Selective Sampling of Phosphopeptides for Detection by MALDI Mass

Selective sampling was also applied to the tryptic digest of β- and α-caseins to .... α-cyano-4-hydroxycinnamic acid,10,14,25 2',4',6'-trihydroxyac...
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Anal. Chem. 2005, 77, 6078-6084

Technical Notes

Selective Sampling of Phosphopeptides for Detection by MALDI Mass Spectrometry Haixia Zhang,† Cunjie Zhang,‡ Gilles A. Lajoie,†,‡ and Ken K.-C. Yeung*,†,‡

Department of Chemistry and Department of Biochemistry, The University of Western Ontario, London, Ontario, Canada

The analysis of phosphopeptides by mass spectrometry (MS) is one of the most challenging tasks in proteomics. This is due to the lower isoelectric point (pI) of phosphopeptides, which leads to inefficient sample ionization in MS, particularly when competing with other peptides. The problem is compounded by the typical low abundance of phosphopeptides in biological samples. We describe here a simple nonsorptive method to isolate phosphopeptides based on their pI. A voltage is applied to selectively migrate the phosphopeptides into a capillary, which are negatively charged at acidic pH. The selectively sampled fraction is directly deposited onto MALDI sample target in nanoliter volumes (7-35 nL) for highly sensitive MS detection. No significant sample loss is evident in this procedure; hence, the MS was able to detect the isolated phosphopeptides at trace quantity. In this case, attomolelevel detection limit is achieved for synthetic phosphopeptides (nM concentration and nL volume), from a mixture containing other peptides at up to 1 million times higher in concentration. Selective sampling was also applied to the tryptic digest of β- and r-caseins to reveal the multiple phosphorylated peptides at the low-femtomole level using MALDI MS. Knowledge of pI based on the rejection/injection of peptides was found to be useful in peak assignment. To confirm the sequence of the selectively sampled peptides, fraction collection was performed for offline ESI MS/MS analysis. Phosphorylation of proteins has been traditionally measured by highly specific and sensitive techniques such as autoradiography with 32P-labeling or immunoprecipitation using phosphospecific antibodies.1 However, with the ever-increasing needs of high sample throughput in proteomics, phosphorylation is more commonly analyzed with mass spectrometry (MS).2-4 The addition of phosphate significantly reduces the isoelectric point (pI) of a * To whom correspondence should be addressed. Facsimile: 519 661 3022. E-mail: [email protected]. † Department of Chemistry. ‡ Department of Biochemistry. (1) Yan, J. X.; Packer, N. H.; Gooley, A. A.; Williams, K. L. J. Chromatogr., A 1998, 808, 23-41. (2) Mann, M.; Jensen, O. N. Nat. Biotechnol. 2003, 21, 255-261. (3) Zeller, M.; Konig, S. Anal. Bioanal. Chem. 2004, 378, 898-909. (4) McLachlin, D. T.; Chait, B. T. Curr. Opin. Chem. Biol. 2001, 5, 591-602.

