Phosphopeptide Derivatization Signatures To Identify Serine and

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Anal. Chem. 2001, 73, 5387-5394

Phosphopeptide Derivatization Signatures To Identify Serine and Threonine Phosphorylated Peptides by Mass Spectrometry Mark P. Molloy† and Philip C. Andrews*

Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0606

The development of rapid, global methods for monitoring states of protein phosphorylation would provide greater insight for understanding many fundamental biological processes. Current best practices use mass spectrometry (MS) to profile digests of purified proteins for evidence of phosphorylation. However, this approach is beset by inherent difficulties in both identifying phosphopeptides from within a complex mixture containing many other unmodified peptides and ionizing phosphopeptides in positive-ion MS. We have modified an approach that uses barium hydroxide to rapidly eliminate the phosphoryl group of serine and threonine modified amino acids, creating dehydroamino acids that are susceptible to nucleophilic derivatization. By derivatizing a protein digest with a mixture of two different alkanethiols, phosphopeptide-specific derivatives were readily distinguished by MS due to their characteristic ion-pair signature. The resulting tagged ion pairs accommodate simple and rapid screening for phosphopeptides in a protein digest, obviating the use of isotopically labeled samples for qualitative phosphopeptide detection. MALDI-MS is used in a first pass manner to detect derivatized phosphopeptides, while the remaining sample is available for tandem MS to reveal the site of derivatization and, thus, phosphorylation. We demonstrated the technique by identifying phosphopeptides from β-casein and ovalbumin. The approach was further used to examine in vitro phosphorylation of recombinant human HSP22 by protein kinase C, revealing phosphorylation of Thr-63. Reversible protein phosphorylation is an energetically efficient mechanism of modulating cellular protein activity in response to environmental stimuli. The significance of protein phosphorylation as a signaling system has been established in the control of many diverse, fundamental cellular events including cell cycle,1 apoptosis,2 metabolism,3 cellular morphology,4 and protein synthesis.5 * Corresponding author: (tel) 734 763 3130; (fax) 734 647 0951; (e-mail) [email protected]. † Current address: Pfizer Global Research and Development, Ann Arbor, MI, 48105. (1) Helmbrecht, K.; Zeise, E.; Rensing, L. Cell Proliferation 2000, 33, 341365. (2) Cross, T. G.; Scheel-Toellner, D.; Henriquez, N. V.; Deacon, E.; Salmon, M.; Lord, J. M. Exp. Cell Res. 2000, 256, 34-41. (3) Herrington, J.; Smit, L. S.; Schwartz, J.; Carter-Su, C. Oncogene 2000, 19, 2585-2597. 10.1021/ac0104227 CCC: $20.00 Published on Web 10/16/2001

© 2001 American Chemical Society

Furthermore, perturbations of phosphorylation machinery have been linked to the development of several pathological states, emphasizing the importance of protein phosphorylation in cellular homeostasis. The postgenome era has realized the possibilities of analyzing the transcriptional activity of many genes through gene-expression profiling6 and the identification of expressed proteins by proteomics.7 Important criteria encompassed by these techniques are rapid, large-scale, high-throughput analyses. For the study of phosphoproteins, investigations are best conducted at the protein level, where the analyte is found in its posttranslational, chemically modified form. However, a number of technical difficulties arise in efforts to develop generalized analytical tools and techniques for global investigations of protein phosphorylation. Traditional approaches for monitoring phosphoproteins rely on autoradiography using either metabolic radiolabeling or antibodies directed toward phosphoamino acid-containing epitopes. However, various drawbacks are associated with both of these approaches including the costs associated with antibody production, limited affinity of many antibodies, dilution of isotopes by endogenous orthophosphate, and limitations in radiolabeling certain sample types (e.g., live animals). Mass spectrometry (MS) has emerged as a powerful alternative technique for studying protein posttranslational modifications.8-10 Most approaches involve enzymatic digestion followed by mass analysis of the peptide fragments. Typically, the presence of phosphorylated peptides and the site of phosphorylation can only be deduced with very intensive interrogation of the mass spectra. However, for proteome studies, it will be necessary to identify phosphorylated peptides in a high-throughput manner from within a heterogeneous digestion mixture. Current MS approaches include the use of parent ion scanning with triple-quadropole instruments11-13 or MALDI-MS14,15 to detect peptide mass shifts (4) Kuhn, T. B.; Meberg, P. J.; Brown, M. D.; Bernstein, B. W.; Minamide, L. S.; Jensen, J. R.; Okada, K.; Soda, E. A.; Bamburg, J. R. J. Neurobiol. 2000, 44, 126-144. (5) Dufner, A.; Thomas, G. Exp. Cell Res. 1999 253, 100-109. (6) Lockhart, D. J.; Winzeler, E. A. Nature 2000, 405, 827-836. (7) Harry, J. L.; Wilkins, M. R.; Herbert, B. R.; Packer, N. H.; Gooley, A. A.; Williams, K. L. Electrophoresis 2000, 21, 1071-1081. (8) Qin, J.; Chait, B. T. Anal. Chem. 1997, 69, 4002-4009. (9) Wilm, M. Adv. Protein Chem. 2000, 54, 17-23. (10) Larsen, M. R.; Roepstorff, P. Fresenius J. Anal. Chem. 2000, 366, 677-690. (11) Wilm, M.; Neubauer, G.; Mann, M. Anal. Chem. 1996, 68, 527-533. (12) Carr, S. A.; Huddleston, M. J.; Annan, R. S. Anal. Biochem. 1996, 239, 180192.

