ethanol esterification was employed to facilitate the identification of gel-separated proteins. Esterification has been widely employed in mass spectrometric research. Peptides obtained from enzymatic digestions were esterified with methanolic HCl and then acetylated in order to make them volatile enough for sequence analysis by gas chromatography/mass spectrometry (GC/MS).1-3 Microscale esterification was developed to determine C-terminal amidation in peptides by matrix-assisted desorption/ionization time-of-flight (MALDI-TOF).4 To avoid oxidation of tryptophan or methionine or other side reactions, esterification was performed in the presence of β-mercaptoethanol. Kowalak and Walsh utilized esterification to identify a novel post-translational modification of E. coli ribosomal protein S12 with MALDI-TOF MS.5 By esterifying an Asp-containing peptide, they showed that the β-carboxyl group was not modified, but demonstrated that Asp-88 is converted to β-methylthio-aspartic acid. Esterification of peptides has been employed in order to overcome ion yield suppression. Conversion of carboxyl groups into corresponding neutral ester groups dramatically reduces spectral suppression for peptides having negative net charge in plasma desorption mass spectra.6 Esterification has been shown to improve sensitivity for hydrophilic peptides in fast atom bombardment (FAB)7 and liquid secondary * To whom correspondence should be addressed. E-mail: [email protected]. † Department of Chemistry. ‡ Department of Biology. (1) Agarwal, K. L.; Kenner, G. W.; Sheppard, R. C. J. Am. Chem. Soc. 1969, 91, 3096-3097. (2) Caprioli, R. M.; Seifert, W. E., Jr.; Sutherland, D. E. Biochem. Biophys. Res. Commun. 1973, 55, 67-75. (3) Young, M. A.; Desiderio, D. M. Anal. Biochem. 1976, 70, 110-123. (4) Fischer, W. H.; Craig, A. G. J. Protein Chem. 1994, 13, 452-453. (5) Kowalak, J.; Walsh, K. A. Protein Sci. 1996, 5, 1625-1632. (6) Schmitter, J.-M. J. Chromatogr. 1991, 557, 359-368. (7) Naylor, S.; Findeis, A. F.; Gibson, B. W.; Williams, D. H. J. Am. Chem. Soc. 1986, 108, 6359-6363. 10.1021/ac0481250 CCC: $30.25 Published on Web 05/17/2005

© 2005 American Chemical Society

ion mass spectrometry (LSIMS).8 The number of free carboxylic groups in biological molecules has been determined from mass shifts induced by esterification.9,10 Esterification has been applied to de novo sequencing of peptides by Hunt et al. through a comparison of tandem mass spectra of nonesterified and esterifed peptides.11 All peaks arising from C-terminal peptide fragments or from fragments that contain Asp and Glu residues shift by +14 Da units upon esterification with methanol, whereas N-terminal fragments with no acidic residues show the same mass in both spectra. Recently, Goodlett and co-workers proposed differential isotope esterification of peptides for relative quantitation by using both d0- and d3-methanols.12 They demonstrated that this method can also be exploited for de novo sequencing by comparing the spectra obtained from d0- and d3-methyl esterified samples to identify y-ion fragments. Finally, tryptic peptides are often converted to methyl esters prior to immobilized metal-affinity chromatography so as to reduce nonspecific binding of phosphopeptides in complex mixtures.13-15 The value of a derivatization reaction is affected by the speed and completeness of the reaction, the occurrence of undesirable byproducts, and ease of reagent removal. These factors can be particularly critical when a derivatization is applied to a complex system, for instance, a mixture of tryptic protein digests. Esterification appears to be attractive because of its simplicity. Methyl esterification, however, causes solvolysis, the conversion of the amide side chains of asparagine and glutamine to the corresponding alcoholic esters. Young and Desiderio demonstrated that solvoysis occurs to a greater extent when higher concentrations of HCl, higher temperatures, and/or longer reaction times are used.3 Whereas esterification at room temperature for 4 h avoided solvolysis of Asn and Gln, incubation of peptides at 45 °C for 16 (8) Falick, A. M.; Maltby, D. A. Anal. Biochem. 1989, 182, 165-169. (9) Ferone, R.; Hanlon, M. H.; Singer, S. C.; Hunt, D. F. J. Biol. Chem. 1986, 261, 16356-16362. (10) Arnold, R. J.; Reilly, J. P. Anal. Biochem. 2000, 281, 45-54. (11) Hunt, D. F.; Yates, J. R., III; Shabanowitz, J.; Winston, S.; Hauer, C. R. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 6233-6237. (12) Goodlett, D. R.; Keller, A.; Watts, J. D.; Newitt, R.; Yi, E. C.; Purvine, S.; Eng, J. K.; Von Haller, P.; Aebersold, R.; Kolker, E. Rapid Commun. Mass Spectrom. 2001, 15, 1214-1221. (13) Ficarro, S. B.; McCleland, M. L.; Stukenberg, P. T.; Burke, D. J.; Ross, M. M.; Shabanowitz, J.; Hunt, D. F.; White, F. M. Nat. Biotechnol. 2002, 20, 301-305. (14) Dikler, S.; Saenz-Vash, V.; Qui, H.; Stoerker, J.; Grant, K. L. 51st ASMS Conference on Mass Spectrometry and Allied Topics; Montreal, Quebec, Canada, June 8-12 2003; MPS 366. (15) Stupak, J.; Wang, Z.; Huaizhi, L.; Li, L. 51st ASMS Conference on Mass Spectrometry and Allied Topics; Montreal, Quebec, Canada, June 8-12 2003; MPS 369.