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peptide, and this leads to poor ionization efficiency of phosphopeptides in both electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). Furthermore, the presence of other peptides with higher pIs will further suppress the already poor ionization of phosphopeptides. As a result, it has been very difficult to detect multiphosphorylated species or phosphorylation that occur at low stoichiometry. A number of approaches had been reported to enhance the ionization of phosphopeptides. They include the addition of ammonium salts5,6 or phosphoric acid7,8 to the samples, as well as the use of negative ion mode MS.9-11 Considerable efforts in the separation or isolation of phosphopeptides prior to MS analysis had also been reported. All of these involved sorptive-based techniques, such as reversed-phase HPLC,7,8,11,12 immobilized metal affinity chromatography (IMAC),12-16 chemical reactionbased affinity,17,18 and the recently reported titanium oxide19 and graphite20 columns. Nevertheless, sorption-based techniques generally suffer from sample loss through irreversible sorption and dilution in the elution/recovery step, making it difficult to detect phosphopeptides at ultralow levels (subfemtomole). We (5) Asara, J. M.; Allison, J. J. Am. Soc. Mass Spectrom. 1999, 10, 35-44. (6) Yang, X.; Wu, H.; Kobayashi, T.; Solaro, R. J.; van Breemen, R. B. Anal. Chem. 2004, 76, 1532-1536. (7) Kjellstrom, S.; Jensen, O. N. Anal. Chem. 2004, 76, 5109-5117. (8) Kim, J.; Camp II, D. G.; Smith, R. D. J. Mass Spectrom. 2004, 39, 208215. (9) Janek, K.; Wenschuh, H.; Bienert, M.; Krause, E. Rapid Commun. Mass Spectrom. 2001, 15, 1593-1599. (10) Ma, Y.; Yun, L.; Zeng, H.; Ron, D.; Mo, W.; Neubert, T. A. Rapid Commun. Mass Spectrom. 2001, 15, 1693-1700. (11) Beck, A.; Deeg, M.; Moeschel, K.; Schmidt, E. K.; Schleicher, E. D.; Voelter, W.; Haring, H. U.; Lehmann, R. Rapid Commun. Mass Spectrom. 2001, 15, 2324-2333. (12) 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. (13) Liu, H.; Stupak, J.; Zheng, J.; Keller, B. O.; Brix, B. J.; Fliegel, L.; Li, L. Anal. Chem. 2004, 76, 4223-4232. (14) Stensballe, A.; Jensen, O. N. Rapid Commun. Mass Spectrom. 2004, 18, 1721-1730. (15) Jin, W.; Dai, J.; Zhou, H.; Xia, Q.; Zou, H.; Zeng, R. Rapid Commun. Mass Spectrom. 2004, 18, 2169-2176. (16) Cao, P.; Stults, J. S. Rapid Commun. Mass Spectrom. 2000, 14, 1600-1608. (17) Zhou, H.; Watts, J. D.; Aebersold, R. Nat. Biotechnol. 2001, 19, 375-378. (18) Oda, Y.; Nagasu, T.; Chait, B. T. Nat. Biotechnol. 2001, 19, 379-382. (19) Pinkse, M. W. H.; Uitto, P. M.; Hilhorst, M. J.; Ooms, B.; Heck, A. J. R. Anal. Chem. 2004, 76, 3935-3943. (20) Larsen, M. R.; Graham, M. E.; Robinson, P. J.; Roepstorff, P. Mol. Cell. Proteomics 2004, 3, 456-465. 10.1021/ac050565j CCC: $30.25

© 2005 American Chemical Society Published on Web 08/11/2005

recently reported a selective sampling technique for peptides based on their net charges using capillary electrophoresis (CE), which does not involve sample extraction by sorption.21 The sample remains in solution throughout the process, and it can be deposited in very small volume (nanoliters) on MALDI plates to allow sensitive MS analysis. In this work, we describe the application of selective sampling for the analysis of phosphopeptides. On the basis of the acidic pI of phosphopeptides, they can be selectively isolated from the bulk mixture even at trace quantity, and in turn perform MS analysis. Development of selective sampling at various sampling pH conditions is conducted to facilitate the selection of phosphopeptides with various pIs. The effectiveness of the selective sampling technique is illustrated by using a variety of phosphopeptides, with different number of phosphorylation sites and from various sources (synthetic peptides to tryptic digest of commercially available model proteins). EXPERIMENTAL SECTION Chemicals. Zwitterionic surfactant, 1,2-dimyristoyl-sn-glycero3-phosphocholine (DMPC), and anionic surfactant, 1,2-dimyristoylsn-glycero-3-phosphate (monosodium salt) (DMPA), were from Avanti Polar Lipids, Inc. (Alabaster, AL). Reagent grade phosphoric acid, hydrochloric acid, and ammonium and sodium hydroxide were purchased from EM Science (Gibbstown, NJ). Mesityl oxide, citric acid, ammonium bicarbonate, ammonium citrate dibasic salt, calcium chloride, dithiothreitol (DTT), iodoacetamide, sodium dodecyl sulfate (SDS), and Tris-HCl were obtained from SigmaAldrich (Markham, ON, Canada). Urea was obtained from Merck KGaA (Darmstadt, Germany). Citric acid (15 mM) was used to prepare buffers with various pH adjusted by ammonium hydroxide. 2,5-Dihydroxybenzoic acid (DHB, from Sigma) was used as received. HPLC grade acetone and ethanol (100%) were from Fisher Scientific Ltd. (Nepean, ON, Canada). Deionized water (Millipore, MA) was used in preparation of all solutions. Synthesis of Phosphopeptide and Calculation of pI. The synthetic peptides, DSSLLLK (MW ) 774.45, pI 6.06, P0), DpSSLLLK (MW ) 854.42, pI 3.75, P1) and DpSpSLLLK (MW ) 934.38, pI 2.29, P2) were prepared by a solid-phase method. The protected amino acids were obtained from Nova Biochem. (EMD Biosciences, Inc., San Diego, CA). The pI values of all peptides used in this work were calculated using the Demo version of Protein Tools (ChemSW, Fairfield, CA). For the phosphate in phosphorylated serine, the pKa1 and pKa2 values are 0.9 and 6.1 respectively.22 Phosphoproteins and Trypsin Digestion. β-Casein and R-casein (bovine milk) were purchased from Sigma-Aldrich. Modified sequence grade trypsin was from Promega Corp. (Madison, WI). In-solution digestion of proteins was performed in 50 mM ammonium bicarbonate (pH 8) with 2 mM CaCl2. An enzyme-to-protein ratio of 1:100 (w/w) was used, and the digestion was allowed to proceed overnight at 37 °C. Then the digest solution was dried by a vacuum concentrator to remove the ammonium bicarbonate. The remaining solid sample was redissolved in water and stored in a freezer prior to use. Electroosmotic Flow (EOF) Suppression. The control of EOF to prevent the nonselective sweeping of the bulk sample