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of 80 Da corresponding to putative phosphopeptides. Tandem MS approaches are then required to confirm the phosphorylation status of these peptides. However, an inherent problem limiting analysis of tryptic phosphopeptides is poor ionization in positiveion MS, presumably due to proton sequestration by the phospho acid groups. The outcome is that many phosphopeptides remain undetected when analyzed in this manner. The recent work by Annan et al.16 heads toward addressing this issue by employing a multidimensional scanning technique for mapping phosphopeptides that includes negative-ion precursor scans of LC-separated peptides, to detect putative phosphopeptides and determine their mass, and positive-ion nanoelectrospray tandem MS for detailed characterization. Several other approaches for MS-based analysis of phosphopeptides have been described. One approach involves using immobilized metal affinity chromatography (IMAC) in attempts to enrich for phosphopeptides from digestion mixtures prior to MS.17,18 However, this method suffers from limited selectivity compared to conventional affinity techniques and may be best used as a general enrichment step prior to analysis. A different approach involves screening samples by MALDI, both before and after incubation with a nonspecific phosphatase.19,20 The subsequent loss/appearance of peptide ions has been used to infer the phosphorylation status of peptides. Aebersold’s laboratory has embarked on a novel approach that involves a complex series of protection/deprotection reactions to derivatize phosphoamino acids and use them in affinity purification techniques.21 While this approach appears promising, a simpler method for tagging phosphopeptides is needed. Jaffe et al.22 described a method for MS analysis of derivatized phosphopeptides based on a technique adopted for producing stable phosphoamino acids amenable with detection in Edman sequencing.23,24 The phosphoryl group of serine and threonine phosphorylated amino acids is β-eliminated by exposure to base (∆, -98 Da), generating a dehydrated amino acid susceptible to nucleophilic addition25-29 by ethanethiol (∆, +62 Da). Phosphotyrosine is unaffected by this treatment, remaining intact. The approach provides two key advantages. First, a negative charge (13) Dreger, M.; Otto, H.; Neubauer, G.; Mann, M.; Hucho, F. Biochemistry. 1999, 38, 9426-9434. (14) Kalo, M. S.; Pasquale, E. B. Biochemistry 1999, 38, 14396-14408. (15) Muller, D. R.; Schindler, P.; Coulot, M.; Voshol, H.; van Oostrum, J. J. Mass Spectrom. 1999, 34, 336-345. (16) Annan, R. S.; Huddleston, M. J.; Verma, R.; Deshaies, R. J.; Carr, S. A. Anal. Chem. 2001, 73, 393-404. (17) Posewitz, M. C.; Tempst, P. Anal. Chem. 1999, 71, 2883-2892. (18) Cao, P.; Stults, J. T. Rapid Commun. Mass Spectrom. 2000, 14, 1600-1606. (19) Wang, Y. K.; Liao, P.-C.; Allison, J.; Gage, D. A.; Andrews, P. C.; Lubman, D. M.; Hanash, S. M.; Strahler, J. R. J. Biol. Chem. 1993, 268, 1426914277. (20) Liao, P.-C, Leykam, J.; Andrews, P. C.; Gage, D. A.; Allison, J. Anal. Biochem. 1994, 219, 9-20. (21) Zhou, H.; Watts, J. D.; Aebersold, R. Nat. Biotechnol. 2001, 19, 375-378. (22) Jaffe, H.; Veeranna, Pant, H. C. Biochemistry 1998, 37, 16211-16224. (23) Meyer, H. E.; Hoffmann-Porsorke, E.; Korte, H.; Heilmeyer, L. M., Jr. FEBS Lett. 1986, 204, 61-66. (24) Meyer, H. E.; Hoffmann-Posorske, E.; Heilmeyer, L. M., Jr. Methods Enzymol. 1991, 201, 169-185. (25) Holmes, C. F. FEBS Lett. 1987, 215, 21-24. (26) Mega, T.; Nakamura, N.; Ikenaka, T. J. Biochem. (Tokyo) 1990, 107, 6872. (27) Byford, M. F. Biochem. J. 1991, 280, 261-265. (28) Fadden, P.; Haystead, T. A. Anal. Biochem. 1995, 225, 81-88. (29) Resing, K. A.; Johnson, R. S.; Walsh, K. A. Biochemistry, 1995, 34, 94779487.