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h converted Asn and Gln to methanolic esters of Asp and Glu. They exploited these different conditions to distinguish aspartyl and glutamyl dipeptides from asparaginyl and glutaminyl dipeptides in GC/MS studies. To distinguish the isobaric immonium ions of Gln and Lys in peptide sequencing by LSIMS, Kausler et al. exploited the solvolysis of Gln at 100 °C.16 Despite the extensive occurrence of solvolysis, methanolic HCl has mainly been employed in mass spectrometric studies involving esterification. Because it offers the best combination of ion yield and sample cleanup, hexanol has been proposed as the optimal esterifying reagent.8 Nevertheless, it also exhibits incomplete reactions for peptides with multiple carboxylic groups. This is important because incomplete derivatization and side reactions increase the complexity of data analysis and decrease the sensitivity of experiments. In this work MALDI-TOF MS is employed to examine the esterification of tryptic peptides with alcohols. The effect of esterification on intensities of tryptic peptides was investigated to help improve our understanding of MALDI peptide ionization yield. Mass spectra of mixtures that consist of equivalent amounts of unesterified and esterified tryptic digests are compared for quantitative analysis. The effect of alcohol size on peak intensity is also probed. Karty et al. reported the impact of guanidination on proteomic analysis with proteins isolated from Caulobacter crescentus stalk by 2D gel eletrophoresis.17 They utilized the number of lysine residues of typtic peptides to make a protein’s identification more definitive. Likewise, we can also use information about the number of free carboxylic groups in a tryptic protein digest to reduce the number of false protein assignments. Pappin also demonstrated that esterification can be employed to obtain compositional information of peptides (number of acidic residues) in peptide mass mapping.18 Ethanol esterification is utilized to facilitate the identification of proteins separated in a 1D gel band with Prodigies (Protein Digest Identification and Elucidation Software),17,19 a data analysis program written in-house. The effect of tryptic digest esterification on peptide mass mapping database searching is also discussed. EXPERIMENTAL SECTION Materials. Human hemoglobin, bovine serum albumin (BSA), tosyl-L-phenylalanine chloromethyl ketone (TPCK)-treated bovine trypsin, R-cyano-4-hydroxycinnamic acid (CHCA), and Zorbax C18 were purchased from Sigma Chemical Co. (St. Louis, MO). Acetyl chloride, trifluoroacetic acid (TFA), dithiothreitol (DTT), and iodoacetic acid were supplied by Aldrich Chemical Co. (Milwaukee, WI). Methanol, n-propanol, ethylene glycol, acetonitrile, and 3 Å molecular sieve were obtained from Fisher (Fair Lawn, NJ). Ammonium bicarbonate was supplied by EM Science (Gibbstown, NJ). Ethanol was purchased from Aaper Alcohol and Chemical Co. (Shelbyville, KY). The sequencing grade trypsin was supplied by Promega Co. (Madison, WI). All alcohols were dried by using 3 Å molecular sieve prior to mixing with acetyl (16) Kausler, W.; Schneider, K.; Spiteller, G. Biomed. Environ. Mass Spectrom. 1988, 17, 15-19. (17) Karty, J. A.; Ireland, M. M. E.; Brun, Y. V.; Reilly, J. P. J. Proteome Res. 2002, 1, 325-335. (18) Pappin, D. J. Methods Mol. Biol. 1997, 64, 165-173. (19) Karty, J. A.; Kim, T.-Y.; Reilly, J. P. In Sample Preparation for Hyphenated Analytical Techniques; Rosenfeld, J. A., Ed.; Blackwell Publishing Ltd., CRC Press: Oxford, U.K., 2004; pp 52-79.

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chloride. Water used in this work was double-distilled using a Millipore apparatus. Microcon 30 kDa molecular weight cutoff (MWCO) centrifugal filter devices were obtained from Amicon, Inc. (Beverly, MA). Tryptic Digestion of Human Hemoglobin. Seventy-five microliters of hemoglobin solution (1 g/L) and 25 µL of 100 mM ammonium bicarbonate solution were added to a tube containing 5 µg of trypsin. The mixture was vortexed and incubated at 37 °C for 4 h. The reaction was terminated by adding 10 µL of 10% (v/ v) TFA in water to the mixture. The solution was divided into four 25 µL aliquots that were then dried in a speed-vac. Tryptic Digestion of BSA. A 440 pmol/µL aqueous solution of BSA was mixed in equal volume with a stock solution of 50 mM DTT in 100 mM ammonium bicarbonate. Reduction of disulfide bonds was carried out at 70 °C for 45 min. Carboxymethylation was performed by mixing 20 g/L aqueous iodoacetic acid in equal volume with the reduction mixture and incubating it at room temperature for 30 min in the dark. The reduction mixture was purified by 30 kDa MWCO filtration prior to digestion so that excess reagents would not also modify the cysteines of trypsin. A fresh MWCO membrane was initially washed twice with 500 µL aliquots of double-distilled water. The reduced protein mixture was then transferred to the washed MWCO filter and centrifuged at 10 000g for 10 min. Then 100 µL of 15 mM ammonium bicarbonate solution was added and the purified/ reduced protein was reconstituted by sonicating and vortexing. Digestion was initiated by adding 2 pmol/µL trypsin in 25 mM ammonium bicarbonate in 1:1 volumetric ratio to the reduced solution. This solution was incubated at 37 °C for 16 h. 10% (v/v) TFA aqueous solution was added to the solution to terminate the reaction. Four 25 µL aliquots were made from the quenched solution and then evaporated to dryness in a speed-vac. Gel Spot Destaining and Protein Digestion. 1D gel bands of Caulobacter crescentus stalk protein were obtained from the laboratory of Dr. Yves Brun. Gel spots were destained and digested in the manner described by Karty et al.17 Gel spots were destained by adding 100 µL of 50% (v/v) 100 mM ammonium bicarbonate in acetonitrile and shaking for 20 min. This step was repeated after decanting the liquid. Then 100 µL of distilled water was added to the decanted tube and shaken for 15 min. After decanting, another 100 µL of water was added and the gel spots were allowed to stand for 5 min. The water was decanted, and the spots were soaked in 100 µL of acetonitrile for 5 min. The destained gel spots were dried in a speed-vac. To each dried gel band, 15 µL of 16.67 mg/L sequencing grade trypsin, 3 µL of 16.16 mg/L TPCK-treated trypsin in 10 mM ammonium bicarbonate, and 20 µL of distilled water were added and incubated for 12-16 h at 37 °C. Peptide Extraction. The digestion was quenched by adding 100 µL of 0.1% (v/v) TFA. The solution was sonicated for 20 min. Liquid was separated from the intact piece of gel using a micropipet and then saved in a separate tube. Then 100 µL of 30% (v/v) acetonitrile were added to the gel spot and the solution was sonicated for 20 min. The liquid was extracted once again. Finally, this was repeated with 100 µL of 60% (v/v) acetonitrile. The liquid obtained from these three steps was combined and centrifuged to dryness. Eight microliters of water was added to resuspend the peptides.