solution into the capillary is critical to the success of selective sampling. Coating the capillary inner walls with the zwitterionic surfactant DMPC was found to be effective in suppressing the EOF (1 µL) is required for offline ESI using an emitter tip (Proxeon Biosystems). To accumulate a larger volume, the sampling and collection procedure was repeated four times into the same vial, giving a total sample volume of ∼1.5 µL. Prior to MS analysis, an equal volume of 0.1% formic acid in acetonitrile was added to the collected sample, and the mixture (3 µL) was loaded into the ESI emitter. For all ESI mass spectral analyses, higher concentrations were also used for the trypsin digestion samples, to ensure good sequence coverage in the MS/ MS mode. The estimated peptide concentration in the final mixture loaded into the nanoESI emitter was 25 pmol/µL for β-casein and 15 pmol/µL for R-casein. ESI-MS and MS/MS measurements were performed on a Q-TOF 2 mass spectrometer operating in positive ion mode (Waters/Micromass, Milford, MA). (25) Huang, S.; Hsu, J.; Morrice, N. A.; Wu, C.; Chen, S. Proteomics 2004, 4, 1935-1938. (26) Wei, H.; Nolkrantz, K.; Powell, D. H.; Woods, J. H.; Ko, M.; Kennedy, R. T. Rapid Commun. Mass Spectrom. 2004, 18, 1193-1200. (27) Keller, B. O.; Li, L. J. Am. Soc. Mass Spectrom. 2001, 12, 1055-1063.