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is converted to a neutral group, reducing the suppression effects normally observed for phosphopeptides in positive-mode ionization. Second, the resultant change in expected peptide mass (∆, -36 Da) is interpreted as evidence of a phosphorylation site within that peptide. During the final preparation of this paper, Oda et al.30 illustrated the promise of a chemical derivatization approach for affinity enrichment of phosphopeptides prior to MS analysis. In the present report, we have extended an investigation into the chemical derivatization of phosphopeptides and examined the resultant effects on peptide ionization by MALDI-MS. This involved refining the methodology to markedly improve derivatization reaction times and led to the development of strategies for unambiguous assignment of phosphorylated peptides in digestion mixtures. We tested this approach using model phosphopeptides, as well as the phosphoproteins β-casein and ovalbumin. Finally, we applied the methodology to investigate the phosphorylation status of the recently described human small heat shock protein HSP22 (NCBI Accession No. AF250138) after in vitro phosphorylation by protein kinase C (PKC). EXPERIMENTAL SECTION Reagents. Alkanethiols (ethanethiol, propanethiol, butanethiol, pentanethiol, hexanethiol, heptanethiol, octanethiol) were from Aldrich. Serine phosphorylated peptide UOM9B (KRPpSQRHGSKY-NH2, 1423 Da) and threonine phosphorylated peptide UOM11 (KRpTIRR-OH, 909 Da) were purchased from the University of Michigan Protein Structure Core Facility (www.bref.med. umich.edu). Ovalbumin and β-casein were purchased from Sigma. In Vitro Phosphorylation of HSP22 by PKC. Recombinant His-tagged human HSP22 was expressed in Escherichia coli and purified using a nickel affinity column.31 HSP22 was in vitro phosphorylated by incubation of 0.5 µg of substrate with 5 ng of PKC, catalytic subunit from rat brain (Calbiochem) in a buffer containing 20 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 0.5 mM CaCl2, 1 mM dithiothreitol, and 1 mM ATP. β-Elimination and Derivatization of Test Phosphopeptides. A 2.5-µg sample of UOM9B (pSer; 5 µg/µL) or UOM11 (pThr; 5 µg/µL) in 10 mM Tris-HCl, pH 8.5, was added to a solution, with final concentrations of 30% (v/v) 1-propanol (9 µL), 20 mM Ba(OH)2 (3µL), and 0.5 M appropriate alkanethiol, with water added to a final volume of 30 µL. The reaction solution was freshly prepared for each experiment. Derivatization was conducted in a water bath at 45 °C for 1-5 h as indicated in the text. A 5-µL aliquot of the reaction was mixed with 2.5 µL of a 1 M ammonium sulfate solution to form a barium salt, ending the reaction. The barium sulfate was removed after a 30-s centrifugation step in a desktop centrifuge. A 1-µL sample of supernatant was collected and mixed with 9 µL of 60% (v/v) acetonitrile (MeCN), 0.1% (v/v) TFA for MALDI analysis. Sulfhydryl Protection. Protein sulfhydryl residues were protected by reduction for 30 min with 20 mM DTT in 10 mM Tris-HCl, pH 8.5, followed by carboxyamidomethylation with 60 mM iodoacetamide in 10 mM Tris-HCl, pH 8.5. for 1 h at 30 °C. Excess reagents were removed by dialysis for 4 h against 10 mM Tris-HCl, pH 8.5. (30) Oda, Y.; Nagasu, T.; Chait, B. T. Nat. Biotechnol. 2001, 19, 379-382. (31) Benndorf, R.; Sun, X.; Gilmont, R.; Biederman, K. J.; Molloy, M. P.; Goodmurphy, C. W.; Cheng, H.; Andrews, P. C.; Welsh, M. J. J. Biol. Chem. 2001, 276, 26753-26761.