Figure 1. MALDI TOF mass spectra of (A) unmodified, (B) methyl esterified, (C) ethyl esterified, (D) n-propyl esterified, and (E) ethylene glycol esterified human hemoglobin tryptic digests. Fully esterified peaks are labeled with a [*] and peaks corresponding to incomplete esterification or to larger than expected esterification shifts are labeled with [-] and [+], respectively.

Esterification of Tryptic Digest. Alcoholic HCl was prepared by the dropwise addition of 15 µL of acetyl chloride to 85 µL of dried alcohol as in the work of Kowalak et al.5 For tryptic protein digests, 100 µL of alcoholic HCl was added to the dried aliquot of tryptic digest and allowed to react at 37 °C for 4 h. (Different esterification conditions were also tried, but a higher temperature increased the amount of solvolysis of Asn/Gln and a lower temperature yielded less complete esterification.) The reaction mixture was dried in a speed-vac and diluted with 10 µL of doubledistilled water. For the gel band analysis, another 3 µL of extract was dried in a speed-vac. Then, 100 µL of alcoholic HCl was added to this dried aliquot of gel band extract. Mass Spectrometry. For tryptic protein digests, MALDI samples were prepared by mixing 1 µL of peptide suspension with 9 µL of matrix solution (CHCA in 1:1 (v/v) 0.1% TFA aqueous solution/acetonitrile), and then 0.65 µL of matrix/analyte solution were applied to the sample probe. Esterified samples obtained from the gel band digests were purified using micropipet tips packed with C18 stationary phase and then peptides were eluted with 2 µL of matrix solution (10 g/L CHCA in 50% (v/v) acetonitrile/0.1% (v/v) TFA). Mass spectra were recorded on a Bruker Reflex III reflectron TOF mass spectrometer. One hundred shots of 337 nm light from a 200 µJ N2 laser were irradiated to record each mass spectrum. Database Searches. Expected monoisotopic masses and pI values of tryptic protein digests were obtained from the web-based Expasy search engine.20 In the analysis of data involving both underivatized and derivatized samples using our Prodigies software,17,19 a peptide mass error was assumed to be 0.15 Da. We also allowed for up to two missed trypsin cleavage sites, partial single oxidation of methionine residues, partial loss of protein N-terminal methionines, complete reduction and alkylation of cysteines, and partial conversion of peptide N-terminal glutamine residues to pyroglutamic acid. (20) http://www.expasy.org.

RESULTS AND DISCUSSION Reactivities of Different Alcohols. In an attempt to probe the completeness of the derivatization, hemoglobin tryptic digests were esterified with three different alcohols, methanol, ethanol, and n-propanol. Esterification was accomplished through incubation of the peptides in alcoholic HCl at 37 °C for 4 h. Figure 1A displays a MALDI mass spectrum of underivatized hemoglobin tryptic peptides. MALDI mass spectra of hemoglobin tryptic digests esterified with methanol, ethanol, n-propanol, and ethylene glycol are shown in Figure 1B, C, D, and E, respectively. The sequences and masses of hemoglobin tryptic peptides appearing in Figure 1 are summarized in Table 1. With methanol and ethanol, the esterification of most peptides is complete. With n-propanol, however, peaks associated with full esterification (for which every Asp, Glu, and C-terminus is esterified) cannot be seen for a few peptides. Fully esterified peptides are labeled with a [*]. Peaks corresponding to incomplete esterification or to larger than expected esterification shifts are labeled with [-] and [+], respectively. The number of incompletely esterified peptides increases from one (g-) with methanol (Figure 1B) to three (d-, g-, and l-) with ethanol (Figure 1C) to nine (d-, e-, g-, h-, k-, l-, m-, n-, and o-) with n-propanol (Figure 1D). With n-propanol there is even evidence of underivatized peptides (labeled with no sign, a, c, d, e, g, k, l, m, and n in Figure 1D) as well as partially esterified peptides (d-, g-, h-, l-, m-, n-, and o-). These observations indicate that different alcohols exhibit significantly different esterification reactivities. Esterification shifts that are larger than expected result from solvolysis of the amide group in the side chain of Asn or Gln to the corresponding ester. This phenomenon induces mass shifts of +15 Da for methanol, +29 Da for ethanol, and +43 Da for n-propanol. In esterification with methanol (Figure 1B), solvolysis product peaks (labeled with [+]) are found for almost all peptides that contain an Asn or Gln residue. For peptides containing two Asn or Gln within their sequences, both singly and doubly Analytical Chemistry, Vol. 77, No. 13, July 1, 2005

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Table 1. Comparison of MALDI Ionization of Hemoglobin Tryptic Peptides Esterified with Different Alcohols relative peak intensityc ID

sequence range

sequence

a b c d e f g h i j k l m n o

R92-R100 β97-β105 β32-β41 β19-β31 β122-β133 β134-β147 R18-R32 β68-β83 β106-β121 β67-β83 R42-R57 β42-β60 β84-β105 R63-R91 R62-R91