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The peptide sequences were first determined from the resulting singly charged, deisotoped MS/MS spectra (Max-Ent3) using PepSeq in MassLynx 4.0 (Waters/Micromass) or PEAKS (Bioinformatics Solutions Inc, Waterloo, ON, Canada) and then further confirmed manually. RESULTS AND DISCUSSION Selective Sampling of Synthetic Phosphopeptides. The effectiveness of the selective sampling technique in isolating the phosphopeptides was first evaluated using a mixture of three synthetic peptides: DSSLLLK (P0), DpSSLLLK (P1), and DpSpSLLLK (P2). The phosphopeptide concentrations (P1 and P2) were kept constant at 28.6 nM, while the P0 concentration was varied from 1000 to 1 000 000 times higher. The selective sampling of P1 and P2 (pI of 3.75 and 2.29, respectively) was performed at pH 5.2. The fraction was first analyzed with positive ion mode MALDI MS (Figure 2A-C). In all three cases, the (M + H)+ signals from P1 and P2 were clearly observed. The intensity of P1 and P2 remained generally constant up to 105-fold of excess P0 (mass spectra not shown). At 106-fold of excess P0, the observed P1 and P2 signal intensity was relatively lower (Figure 2C). Similar behavior was reported in our initial study of selective sampling.21 It was attributed to the excessively high peptide concentration in the sample mixture solution (P0 concentration was 29 mM or 22 mg/ mL in this case). At this sample concentration, the solution became noticeably more viscous than other solutions. In addition, the ionic strength was expected to increase substantially, which could lower the local field strength within the sample vial. Both effects could retard the migration of the phosphopeptides into the capillary, thus resulting in lower S/N. However, significant improvement was evident when compared to the mass spectrum of the original mixture (Figure 2D, sample composition as in Figure 2A), in which only the (M + H)+ signal from P0 was observed. Next, the selectively sampled fractions were analyzed by MALDI MS in the negative ion mode. In agreement with previous reports,9-11 higher relative peak intensity was observed for phosphopeptides in the negative ion mode, such that small signals from P1 and P2 were detected even prior to selective sampling (Figure 2H). However, selective sampling allowed a much more favorable introduction of P1 and P2 and completely eliminated the signal from P0 even at the highest P0 concentration (Figure 2G). Detection Limits and Sample Recovery. The detection limits of the selectively sampled P1 and P2 were determined for our MALDI MS instrument, based on the sample concentration and volume spotted on the target. The signal-to-noise ratio (S/N) was recorded from samples of decreasing concentration and spot volume, in replicates of six under each condition. All P1 and P2 solutions contained concentrations of P0 that were 1000 times higher. In the positive ion mode, an average S/N of 4 was recorded from 40 amol of P1 (5.7 nM and 7 nL), and an average S/N of 5 was observed from 80 amol of P2 (5.7 nM and 14 nL). In the negative ion mode, the observed S/N was generally lower. An average S/N of 7 was recorded for 200 amol for both P1 and P2 (14 nM and 14 nL). More important than the sensitivity is whether this sampling technique contributes to any significant sample loss or dilution. A recovery study was conducted by comparing the S/N of 500 amol of P2 (14 nM and 35 nL) in the positive ion mode from the selectively sampled fraction with that from a pure solution of P2

Figure 2. MALDI mass spectra of selectively sampled fractions from mixtures of P0, P1, and P2 at concentration ratios of (A,E) 103:1:1; (B,F) 104:1:1, and (C,G) 106:1:1. (D,H) MALDI mass spectra of the original mixture, without selective sampling, at a concentration ratio of 103:1:1. Positive ion mode in (A-D) and negative ion mode in (E-H). Sample quantity spotted was 35 nL containing 1 fmol each of P1 and P2. The signals at m/z of 772 (A-C), 83 Da less than P1, was a result of the postsource loss of HPO3, which could not be properly calibrated in the reflectron mode according to Bruker technical support. The same phenomenon was also observed for other phosphopeptides studied in this work.

spotted from the capillary. Fifteen replicates were performed. An average S/N of 5.9 was obtained in the pure P2 solution, and an average S/N of 5.8 was recorded from the selectively sampled fraction from a mixture. This resulted in an excellent recovery of 98%. Recognizing that a MALDI MS signal is only semiquantitative, one can still conclude that significant sample loss or dilution was not happening in the selective sampling step. It should be noted that sample enrichment or concentration did not occur either. Our technique simply isolates the phosphopeptides at the same concentrations as in the original sample mixture.21

Selective Sampling from a Tryptic Digest of β-Casein. The selectivity of the sampling technique at different pH conditions is illustrated with the tryptic digest of β-casein. Using positive ion mode MALDI MS, the tetraphosphorylated β1, the monophosphorylated β2, and six nonphosphorylated peptides with pI ranging from 4 to 10 were detected from the original tryptic digest (Table 1). When selective injection was performed at increasing sampling pH from 3.0 to 6.5, peptides with pI below the sampling pH were introduced into the capillary and in turn detected by MALDI MS (positive ion mode, Figure 3A-C). The last two peaks listed in Analytical Chemistry, Vol. 77, No. 18, September 15, 2005

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Table 1. Peptides Identified from the Tryptic Digest of β-Casein, in the Order of Calculated pI peptide ID T1-2(β1) T6(β2) T15 β-casein B T16 T11 T14 T13 a

residue

sequence

1-25

RELEELNVPGEIVEpSLpSpSpSEESITR FQpSEEQQQTEDELQDK DMPIQAFLLYQEPVLGPVR YPVEPFTER GPFPIIV EMPFPK AVPYPQR VLPVPQK

33-48 184-202 67-75 203-209 108-113 177-183 170-176

mass MH+a

calc pI

3122.26

1.37

2061.82 2186.16 1137.55 742.44 748.36 830.44 780.49

3.29 4.32 4.48 5.75 6.22 9.00 9.00

Theoretical monoisotoptic masses.