β-Elimination and Derivatization of Proteins. A 5-µg sample of β-casein, ovalbumin, or HSP22 (1 µg/uL in 10 mM Tris-HCl, pH 8.5) was digested overnight at 37 °C with porcine trypsin (Promega; 1:40 ratio of enzyme/substrate). The digested sample was mixed directly with a freshly prepared solution of final concentration 30% (v/v) 1-propanol, 20 mM Ba(OH)2, and 0.5 M each alkanethiol. The reaction was conducted in a water bath at 45 °C for 3 h. The reaction was stopped by the addition of ammonium sulfate and prepared for MALDI-MS as described above. Matrix Preparation. (a) Dried-Droplet Method. An 0.8-µL sample of analyte was spotted onto a gold target plate and 0.8 µL of MALDI matrix (10 mg/mL R-cyano-4-hydroxycinnamic acid in 50% (v/v) MeCN, and 0.1% (v/v) TFA) added and allowed to airdry. (b) Thin-Layer Method. A matrix solution of 10 mg/mL R-cyano-4-hydroxycinnamic acid was prepared in acetone/0.1% TFA as previously described.32 An 0.8-µL aliquot of matrix was quickly applied to the gold target plate, and after matrix crystals had formed, 0.8 µL of analyte was applied. The sample was washed with 5 µL of 0.1% TFA for 10 s and then air-dried. MALDI-MS. Spectra were acquired using a PerSeptive Biosystems Voyager DE-STR MALDI operating in delayed extraction reflector mode. Tandem MS. Samples were prepared for MS/MS by desalting the derivatized peptide mixture using a C18 ZipTip (Millipore, Bedford, MA) following the manufacturer’s guidelines. Peptides were eluted into 3 µL of 70% (v/v) MeCN/0.1% (v/v) formic acid. The peptides were introduced into a Micromass Q-TOF MS using a static nanoflow probe (Micromass, Manchester, U.K.) for nanoelectrospray ionization. MS/MS spectra were obtained by fragmenting selected ions using collision energies of 20-35 V. RESULTS AND DISCUSSION Barium Hydroxide Catalyzes Rapid Derivatization of Phosphopeptides. Under strong base conditions, phosphoserine and phosphothreonine undergo dehydration through β-elimination, forming dehydroalanine and dehydroamino-2-butyric acid, respectively. The dehydrated amino acids are suitable targets for nucleophilic derivatization via Michael addition. We set out to exploit this reaction to develop simple methods for rapid screening of phosphopeptides by MS. Methods to produce stable phosphoamino acid derivatives detectable in Edman sequencing have been described.23-24 More recently, the approach was rekindled, allowing detection of these derivatives by electrospray MS.22 The method employed in both of these approaches entails reacting the β-eliminated, dehydrated amino acids with ethanethiol to yield stable S-ethylcysteine (for phosphoserine) or β-methyl-S-ethylcysteine (for phosphothreonine). In these reports, the highly alkaline conditions required for β-elimination were established using NaOH; however, the slow kinetics of this reaction necessitates incubating the reaction overnight. In an effort to shorten the analysis time window, we modified this approach, using Ba(OH)2 27 to catalyze the derivatization of two test peptides UOM9B (pSer) and UOM11 (pThr). This required establishing a protocol with an appropriate solvent compatible with Ba(OH)2 solubility, solubilization of the al(32) Vorm, O.; Roepstorff, P.; Mann, M. Anal. Chem. 1994, 66, 3281-3287.

Figure 1. Model phosphopeptides derivatized with alkanethiols of increasing carbon chain length for 1, 3, and 5 h (n ) 3) and assayed by MALDI-MS. (A) UOM9B (pSer); (B) UOM11 (pThr). Note in (A) the 5-h time point for C7/C8 could not be accurately determined as the intensity of the underivatized sample was extremely small.

kanethiol nucleophiles, and suitability of matrix crystallization for MALDI-MS. Various solvents were tested at different concentrations including dimethyl sulfoxide, dimethyl formamide, 1-methyl2-pyrrolidinone, and 2-propanol, but all were excluded due to their incompatibility for solubilization or matrix crystal formation. On the other hand, 1-propanol and acetonitrile at concentrations of 30-40% were found to be suitable solvents for this application. β-Elimination and nucleophilic addition were conducted in a “onepot” reaction, and aliquots removed at various time points for assaying by MALDI-MS (Figure 1). In as few as 60 min, derivatized phosphopeptide product could be detected for both UOM9B and UOM11, although a substantial improvement in detection of the derivatized product was achieved by extending the reaction to 3-5 h. On average, derivatization of the UOM9B peptide occurred at 1.3 times the rate of UOM11 derivatization. The slower derivatization of pThr with barium hydroxide-catalyzed β-elimination is consistent with the structure of pThr and with previous reports;27 however, this effect did not compromise our ability to detect the phosphothreonine-containing peptide. As an aside, we note that O-linked carbohydrates are also susceptible to β-elimination via this approach; however, their reaction rate is Analytical Chemistry, Vol. 73, No. 22, November 15, 2001