LRVDPVNFK LHVDPENFR LLVVYPWTQR VNVDEVGGEALGR EFTPPVQAAYQK VVAGVANALAHKYH VGAHAGEYGAEALER VLGAFSDGLAHLDNLK LLGNVLVCVLAHHFGK KVLGAFSDGLAHLDNLK TYFPHFDLSHGSAQVK FFESFGDLSTPDAVMGNPK GTFATLSELHCDKLHVDPENFR VADALTNAVAHVDDMPNALSALSDLHAHK KVADALTNAVAHVDDMPNALSALSDLHAHK

(M + H)+ PIa 1087.63 1126.56 1274.73 1314.67 1378.70 1449.80 1529.73 1669.89 1719.97 1797.99 1833.89 2058.95 2529.22 2996.49 3124.58

H.I.b

MeOH

8.8 -4.8 1.2 ( 0.4 5.3 -9.0 2.1 ( 0.8 8.8 3.5 0.05 ( 0.01 4.1 -1.5 4.0 ( 1.8 6.1 -9.0 0.28 ( 0.05 8.6 8.1 1.2 ( 0.4 4.8 -5.5 3.9 ( 1.4 5.2 6.6 0.98 ( 0.37 8.2 20.3 0.77 ( 0.10 6.8 8.2 1.1 ( 0.4 6.6 -7.5 0.26 ( 0.09 4.0 -4.1 0.16 ( 0.04 5.3 -11.0 0.37 ( 0.11 5.1 3.9 0.30 ( 0.10 5.7 0.0 0.43 ( 0.16

EtOH

PrOH

EtGly

3.2 ( 0.1 1.2 ( 0.5 0 13 ( 2 0.34 ( 0.21 0.90 ( 0.30 8.6 ( 4.7 1.7 ( 0.4 0.65 ( 0.06 1.5 ( 0.6 0.71 ( 0.28 0.19 ( 0.07 0.48 ( 0.25 0.52 ( 0.13 0.35 ( 0.08

1.4 ( 0.1 1.7 ( 0.2 0.36 ( 0.01 9.2 ( 1.9 0.98 ( 0.06 1.0 ( 0.1 29 ( 3 0.98 ( 0.04 0.57 ( 0.09 0.97 ( 0.03 0.93 ( 0.04 0.95 ( 0.02 0.83 ( 0.03 d d

1.2 ( 0.4 1.4 ( 0.3 0.19 ( 0.07 0.75 ( 0.20 0.23 ( 0.03 0.78 ( 0.06 0.99 ( 0.11 0.58 ( 0.15 0.63 ( 0.19 0.54 ( 0.23 0.38 ( 0.14 0.13 ( 0.09 0.30 ( 0.04 0.27 ( 0.03 0.43 ( 0.13

a pI values were calculated by using the web-based Expasy program.20 b Hydropathy Indices (H.I.) were calculated the values of individual amino acids as defined elsewhere.21 A negative value in H.I. means that the peptide is hydrophobic and a positive value means that it is hydrophilic. c The relative peak intensity after esterification is defined as the ratio of peak intensity for the fully esterified peak to that for the unreacted one in a spectrum obtained from an equivalent mixture of unmodified and esterified peptides. The values in the table represent an average of four-time measurements with a standard deviation. d The relative peak intensities could not be calculated for two cases since unmodified peptides were not detectable in mass spectra of a 1:1 mixture of underivatized and esterified peptides.

solvolized peaks appear. Peak intensities of solvolysis products are comparable to those arising from fully esterified peptides without solvolysis. To confirm the solvolysis interpretation, the peaks labeled d* and d+ (VNVDEVGGEALGR) in Figure 1B were fragmented in a tandem-TOF mass spectrometer and daughter ion distributions for each were recorded (data not shown). Except for b1 all b-type ions arising from d+ were 15 Da heavier than those derived from d*, whereas the masses of corresponding y-, v-, and w-type ions in the two spectra were identical. This demonstrates that solvolysis occurred at the Asn residue in the peptide. In esterification with ethanol (Figure 1C), solvolysis peaks are much smaller than those corresponding to fully esterified peptides. Likewise, no doubly solvolized peptide appears in Figure 1C. Only three solvolysis product peaks of very low intensities (b+, d+, and l+) are observed in esterification with n-propanol (Figure 1D). The extent of esterification and solvolysis appears to depend on differences in reactivities of alcohols that are induced by their different nucleophilicities. The existence of an electron-donating alkyl group is beneficial for providing electron density necessary for nucleophilic attack. Steric effects, however, must also play a role. Methanol is a less hindered nucleophile than ethanol or n-propanol. Thus, the fact that methanol yields the most complete esterification and the most frequent solvolysis among alcohols implies that the steric effect is more crucial for this reaction. Falick et al. found no evidence of solvolysis of Asn or Gln residues in esterification experiments using n-hexanol, even with harsher reaction conditions.8 This is consistent with our observations. MALDI Ion Yields of Esterified Peptides. Ion signals associated with individual peptides often change upon esterification. In Figure 1A, for instance, the most intense peak is at m/z 1274.73 (peak c) and the next one is at m/z 1529.73 (peak g). After methyl esterification, the most intense peak is at m/z 1585.78 corresponding to the methylated R18-R32 (peak g*). The next most intense peak in Figure 1B is the methyl esterified peak d*, 4188 Analytical Chemistry, Vol. 77, No. 13, July 1, 2005