Figure 3. Positive ion MALDI mass spectra of selectively sampled fractions from a tryptic digest of β-casein adjusted at various pH: 3.0, 4.0, 5.2, and 6.5, respectively, in (A-D). K denotes possible assignments to keratin peptide based on m/z. U denotes unassigned signals. “r” indicates the 83-Da difference due to postsource loss of phosphate. Sample quantity, 14 fmol in 35-nL spot, based on initial protein concentration in digest.

Table 1, T14 and T13, had pIs higher than pH 6.5, and so they were not observed in any of the selectively sampled fractions. Two interesting observations should be noted here. First, the introduction and rejection of peptides occurred with only ∼0.3 unit difference between the sampling pH and the pI: T11 (pI 6.22) was included at sampling pH 6.5; β2 (pI 3.29) was rejected at sampling pH 3.0. Second, selective sampling reveals the pI information of 6082 Analytical Chemistry, Vol. 77, No. 18, September 15, 2005

an injected peptide, which can be helpful in peak assignment. For example, the m/z 1137.6 signal (YPVEPFTER from β-casein B) was initially suspected to be from another peptide of β-casein (VKEAMAPKHK, residue 98-107, m/z 1138.6, pI 9.71), but its pI was too high for it to be injected at pH 5.2 (Figure 3C). To confirm the sequence of this peptide, the pH 5.2 fraction was collected and analyzed with ESI MS/MS. In a similar manner, collection was also carried out on the pH 4.0 fraction for ESI MS/ MS, to obtain sequence information and confirm the phosphorylation sites of β1 and β2 (MS/MS spectra are shown in Supporting Information as Figures S-1 and S-2). The selectively sampled fractions were also analyzed by the negative ion mode MALDI MS. The negative ion mode favors the detection of highly acidic peptides, as observed by others;9-11 hence, all spectra were dominated by signals from the two phosphopeptides and the most acidic peptide T15. The main drawback of the negative ion mode, however, is the generally poor S/N compared to that from the positive ion mode. The S/N of β1 and β2 was compared between the positive and negative ion modes, from the pH 4.0 selectively sampled fractions. Based on five replicates, an average S/N of 10 was obtained from 1.4 fmol of β1 in the positive mode (200 nM and 7 nL). An average S/N of 4 was recorded from 7 fmol of β1 in the negative mode (200 nM and 35 nL). For β2, an average S/N of 7 from 140 amol of β2 was obtained in the positive ion mode (20 nM and 7 nL). In the negative ion mode, the average S/N was 8 from 700 amol (20 nM and 35 nL). Hence, ∼5-fold increase in S/N was observed for both β1 and β2 when analyzed in the positive ion mode. The detection of phosphopeptides from β-casein reported in the literature ranged from low-picomole to mid-femtomole,7-10,16 with 18 fmol (for both of β1 and β2) being the lowest reported to date.10 In these reports, the sample concentrations ranged from 1 µM9 to 40 nM10 when MALDI MS was used. In ref 8, LC-ESI MS was used, which allowed partial sample concentration by trapping on the head of column. Detection of the singly phosphorylated β2 from a lower concentration of 5 nM was reported (10 µL injected, 50 fmol), but the detection of the tetraphosphorylated β1 required a much higher concentration (5 µM or 50 pmol in 10 µL injection). In summary, our selective sampling with nanoliter MALDI spotting provided superior mass detection limit, while offering comparable or slightly better concentration detection limit. Limitation of Sample Complexity on Selective Sampling. The previous example of selective sampling was performed on the digest of pure β-casein solutions. To mimic more closely conditions used in proteomics, in which various reagents are added to solubilize and unfold the proteins, selective sampling was performed on digested β-casein solution with the following reagents present: 50 mM DTT, 25 mM iodoacetamide, 2.5 M urea, 0.05% SDS, and 20 mM Tris-HCl. The concentration of these reagents is similar to the tolerance level of trypsin recommended by the supplier (Promega). The sample concentration (0.4 µM) and sampling pH (4.0) are kept the same as in Figure 3B. In general, selective injection remains as effective in isolating the phosphopeptides β1 and β2 in the presence of the background reagents. As one would expect, the MALDI MS intensities were lower than those from the pure sample without the additives. Based on the results from 12 replicates, the intensities of both β1 and β2 were reduced by ∼50% due to the added reagents (mass