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Figure 2. (A) MALDI analysis of UOM9B after 3 h of competitive derivatization using C3 (arrow) and C7 (asterisk) alkanethiols. m/z 1423.6 is the underivatized peptide. (B) UOM9B derivatized separately with C3 (arrow) or C7 (asterisk) alkanethiols and then mixed for MALDI analysis.

significantly slower (20 times)27 and is unlikely to confuse analyses focusing on phosphopeptides. Alkanethiol Aliphaticity and Matrix Preparation Influences Ionization by MALDI-MS. We evaluated the approach of increasing the alkanethiol carbon chain length used for phosphopeptide derivatization with a future goal of using this method to selectively enrich for derivatized phosphopeptides in a protein digest prior to MS analysis. Test phosphopeptides UOM9B and UOM11 were derivatized with equal concentrations of alkanethiols of increasing carbon chain length, ranging from ethanethiol (C2) to octanethiol (C8). The reaction was assayed by MALDI using the dried-droplet method of matrix preparation33 at 1-, 3-, and 5-h time points, and the ratio of the peak areas of the derivatized versus underivatized peptide was recorded. Converse to our initial expectations, both pSer- and pThrderivatized peptides exhibited markedly increased peak area ratios with increasing alkanethiol carbon chain length (Figure 1A and B, respectively). The differences in peak area ratio observed between a peptide reacted with a C2 thiol and a C8 thiol was greater than 50-fold (n ) 3). One interpretation of this result implies more complete derivatization with the use of longer alkanethiols, although kinetics would suggest that this is unlikely. An alternative explanation is that although the longer chain-length thiols might react more slowly, the resultant derivatized peptides of greater aliphaticity exhibit superior ionization efficiencies in MALDI-MS compared to more hydrophilic derivatized peptides when prepared in a similar manner. However, we could find only a few studies relevant to our hypothesis that briefly discussed a connectionbetweenpeptidehydrophobicityandMALDIionization.34-36 5390 Analytical Chemistry, Vol. 73, No. 22, November 15, 2001

To clarify our observations, we designed several experiments to investigate the nature of our findings. Figure 2A shows MS analysis for a reaction where two separate, equimolar concentrations of alkanethiol nucleophiles of different carbon chain lengths (C3 and C7) compete for the Michael substrate residue within the same reaction mixture. In this experiment, where both thiols are competing for the same site, MALDI ionization signals (peak areas) were far greater for the shorter carbon chain-length derivative (6.8-fold). This is in contrast to the situation when the reactions are conducted and analyzed separately. In the case of separate analyses, longer carbon chain-length derivatives show greater ionization efficiencies than those peptides derivatized with shorter carbon chain-length alkanethiols (see Figure 1). Our interpretation of these observations is that the kinetics of derivatization with the smaller alkanethiol is faster than for the longer carbon chain-length compound. This suggests that the basis for the difference in ion intensity in Figure 1A and B is not due to faster reaction kinetics or more complete derivatization by the longer carbon chain-length thiol. Furthermore, the conclusion that more hydrophobic tags increase the MALDI detection sensitivity was strengthened when we analyzed peptide samples that were derivatized separately and then combined in equimolar amounts for MS analysis using the dried-droplet sample preparation method (Figure 2B). In this case, we observed greater peak intensity for (33) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. (34) Beavis, R. C.; Bridson, J. N. J. Phys. D: Appl. Phys. 1993, 26, 442-447. (35) Amado, F. M. L.; Domingues, P.; Graca Santana-Marques, M.; Ferrer-Correia, A. J.; Tomer, K. B. Rapid Commun. Mass Spectrom. 1997, 11, 1347-1352. (36) Cohen, S. L.; Chait, B. T. Anal. Chem. 1996, 68, 31-37.