which is not prominent in the underivatized spectrum. However, the signal for methylated β32-β41 (peak c*) is much weaker than before esterification. This trend also follows in Figure 1C. These observations indicate that esterification with alcohols can affect MALDI ion yields in unpredictable ways. To investigate the effect of esterification on absolute signal intensities, mass spectra of a 1:1 mixture of unreacted and esterified tryptic hemoglobin peptides were recorded after reaction with methanol, ethanol, n-propanol, and ethylene glycol. In these spectra, each underivatized peptide serves as an internal standard for quantitative comparison with its esterified counterpart. The relative peak intensity for esterification is defined as the ratio of peak intensity for a fully esterified peptide to that for the unreacted one within the same spectrum. (Values greater than 1 correspond to peak intensities that increase upon derivatization.) The observed relative peak intensities for tryptic digests of hemoglobin esterified with methanol, ethanol, n-propanol, and ethylene glycol are summarized in Table 1. (The values in the table represent an average of four-time measurements with a standard deviation.) Some peptides appear more intense after esterification while others are diminished. In an attempt to understand these variations, the pI values and hydropathy indices for underivatized tryptic hemoglobin peptides were calculated using the web-based Expasy search engine20 and the hydropathy scales for the 20 amino acids defined by Kyte et al.21 These molecular properties were considered since the replacement of a charged carboxylic acid group with a neutral ester affects both the pI and the hydrophobicity of a peptide (Table 1). Figures 2A and 2C shows plots of relative peak intensities vs pI values for methanol and ethanol, respectively. Plots of relative peptide peak intensities vs hydropathy indices for esterification with methanol and ethanol are displayed in Figures 2B and 2D, respectively. A negative value in hydropathy index means that the peptide is hydrophobic and (21) Kyte, J.; Doolittle, R. F. J. Mol. Biol. 1982, 157, 105-132.

Figure 2. Plots of relative peak intensity vs pI for hemoglobin tryptic digests esterified with (A) methanol and (C) ethanol. Plots of relative peak intensity vs hydropathy index for hemoglobin tryptic digests esterified with (B) methanol and (D) ethanol. The data shown in Figures 2 and 3 are an average of four measurements.

a positive value means that it is hydrophilic. The peptides showing significant peak enhancement after esterification generally have low pI values and are hydrophilic before derivatization. Nevertheless, there is no clear correlation between these properties. For example, although FFESFGDKSTPDAVMGNPK (peptide l) has a pI value of 4.0 and a hydropathy index of -4.1, it shows a decrease in peak intensity after esterification with methanol and ethanol. Detection of hydrophilic peptides is typically suppressed by the presence of hydrophobic peptides in FAB and LSIMS. Because the matrixes such as glycerol used in these experiments are hydrophilic liquids, hydrophobic peptides tend to reside on their outer surfaces. Ion suppression can be overcome by esterification of polar carboxylic groups.7,8,22 It is unclear whether a similar effect will occur in MALDI matrixes. In addition, there is no general agreement on whether residence on the surface of a MALDI spot facilitates the desorption/ionization process.22 Thus, the lack of a strong correlation between MALDI ion yield and peptide hydrophobicity may not be surprising. Although hydrophobicity of a peptide is not the dominant factor that determines MALDI ion yield, an increase in hydrophobicity of a peptide may help enhance MALDI signal. For the peptides showing a significant increase in the ion yield after esterification, a more hydrophobic alcohol tends to enhance this effect. The fact that solvolysis byproducts occur for some peptides, however, can introduce complexity, for the existence of byproducts prevents us from exactly calculating relative peak intensities. For this reason, peptides that do not induce solvolysis byproducts are simplest to interpret. VGAHAGEYGAEALER (peptide g) does not contain Asn or Gln in its sequence and therefore produces no solvolysis byproducts. Thus, it is a good choice for studying the effect of esterification on peak intensities. As shown in Table 1, peak enhancement after esterification for this peptide increases (22) Dreisewerd, K. Chem. Rev. 2003, 103, 395-425.

from methanol to n-propanol. In contrast, VNVDEVGGEALGR (peptide d) displays a significant signal enhancement after esterification, but the n-propyl esterified peptide shows a smaller relative peak intensity than the ethyl esterified one. This may result from existence of multiple partially esterified peaks. Along similar lines, esterification with ethylene glycol (Figure 1E) decreases peak intensities of almost all tryptic hemoglobin peptides. Since ethylene glycol has an additional hydrophilic hydroxyl group, it does not significantly change the hydrophobicity of a peptide. Although ethylene glycol dries at a slower rate than the other alcohols in a speed-vac, it evaporates in 90 min with heating. In conclusion, for peptides in which esterification increases the ion yield significantly, a more hydrophobic alcohol tends to enhance this effect. This trend is consistent with that observed by Falick et al. who compared the absolute signals in LSIMS after esterification with alcohols.8 They demonstrated that if the ion yield of a hydrophilic peptide is enhanced upon esterification, longer aliphatic chain alcohols tend to produce larger signal improvements. A tryptic BSA digest was also studied to investigate the effect of esterification on peak intensities. Figures 3A and 3C shows plots of relative peak intensities of tryptic BSA peptides vs their pI values for methanol and ethanol, respectively. Plots of relative peak intensities of the peptides vs their hydropathy indices for esterification with methanol and ethanol are displayed in Figures 3B and 3D, respectively. (The data in Figure 3 were obtained from four-time analyses.) As in the previous experiment when esterification enhances a peptide’s ion yield, an ethylated peptide gives stronger signals than a methylated one. However, these effects are not straightforward to predict. Note that peptides with larger relative peak intensities generally show larger errors in Figures 2 and 3. This may be attributed to the definition of the relative peak intensity. Since the relative peak intensity is defined as the ratio of peak intenAnalytical Chemistry, Vol. 77, No. 13, July 1, 2005

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Figure 3. Plots of relative peak intensity vs pI for BSA tryptic digests esterified with (A) methanol and (C) ethanol and plots of relative peak intensity vs hydropathy index for BSA tryptic digests esterified with (B) methanol and (D) ethanol.