Figure 4. MALDI mass spectra of the tryptic digest of R-casein: (A,B) original sample without selectively sampling. (C,D) selectively sampled fraction at pH 5.2. (A,C) in positive ion mode. (B,D) in negative ion mode. Sample quantity, 35 fmol in 35-nL spot, determined based on the initial total R-casein (s1 + s2) concentration in digest. U denotes unassigned signals. “r” indicates the 83-Da difference due to postsource loss of phosphate.

spectra not shown). When these reagents were studied individually, we found the effect of SDS to be the most detrimental. Being an anionic surfactant, SDS was suspected to comigrate with the phosphopeptides and in turn suppress the MALDI MS signals. In comparison to these reagents, selective injection is much less affected by the presence of other peptides in the background as illustrated in Figure 2. The injection/rejection of peptides in this method is strictly based on pI, so that the phosphopeptides can be selectively isolated as long as the background nonphosphorylated peptides have higher pI values. Selective Sampling from a Tryptic Digest of r-Casein. In the next application, selective sampling is used to demonstrate the increase in the sequence coverage in the tryptic peptide mapping of another phosphorylated protein, R-casein. According to the MALDI mass spectra of the original tryptic digest of R-casein (1 µM solution, Figure 4A,B), both R(s1)- and R(s2)caseins were present in this commercially available sample (SigmaAldrich). Table 2 lists the tryptic peptides that were identified based on their m/z. Fifteen fragments were detected in the positive ion mode, and 10 were detected in the negative ion mode. This corresponds to a sequence coverage of 45% for both R-caseins. From the 15 fragments detected in the positive ion mode, only 4 were phosphopeptides, which covered only 4 of the 19 known phosphorylation sites. One additional phosphopeptide was detected in the negative ion mode, which revealed another four phosphorylation sites. When selective sampling was performed at pH 5.2, on the same digested solution of 1 µM R-casein (or 35 fmol in a 35-nL spot), 10 phosphopeptides were observed with enhanced intensities compared to that of the original digest (Figure 4C,D). From R(s1)-casein, four phosphopeptides were detected, which showed all eight phosphorylation sites. The other

6 phosphopeptides were from R(s2)-casein, which also covered all 11 known phosphorylation sites. Based on these results, the sequence coverage of R(s1)- and R(s2)-caseins is increased to 59%. MS detection of phosphopeptides from R(s2)-casein in the commercial product of R-casein has been reported in the literature: T16 by Knight et al., from 1 pmol (injected) using LC-ESI MS),28 T14 and T16 by Carr et al., from 0.5 pmol in 2 µL (injected) also using LC-ESI MS,29 and T2 and T7 by Zhou et al., from 1.4 pmol (spotted) using IMAC and MALDI MS.30 The detection in refs 28 and 30 was based on MS (m/z) data and that in ref 29 was based on MS/MS information. Using selective sampling with MALDI MS (based on m/z data), this is the first report of simultaneous detection of up to six phosphopeptides from R(s2)casein, which covered all of the known phosphorylation sites (Figure 4C,D). To further confirm the identity of these phosphopeptides, fraction collection was performed on the selectively injected sample, followed by offline ESI-MS and MS/MS analyses. In the MS mode, all 10 phosphopeptides were detected from the selectively sampled fraction, but only 5 (labeled with ∆ in Table 2) were observed from the original digest at the same concentration. Eight out of the 10 phosphopeptides observed in the ESI MS mode (noted with asterisks in Table 2) had sufficient abundance for MS/MS acquisition. The spectra of these eight peptides with sequence assignment are provided in Supporting Information as Figures S-3-S-10. The expected sequences were (28) Knight, Z. A.; Schilling, B.; Row: R. H.; Kenski, D. M.; Gibson, B. W.; Shokat, K. M. Nat. Biotechnol. 2003, 21, 1047-1054. (29) Carr, S. A.; Huddleston, M. J.; Annan, R. S. Anal. Biochem. 1996, 239, 180-192. (30) Zhou, W.; Merrick, B. A.; Khaledi, M. G.; Tomer, K. B. J. Am. Soc. Mass Spectrom. 2000, 11, 273-282.