Figure 3. (A) MALDI spectrum of an ovalbumin tryptic digest derivatized with pentanethiol. m/z 2096.0 corresponds to tryptic fragment 340359 containing a site of modification at Ser-344 (arrow). (B) Tryptic digest of ovalbumin prior to derivatization. Note that the phosphopeptide tryptic fragment 340-359 of m/z 2090 was not detected.

the longer (C7) chain-length derivative (2.8-fold greater than the C3 thiol derivative). One possible mechanism for the preferential ionization of aliphatic alkanethiol peptide derivatives occurs if hydrophobic interactions promote accumulation of these molecules at the surface or within the growing matrix crystal during solvent evaporation when the dried-droplet method of sample crystallization is performed. Previous investigations have shown that polypeptides attach to the nonpolar surface of growing sinapinic acid matrix crystals,34 and hydrophobic amino acids showed greater relative signal intensity in MALDI compared to more hydrophilic amino acids when the dried-droplet method was used.35 These reports suggest that hydrophobic interactions between analytes and matrix crystals play a role in peptide ionization by MALDI. The sum effect of this interaction, as reported here, may result in increased concentrations of the more hydrophobic derivatized peptides at the crystal surface, leading to greater ionization efficiency. The ability of the sample preparation method to influence ionization was further highlighted when we changed conditions for the matrix preparation. When an equimolar mixture of C3 and C7 derivatives (as used in Figure 2A) was applied to a preformed matrix surface (thin-layer matrix preparation),32 we consistently observed greater ionization intensity for the shorter (C3) chain-length derivative (data not shown), supporting the conclusion that the dried-droplet method of crystallization promotes greater ionization efficiency of hydrophobic peptides. MALDI Analysis of Derivatized Tryptic Peptides. We evaluated the usefulness of the derivatization approach for the rapid detection of phosphopeptides from a tryptic digest of ovalbumin. Prior to overnight tryptic digestion, disulfides were reduced and cysteine residues carboxyamidomethylated to protect these sites from β-elimination. The trypinized sample was derivatized for 3 h in the presence of pentanethiol and assayed by

MALDI. Thirteen peptides were detected corresponding to ovalbumin. They included the unphosphorylated tryptic peptide of residues 340-359 (EVVGSAEAGVDAASVSEEFR), as well as a derivatized homologue of the same peptide fragment derivatized by pentanethiol at a known phosphorylation site, Ser-344 (Figure 3A, m/z 2096.0). Interestingly, prior to derivatization we were unable to detect the phosphorylated fragment 340-359 in an analysis of trypsinized ovalbumin, yet the derivatized homologue of this peptide was readily observed (Figure 3). Detection of the phosphorylated fragment 340-359 through elimination of the phosphoryl group and replacement by a neutral species allowed detection of a derivatized phosphopeptide previously undetected in a peptide mass analysis by MALDI. Another phosphorylation site at Ser-68 was not observed. This could be due to incomplete proteolysis or due to lot-to-lot variation in phosphorylation. The above experiment was also conducted with β-casein, leading to the detection of the phosphoserine-containing tryptic peptide (48-63), modified at Ser-50 (FQSEEQQQTEDELQDK) (data not shown). When ethanethiol was used as the nucleophile, the quadruplely phosphorylated peptide of residues 16-40 was also detected (RELEELNVPGEIVESLSSSEESITR) (data not shown). However, when longer carbon chain-length thiols were used for derivatization, the quadruplely derivatized peptide was not detected, presumably lost via aggregation or absorption to hydrophobic surfaces during the experiment. Similar observations were noted when very long chain-length thiols (e.g., C12) were used to derivatize singly phosphorylated peptides. An alternative explanation is that of steric hindrance due to the proximity of the phosphoamino acid residues to each other that may have affected derivatization. Derivatization Mass Signatures Identify Phosphopeptides in a Digest Mixture. While the derivatization approach was successfully demonstrated for ovalbumin and β-casein, where the presence of phosphoamino acids was known, an improvement to Analytical Chemistry, Vol. 73, No. 22, November 15, 2001

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Figure 4. MALDI analysis of β-casein tryptic digest derivatized with (A) C2 and C3 alkanethiols, (B) C4 and C5 alkanethiols, and (C) C7 and C8 alkanethiols.

the technique was to use derivatization mass signatures to flag attention to phosphopeptides in a digest mixture. Phosphopeptidespecific derivatization mass signatures were established by reacting a β-eliminated protein digest in a competitive manner using two alkanethiol nucleophiles of differing carbon chain length. When the mass of the alkanethiols differed by a methyl group, a 14-Da ion-pair mass signature was observed for the derivatized phosphopeptide (singly phosphorylated) in MALDI. The approach was tested with β-casein, where three separate aliquots were derivatized in the presence of ethanethiol and propanethiol (Figure 4A), butanethiol and pentanethiol (Figure 4B), or heptanethiol and octanethiol (Figure 4C). In each experiment, the derivatized peptides were readily distinguished by observing a pair of peptide ions differing by 14 Da. Using the C2/C3 thiol mixture, tryptic fragment 48-63 containing a site of phosphorylation at Ser-50 was detected at m/z 2026 and 2040, for the C4/C5 mixture at m/z 2054 and 2068, and for the C7/C8 mixture at m/z 2096 and 2110. Any one of these mass signatures was sufficient to recognize the phosphorylated peptide, but when sample is readily available, as is often the case for in vitro kinase assays, multiple derivatization reactions can be used to unequivocally establish the presence of phosphorylated peptides in a digest. This approach obviates the use of isotopes for qualitative detection of phosphopeptides. While we have demonstrated that increasing alkanethiol chain length increases sensitivity in MALDI, and have shown that phosphopeptides derivatized with C7 and C8 chains could be readily detected, we have also raised a concern regarding the concomitant increase in peptide hydrophobicity with this approach. With this in mind, it may be more practical to adhere to 5392