Figure 4. MALDI TOF mass spectra of (A) unmodified and (B) esterified tryptic digests of a 1D gel band extract from Caulobacter crescentus stalk. The underivatized and fully esterified peptides assigned to ORF 362 in the Table 2 are labeled with [#] and [*], respectively. Peaks arising from incomplete esterification, solvolysis, and trypsin autolysis are indicated by [-], [+], and Φ, respectively.

sity for a fully esterified peptide to that for the unreacted one within the same spectrum, variations of peak intensities in different experiments does not significantly change the relative peak intensities for peptides that show comparable or decreased signal intensities after esterification. However, this is not the case for peptides resulting in an increase of signal intensity after esterification because their relative peak intensities are larger than unity. Effect of Esterification on Peptide Mass Mapping. Mass spectrometric observation of multiple peptides from protein digests followed by comparison with proteomic databases is commonly referred to as peptide mass mapping. To improve the confidence with which proteins are identified, it is advantageous to obtain supplemental information about their constituent amino acids. The advantages of employing guanidination to uncover lysine residues were recently reported.17 In that work, information about the total 4190


number of lysine residues in a peptide and the production of enhanced signals for guanidinated peptides facilitated the identification of proteins separated by 2D gel electrophoresis. Esterification can be applied to derive analogous information, viz. the total number of acidic amino acids in a peptide. Comparison of underivatized and esterified mass spectra can, in principle, reduce the number of false protein assignments. One would expect that the mass shifts induced by esterification would be spread wider than those due to guanidination since tryptic peptides typically have zero or one Lys residue, whereas they can have several acidic residues. An ideal modification reagent induces a complete conversion of an intact reactant to a derivatized product without making any byproducts. Based on MALDI mass spectra of esterified tryptic peptides shown above, methanol shows more complete esterification than the other alcohols. However, as discussed above it yields additional peaks of similar intensity that arise from solvolysis of Asn or Gln. n-Propanol leads to numerous partially derivatized peptides under the same reaction conditions, although it gives the best signal enhancement after esterification among alcohols used in this study. Ethanol was finally chosen as the best reagent for esterification of tryptic peptides since it yields almost complete esterification and good signals without substantial side reactions. To examine the value of esterifying tryptic digests in mass mapping experiments, we analyzed 1D gel bands of Caulobacter crescentus stalk proteins. The peptide mass mapping program employed in this experiment is an in-house software package named Prodigies (Protein Digest Identification and Elucidation Software).17,19 A peak report from the mass spectrometer is submitted to the program, which creates a list of experimental masses for database comparison. These data are compared with a theoretical enzymatic digest of a proteome (Caulobacter crescentus, in this case) and the mass mapping results are displayed in a table called a “Master Hit Array (MHA).”

Table 2. Master Hit Arrays Obtained from Interpreting (A) the Underivatized and (B) the Esterified Mass Spectra in Figure 4 ORF number

42 {14}

26 {13}

362 {13}

1142 {11}

1185 {11}

1790 {11}

339 {10}

503 {10}

... ORF ... number

362 {11}

3536 {11}

42 {10}

1185 {9}

1790 {9}

2178 {9}

88 {8}

545 {8}

... ...

A 879.45 1258.66 1129.74 862.43 1036.57 986.61 1529.75 2129.00 1191.53 1201.65 1791.74 1434.75 988.56 1117.55 1410.75 1914.91 894.44 1486.81 1004.53 855.03 1363.66 1550.86 1475.87 2383.99 1107.58 1323.67 1939.98 1433.75 1082.68 1175.54 1938.06 994.18 1234.70 1179.58 958.53 1079.65 2064.04 1855.91 1421.66 1907.95 1513.68 974.52 ...

* * * * * -0.12 * * * * 0.13 -0.15 -0.03 0.09 * -0.07 * 0 0.07 * * * * * * * * * * * * * * 0.01 * -0.07 * 0.13 * * * * ...

* * -0.08 * * -0.09 0.06 * * 0.01 * * * * 0.01 * * * -0.01 * * * * * 0.07 * -0.03 * -0.08 * * * * * 0 * * * * * * * ...

-0.02 * * * * -0.05 * -0.01 * 0.02 * * * * * * * * * * -0.01 * * * * * * * * * 0.02 * * * * -0.08 -0.01 * * -0.02 * 0.01 ...

-0.01 -0.03 * * * * * * 0.13 * * * * * * * * * * * * * * * * * * * -0.03 * * * * * * * * * * * * 0.01 ...

* * -0.12 0.02 0.04 * * * * * * * * 0.03 * * * * -0.05 * * * * * * * * * * * * * * * * * * * 0.04 * * * ...

0.02 0.01 * * -0.01 * -0.03 * * -0.05 * * * * * * * * * * * -0.05 * * * * * 0.02 -0.13 * * * * 0.06 * * * * * * * * ...

* * * * * -0.15 * 0.06 0.11 * 0.12 * * * * 0.11 * -0.08 * * * * * 0.1 * * * * * 0.05 * * * * * * * 0.05 * * * * ...

* * * * * -0.05 * * * * * * * 0.05 * 0.08 * * -0.01 * * * * * * * * * * * * * -0.02 * * * * * * 0.08 * * ...

... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ...

B 1070.71 2329.23 2303.20 1459.78 2022.19 918.50 2241.16 964.53 1157.77 1058.68 2312.20 1120.66 2358.25 1285.75 2224.14 1613.88 2270.15 2341.18 978.55 1016.61 1163.71 2295.17 934.54 1209.64 1970.15 896.78 1011.54 1218.66 981.57 1145.69 1442.81 1042.61 1577.88 1475.82 1449.79 2286.25 1263.65 943.55 801.55 1391.72 1496.79 1694.98 ...