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Table 2. Peptides Identified from the Tryptic Digest of r-Casein, in the Order of Calculated pI peptide observed inb peptide ID

sequence

mass MH+ a

calc pI

T8, s1 T7, s2 T2, s2 T7, s1 T14, s2 T16, s2 T15, s1 T14-15, s2 T16-17, s2 T10, s1 T19, s2 T14-15, s1 T18, s1 T3, s1 T4, s1 T11, s1 T10, s2 T17, s1 T25, s2 T13, s2 T27, s2

QMEAEpSIpSpSpSEEIVPNpSVEQK* NANEEEYSIGpSpSpSEEpSAEVATEEVK NTMEHVpSpSpSEESIIpSQETYK* DIGpSEpSTEDQAMEDIK*∆ EQLpSTpSEENSK* TVDMEpSTEVFTK*∆ VPQLEIVPNpSAEER*∆ EQLpSTpSEENSKK* TVDMEpSTEVFTKK∆ EDVPSER LTEEEK YKVPQLEIVPNpSAEER*∆ EPMIGVNQELAYFYPELFR HQGLPQEVLNENLLR FFVAPFPEVFGK YLGYLEQLLR ALNEINQFYQK EGIHAQQK FALPQYLK NAVPITPTLNR AMKPWIQPK

2720.91 3008.02 2618.91 1927.68 1411.50 1466.60 1660.79 1539.60 1594.71 831.38 748.37 1951.95 2316.13 1759.94 1384.72 1267.70 1367.69 910.47 979.55 1195.67 1098.61

0.72 0.90 1.37 2.16 2.28 3.52 3.62 3.62 4.08 4.09 4.20 4.20 4.20 5.62 6.22 6.22 6.22 7.25 8.83 10.00 10.01

original digest +/-+/-+/++ + +/+/++/-+/++/-+/+ + ++ +

selectively injected sample +/+/+/+/-+/-++/+/+/++/+/+/-

* Sequence verified by ESI MS/MS data; ∆ detected in the ESI MS mode from the original digest (without selective sampling). a Theoretical monoisotoptic masses. b Relative intensity of the peptide signals recorded with MALDI MS: (+) weak in positive ion spectrum; (++) strong in positive ion spectrum; (-) weak in negative ion spectrum; (- -) strong in negative ion spectrum; (blank) not observed.

obtained in all cases. As stated in the Experimental Section, higher sample volumes and concentrations are used to ensure good sequence coverage in the MS/MS data. Detection limit with ESI MS is not determined here, since online ESI coupling is needed for proper sensitivity measurement. CONCLUSIONS The use of selective sampling allows the trace quantity isolation of phosphopeptides based on their unique acidic pI characteristics. It avoids the common problems in chromatographic separations such as sample dilution with mobile phase and sample loss due to irreversible sorption, and thus, nanoliters of phosphopeptide can be sampled and subsequently deposited for sensitive MALDI MS analysis. While a larger sample size of a few microliters is still required to perform the procedure, most of the sample is not consumed and can be further analyzed by other techniques. This technique is effective in selecting phosphopeptides even from a background of highly concentrated nonphosphorylated peptides (up to 106 times, or ∼30 mM). Hence, it should be applicable to complex peptide digests, as long as the background peptides have pIs higher than the phosphopeptides. Selective injection can also be applied to samples containing a high concentration of digestion additives. Even though the signal intensity was reduced, isolation of phosphopeptides remained effective. Selective injection also facilitates the handling of minuscule sample volume and can be readily adopted in the

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development of microfluidic devices. Another merit of this approach is that the selection/rejection of peptides of our sampling technique can provide useful pI information for peak assignment. ACKNOWLEDGMENT The work is supported by the Natural Science and Engineering Research Council of Canada (NSERC), the Ontario Research and Development Challenge Fund (ORDCF), and the University of Western Ontario. The Bruker Reflex IV instrument was funded by the Canada Foundation for Innovation and the Ontario Innovation Trust. The Waters/Micromass Q-TOF 2 instrument was funded by NSERC. The authors also thank the following individuals from the Department of Biochemistry of the University of Western Ontario: Ms. Heidi Liao for synthesizing the phosphopeptides and Dr. Suya Liu for his helpful comments and suggestions. Dr. Liang Li of the University of Alberta and Dr. Bernd Keller of Queen’s University are acknowledged for insightful discussions. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review April 4, 2005. Accepted July 7, 2005. AC050565J