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shorter nucleophiles (e.g., C2-C4) for derivatization, thereby minimizing the potential of peptide losses, particularly for multiply phosphorylated peptides. PKC Phosphorylates Human HSP22 on Thr-63. We applied the strategy of phosphopeptide-specific ion-pair mass signatures to examine in vitro phosphorylation of recombinant human HSP22 PKC. Human HSP22 (NCBI Accession No. AF250138) is a member of the small heat shock family of stress proteins. Recently, HSP22 has been shown to interact with an activated, phosphorylated form of HSP27.31 HSP22 also possesses several putative PKC phosphorylation sites that may be important in modulating its function. To study HSP22 phosphorylation with our approach, a recombinant His-tagged protein was expressed in E. coli, purified, and subjected to in vitro phosphorylation with PKC. A tryptic digest of PKC-treated HSP22 was divided into two aliquots and derivatized with a mixture of ethanethiol and propanethiol in one reaction and a mixture of heptanethiol and octanethiol in a separate reaction. Table 1 lists the MALDI analysis obtained for derivatized HSP22, indicating sites of potential PKCinduced phosphorylation.31 As shown in Figure 5, a pair of peptide ions displaying the characteristic 14-Da mass signature was observed to correspond to tryptic fragment 56-65 (LSSAWPGTLR). Prediction algorithms suggest that this peptide possesses two possible sites of PKC phosphorylation, at Ser-57 and Thr-63; however, the peptide detected by MALDI analysis shows a mass shift corresponding to modification at a single site. Confirmation that tryptic peptide 56-65 was indeed phosphorylated by PKC and analysis of the site of modification was investigated by subjecting the derivatized peptide to MS/MS

Table 1. Theoretical and Experimental MALDI Mass Analysis of in Vitro PKC Phosphorylated Hsp-22 mass (Da)

position

evidencea

2277.96 1540.64 1813.75

(-35)-(-15) (-14)-(-2) (-1)-15

Y Y Yc

534.26 761.38

19-22 23-29

Y Y

2947.36 1087.58

30-55 56-65

Y Y

646.33 673.36

66-71 72-78

Y Y

832.43 1252.63 1858.95

79-86 87-97 98-113

Y Y Ya

953.45 542.26 945.50 509.27 5803.85

116-124 125-128 129-137 138-141 143-196

N N N nd nd

Cys-CAM

(C10) 1870.76

evidencea

N

(S14) 1893.76 + N (C-CAM) 1950.76 (S24/S28) 841.38/921.38 (S57/T63) 1167.58/1247.58 (T41/T76) 753.36/833.36

(C99) 1915.97

Y

N

nd

sequenceb GSHHHHHHGMASMTGGQQMGR DLYDDDDKDPSSR SMADGQMPFSCHYPSR DPFR DSPLSSR

LLDDGFGMDPFPDDLTASWPDWALPR Y 1167.58 and LSSAWPGTLR derivatized forms SGMVPR N GPTATAR

(S104) 1938.95 + N (C-CAM) 1995.97 (S122) 1033.45 N (T140) 589.27

(C195) 5860.87

evidencea

phosphate

nd

FGVPAEGR TPPPFPGEPWK VCVNVHSFKPEELMVK DGYVEVSGK HEEK QQEGGIVSK NFTK IQLPAEVDPVTVFASLSPEGLLIIEAPQ VPPYSTFGESSFNNELPQDSQEVTCT

a Y-Peptide detected by MALDI; N-peptide not observed by MALDI. nd, not determined (outside detection limits). b Italic underlined residues are possible PKC phosphorylation sites. c Y only after derivatization experiment.