-0.05 * * 0 -0.01 * -0.04 * * -0.06 * * * 0.01 * * * * * -0.04 -0.04 * * * * * * * * * * * * * * * * * * * * * ...

* * 0.03 * * * * * * * * * * * * 0.04 * * -0.03 * * * * 0.01 * * * 0 * * 0.05 * * 0.03 * * 0.04 * * * * 0.03 ...

* * * * * * * * -0.06 * * * * * * * 0.12 * * * -0.03 * * -0.01 * * * * -0.02 -0.12 0.06 -0.06 * * * * 0.03 0.04 * * * * ...

-0.07 * * * * 0.01 0.02 * -0.15 * * * * * * * * * * 0 * 0.1 * * * * * * * * 0.09 * * * * * * * * * * * ...

* -0.11 0 * * * * * * * * * * -0.05 * -0.06 * * -0.01 * * * * 0.1 * * * * * * -0.07 * * * * * * * * * * -0.07 ...

* * * * * * * * * -0.08 * * * * -0.04 * * * * * * * * * * * * -0.01 * * * 0 * * * * * * -0.09 0.02 * -0.07 ...

-0.09 * * * * * * * * * * * 0.08 * * * * * * * * * * * * * 0.08 -0.07 * * * 0.05 * * * * * 0.03 * 0 * * ...

-0.06 * * * -0.01 * * * * * * * * * * * * * * -0.05 * * * * * * * 0.03 * * 0.01 * * * * * * * * * 0.13 * ...

... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ...

Figure 4 displays mass spectra of underivatized and esterified tryptic digests of a 1D gel band extract from Caulobacter crescentus stalk. Peaks arising from trypsin autolysis are indicated by Φ. Other peak labels are discussed below. 62 measured masses in the underivatized mass spectrum are submitted to the program for mass mapping. Table 2A shows the top left part of a MHA derived from the underivatized mass spectrum. (The right-side columns and the lower rows of the MHA are deleted to save space, but this does not affect the present discussion.) The top rows of an MHA list the open reading frame (ORF) numbers associated with the proteins most likely to be in the sample. Numbers in braces indicate the total number of matches between experimental data and theoretically predicted peptides for each protein listed. The experimental masses of peptides are displayed in the leftmost column in order of decreasing peak intensity. The fractional numbers in the table are the differences between the measured and theoretical masses, in Da. The * symbol means that an experimental mass does not match any peptide derived from the protein in that column. The ORF with the most matches appears

in the upper left corner of the MHA. 14 measured masses from the underivatized tryptic digest are associated with ORF 42 in Table 2A. However, it is not obvious that this gel band contains ORF 42 because the most intense peaks in the spectrum are not assignable to predicted fragments from this protein. Some of the more intense peaks are associated with ORFs 362, 1142, 1185, and 1790 but the numbers of matching peptides are not convincing enough to establish a definitive interpretation. Derivatized data can help clarify the situation. Table 2B displays the MHA obtained from interpretation of 55 measured masses in the esterified mass spectrum (Figure 4B) of the same gel band tryptic digests. The protein that has the largest number of matches, ORF 362, also appears in the MHA for the underivatized data. Although it seems clear that this protein is in this gel band, others such as ORF 1185, 1790, and 42 are nevertheless also likely to be present. Another tool for peptide mass mapping used in Prodigies involves comparing underivatized and derivatized mass spectra to extract information about constituent amino acids of peptides. Prodigies enables chemical modifications of all peptides in a Analytical Chemistry, Vol. 77, No. 13, July 1, 2005

4191

Table 3. Consistent Master Hit Array Obtained from the Mass Spectra of Figure 4 ORF number

362 {10}

42 {3}

579 {3}

789 {3}

849 {3}

878 {3}

1063 {3}

1238

1244 {3}

1412 {3}

1517 {3}

1788 {3}

... ...

1129.74 862.43 1036.57 986.61 1529.75 2129.00 1201.65 988.56 1117.55 894.44 1363.66 1107.58 1939.98 1938.06 1179.58 958.53 1079.65 1421.66 974.52 1994.03 1333.70 2079.96 2033.99 2112.00 1347.66 2398.15

* * * -0.05 * -0.01 0.02 * * * -0.01 * * 0.02 * * -0.08 * 0.01 * * * * -0.03 0 0.03

* * * -0.12 * * * * * * * * * * 0.01 * -0.07 * * * * * * * * *

-0.08 * * * * * * * * * * * * * * * * * * * * 0.06 * * 0.03 *

* * * * * * -0.06 * * * * 0.02 * * * -0.09 * * * * * * * * * *

* 0.06 * * * * * 0.04 * * * * * * 0.03 * * * * * * * * * * *

* * * -0.07 * * * * * * * 0.01 * * * * -0.08 * * * * * * * * *

-0.06 * * -0.08 * * * * * * * * * -0.03 * * * * * * * * * * * *

* * -0.06 * * * * * 0.13 * * * * * * * * * -0.01 * * * * * * *

* * * * * * * -0.04 * * * 0.08 * * * * * * * * * * * * 0.05 *

* * -0.1 * * * * -0.06 * * * * * -0.05 * * * * * * * * * * * *

* * * * * * * * * 0.02 0.03 * * * * * * * * * * * * * 0.02 *

* * * -0.14 * * * * 0.02 * 0.08 * * * * * * * * * * * * * * *

... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ...

database. Comparison of Prodigies with other web-based database searching programs has been presented elsewhere.17 In this mode, the program generates three MHAs based on data from both underivatized and esterified mass spectra. First, it creates an MHA based on the underivatized mass spectrum as illustrated above. A second MHA is generated based on comparing esterified mass spectra against a database of esterified peptides as also demonstrated above. Next, Prodigies creates a list of pairs of masses in the two spectra that are separated by the esterification mass shift, which must be some multiple of 28.03 Da. (These are called “consistent” masses.) All masses in this table are then compared against the theoretically underivatized tryptic digest to generate a “consistent” MHA. In this comparison, two conditions must be simultaneously satisfied for a match to be counted. First, theoretical peptide masses must match measured masses in both underivatized and derivatized mass spectra. Second, the total number of acidic residues in a peptide must be consistent with that derived from the mass shift between underivatized and derivatized mass spectra. Table 3 presents a consistent MHA obtained from comparison between underivatized and esterified tryptic digest mass spectra of the gel band just considered. Clearly, ORF 362 is now seen as most likely to be present. Several strong signals that are not labeled in Figure 4A suggest that another protein may also be present. In principle, derivatized data should help us to establish whether these peaks arise from other proteins or if they are simply artifacts from nonspecific cleavages or modifications. Although a few strong signals in Figure 4B match peptide masses from other proteins, they do not show consistent matches in the underivatized spectrum. Thus, we could not prove that more than one protein is present in this gel band. As shown in these observations, peptide mass mapping with analyzing “consistent” masses after a derivatization may be advantageous for ambiguous cases since additional information about each 4192


peptide’s constituent amino acids reduces the number of false positive assignments arising from random matches. The underivatized and fully esterified peptides assigned to ORF 362 in the Table 2 are labeled with [#] in Figure 4A and with [*] in Figure 4B, respectively. The peaks arising from incomplete esterification and solvolysis are indicated by [-] and [+] in Figure 4B, respectively. For optimal performance, a peptide modification reaction used in “consistent” mass analysis should be complete and not lead to any side reactions. Unfortunately, esterification does not always satisfy these criteria. As mentioned above, incomplete esterification is often observed for a peptide with multiple carboxylic groups in its sequence and the formation of byproducts from solvolysis of Asn or Gln residue is inevitable. One incompletely esterified peak and three peaks from solvolysis are seen in Figure 4B; however, their signals are comparable to noise. On the other hand, as shown in Figure 1C, these undesirable types of peaks cannot be neglected in mass spectra with a better signal-to-noise ratio. Investigation of esterified mass spectra for all 23 gel bands reveals that 6% of identified peaks are related to incomplete esterification and about 30% of identified Asn/Glncontaining peptides produce solvolysis products.19 In an attempt to perform ethanol esterification without incomplete derivatization and solvolysis, longer reaction times at lower temperatures and varying concentrations of reagents were investigated. Unfortunately, these experiments were not successful. Two major problems arise from partial esterification. First, it reduces sensitivity by dividing peptide ion signals into multiple peaks. Second, false positive assignments can be produced when the number of carboxylic groups in a peptide is incorrectly assigned. Solvolysis of Asn or Gln also causes a decrease of sensitivity and increases complexity in mass spectra, which leads to confusion in the interpretation of “consistent” masses. Furthermore, the mass difference between esterification and solvolysis

is only 1 Da. Thus, if incomplete esterification and solvolysis occur simultaneously in the same peptide, the isotopic distribution of the completely esterified peak will be distorted. Esterification has one more disadvantage in comparison to guanidination. The complete conversion of lysine to homoarginine gives rise to significant enhancement of peak intensity for a lysine-terminated peptide.23-25 Although a more hydrophobic alcohol tends to enhance some peptide ion signals, the effects of esterification are not at all predictable. Variations in peptide peak intensities before and after esterification make it more difficult to uncover the “consistent” masses. Because of these disadvantages, esterification is not generally applicable for the identification of unknown proteins in gel bands. However, it may be helpful in specific cases as demonstrated above. CONCLUSIONS Alcohols vary in their esterification reactivities with peptides. As the size of alcohol molecule increases, the completeness of the reaction decreases but the occurrence of undesirable solvolysis of Asn or Gln also decreases. Esterification reactivities seem to depend more on steric hindrance effects than on the electrondonating character of the alcoholic alkyl groups. Peptides exhibit widely varying MALDI ionization signals after esterification with alcohols. Although there is no apparent correlation between ion yield and hydrophobicity of a peptide, some primarily hydrophilic (23) Brancia, F. L.; Oliver, S. G.; Gaskell, S. J. Rapid Commun. Mass Spectrom. 2000, 14, 2070-2073. (24) Beardsley, R. L.; Karty, J. A.; Reilly, J. P. Rapid Commun. Mass Spectrom. 2000, 14, 2147-2153. (25) Hale, J. E.; Butler, J. P.; Knierman, M. D.; Becker, G. W. Anal. Biochem. 2000, 287, 110-117.

peptides show a significant peak enhancement after esterification. For these peptides, the more hydrophobic alcohols yield greater peak enhancements. Ethanol esterification may be the best choice in peptide mass mapping experiments. Information about the total number of the Asp and Glu residues in a peptide can help improve the identification of proteins by reducing the number of false positive assignments. However, esterification is not always helpful for identifying proteins in a gel band since it is not complete and it yields complicating solvolysis byproducts. Esterification has been widely exploited in mass spectrometric analysis of biological molecules, and methanol has generally been the esterifying reagent utilized. Based on our results, we conclude that ethanol is a better alcohol than methanol from the standpoint of sensitivity. When ethanol is used for esterification, instead of methanol, the extent of partial esterification is very slightly increased. However, a significant decrease of solvolysis of Asn or Gln, which reduces complexity of mass spectra, is more than enough to compensate for loss of sensitivity caused by a slight increase of incomplete modification. Some peptides also yield better peak intensities in MALDI mass spectra when esterified with ethanol compared to methanol. ACKNOWLEDGMENT This work has been supported by grants from the National Science Foundation (CHE 0094579) and National Institutes of Health (GM 061336).

Received for review December 20, 2004. Accepted April 12, 2005. AC0481250


4193

Effects of Tryptic Peptide Esterification in MALDI Mass Spectrometry

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