Figure 5. Derivatization mass signatures obtained when PKC phosphorylated Hsp-22 was trypsinized and derivatized with (A) C2 and C3 alkanethiols and (B) C7 and C8 alkanethiols. The 14-Da derivatization mass signature is indicated.

analysis after ZipTip purification. The doubly charged peptide of m/z 566.2 corresponding to tryptic fragment 56-65 derivatized

with ethanethiol was selected and fragmented. As shown in Figure 6, an extensive y-ion series of derivatized peptide 56-65 was obtained, including the ions y3, y4, y6, and y8. This mass series is consistent with β-methyl-S-ethylcysteine at residue 63, formed by ethanethiol derivatization of β-eliminated pThr. We conclude that Thr-63 is a site of PKC phosphorylation. Moreover, phosphorylation of Thr-63 fits within the sequence motif of S/TXR, which is consistent with phosphorylation by PKC.37 Tandem MS analysis showed no evidence for phosphorylation at other sites within this peptide, consistent with MALDI analysis. The status of other HSP22 sites predicted for PKC phosphorylation remains to be determined. For example, by using MS/ MS, Benndorf et al.31 reported a second PKC-dependent phosphorylation site at Ser-14 that was not observed in our study. In addition, we observed three peptides by MALDI that possess predicted phosphorylation motifs (tryptic peptides 23-29, 7278, and 98-113; cf. Table 1), yet we did not find evidence through chemical derivatization that any of these peptides were phosphorylated. Our goal in this initial study did not involve maximizing detection sensitivity. A substantial improvement in sensitivity could be realized with an appropriate cleanup method following derivatization. The sensitivity of the current protocol is limited by sample dilution steps prior to MALDI to account for excess nucleophile. One approach might involve enriching for the derivatized phosphopeptides, by hydrophobic partitioning prior to MS analysis, or as recently described by biotin-avidin purification.30 In addition, the use of an enrichment step after digestion and prior to derivatization (e.g., IMAC) would have the effect of concentrating target phosphopeptides. This approach would aid the analysis of phosphopeptides that are often present in small quantities by reducing possible ion suppression effects, as well as simplifying subsequent mass spectra by excluding the majority of unwanted, (37) Kennelly, P. J.; Krebs, E. G. J. Biol. Chem. 1991, 266, 15555-15558.

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Figure 6. MS/MS spectrum for doubly charged ion of m/z 566.18 (m/z 1131.5 in Figure 5A). Interpretation of the y ion series of fragment 56-65 identified β-methyl-S-ethylcysteine at residue 63, indicating Thr-63 as a site of PKC phosphorylation.

unphosphorylated peptides. A further point that requires clarification is a perceived loss of some derivatized phosphopeptides, presumably due to hydrophobic adsorption. It would be of interest to note whether this effect was observed when more polar nucleophiles of similar chain length were used. We have experienced only minor problems with loss of singly phosphorylated peptides using the current protocol, although we expect that peptide loss may become a concern for mapping peptides with multiple phosphorylated residues. Depending upon the spatial arrangement of amino acids within such peptides, multiple proteases may be required to limit the presence of derivatized sites within a single peptide. CONCLUSIONS Improved methods for rapid detection of phosphoproteins in digestion mixtures would greatly enhance studies of protein phosphorylation. We have described an approach of derivatizing β-eliminated phosphopeptides with alkanethiols to produce characteristic and specific derivatized phosphopeptide ion-pair mass signatures that provide unambiguous evidence for phosphopeptides by MS. This approach may have broader applications such as in evaluating the phosphorylation state of gel-separated proteins or for assays monitoring in vitro phosphorylation events. With improved sensitivity it may become possible to adopt this approach in screening endogenous proteins for phosphorylation.

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The use of MALDI-MS as a first pass approach to assess protein phosphorylation status has its benefits in the high speed of analysis and the small percent of sample attrition during analysis. The remainder of the derivatized sample is suitable for more detailed analysis by tandem MS techniques, which, as we have demonstrated, can readily establish sites of peptide phosphorylation. ACKNOWLEDGMENT This work was supported by funding to P.C.A. from the NCI (RO1 CA77078-01), NHGRI (RO1HG01709-01) and the Merck Genome Research Institute. We thank Kate Noon for conducting the MS/MS analysis and Rachel Loo for discussions. R. Benndorf and M. J. Welsh (University of Michigan) are acknowledged for kindly providing the phosphorylated HSP22 sample. Note Added in Proof: A recent manuscript described the use of β-elimination of seryl and threonyl phosphates followed by nucleophilic addition of an isotope-labeled affinity tag (Goshe, M. B.; Conrads, T. P.; Panisko, E. A.; Angell, N. H.; Veenstra, T. D.; Smith, R. D. Anal. Chem. 2001, 73, 2578-2586).

Received for review April 11, 2001. Accepted August 17, 2001. AC0104227