MS Analysis

Anal. Chem. 2008, 80, 3007-3015

Detection of Multiphosphorylated Peptides in LC-MS/MS Analysis under Low pH Conditions Hyunwoo Choi, Hye-suk Lee, and Zee-Yong Park*

Department of Life Sciences, Gwangju Institute of Science & Technology 1 Oryong-Dong, Buk-Gu, Gwangju, Korea 500-712

An improved method of detection of multiphosphorylated peptides by RPLC-MS/MS analysis under low pH conditions (pH ∼1.7, 3% formic acid) is demonstrated for the model phosphoproteins, bovine r- and β-casein. Changes in the pH conditions from normal (pH ∼3.0, 0.1% formic acid) to low (pH ∼1.7, 3% formic acid) significantly improved the detection limit of multiphosphorylated peptides carrying negative (-) solution charge states. In particular, bovine β-casein tetraphosphorylated peptide, was detected with a loading amount of only 50 fmol of trypsin-digested bovine β-casein under low pH conditions, which is 200 times lower than necessary to detect the peptide under normal pH conditions. In order to understand the low pH effect, various loading amounts of trypsin-digested bovine r- and β-caseins were analyzed by RPLC-MS/MS analyses under two different pH conditions. The question of whether the low pH condition improves the detection of multiphosphorylated peptides by increasing ionization efficiencies could not be proven in this study because synthetic multiphosphorylated peptides could not be easily obtained by peptide synthesis. Interestingly, increased hydrophilicity resulting from multiple phosphorylation events is shown to negatively affect the peptide retention on reversed-phase column material. It was also demonstrated that the low pH condition could effectively enhance the retention of multiphosphorylated peptides on reversed-phase column material. The usefulness of low pH RPLC analysis was tested using an actual phosphopeptide-enriched sample prepared from mouse brain tissues. Previously, low pH solvents have been used in SCX fractionation and TiO2 enrichment processes to selectively enrich phosphopeptides during the phosphopeptide enrichment procedure, but the improved detection of multiphosphorylated peptides in RPLC-MS/MS analysis under low pH conditions has not been reported before (Ballif, B. A.; Villen, J.; Beausoleil, S. A.; Schwartz, D.; Gygi, S. P. Mol. Cell. Proteomics 2004, 3, 10931101. Villen, J.; Beausoleil, S. A.; Gerber, S. A.; Gygi, S. P. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 14881493. Schlosser, A.; Vanselow, J. T.; Kramer, A. Anal. Chem. 2005, 77, 5243-5250.) Mass spectrometry became a method of choice for protein phosphorylation analysis because it can provide both qualitative * To whom correspondence should be addressed. E-mail: [email protected]. 10.1021/ac7023393 CCC: $40.75 Published on Web 03/18/2008

© 2008 American Chemical Society

and quantitative information about protein phosphorylation without the need for phosphorylation site-specific antibodies.4-11 Many biological systems have been analyzed by mass spectrometry to reveal the role of protein phosphorylation in important biological processes.1,12-16 Mass spectrometric analysis of protein phosphorylation is often not successful, however, because there are certain limitations.4 For example, a low stoichiometry of phosphorylated proteins to unmodified proteins and extensive numbers of phosphorylation sites are known to be the two characteristics of protein phosphorylation that hamper routine analysis.17 In addition, current mass spectrometric techniques usually analyze peptides in positive ion mode to increase the detection of both unmodified and modified peptides. The acidic property, i.e., negative (-) charge of phosphorylated amino acids could lower the degree of protonation; therefore, the ionization efficiencies of phosphorylated peptides in positive ion mode could be adversely affected.4,18 A number of mass spectrometric techniques have been developed to increase the detection limit of phosphorylated (1) Ballif, B. A.; Villen, J.; Beausoleil, S. A.; Schwartz, D.; Gygi, S. P. Mol. Cell. Proteomics 2004, 3, 1093-1101. (2) Villen, J.; Beausoleil, S. A.; Gerber, S. A.; Gygi, S. P. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 1488-1493. (3) Schlosser, A.; Vanselow, J. T.; Kramer, A. Anal. Chem. 2005, 77, 52435250. (4) Garcia, B. A.; Shabanowitz, J.; Hunt, D. F. Methods 2005, 35, 256-264. (5) Pandey, A.; Podtelejnikov, A. V.; Blagoev, B.; Bustelo, X. R.; Mann, M.; Lodish, H. F. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 179-184. (6) Yan, J. X.; Packer, N. H.; Gooley, A. A.; Williams, K. L. J. Chromatogr., A 1998, 808, 23-41. (7) Annan, R. S.; Huddleston, M. J.; Verma, R.; Deshaies, R. J.; Carr, S. A. Anal. Chem. 2001, 73, 393-404. (8) McLachlin, D. T.; Chait, B. T. Curr. Opin. Chem. Biol. 2001, 5, 591-602. (9) Mann, M.; Ong, S. E.; Gronborg, M.; Steen, H.; Jensen, O. N.; Pandey, A. Trends. Biotechnol. 2002, 20, 261-268. (10) Gerber, S. A.; Rush, J.; Stemman, O.; Kirschner, M. W.; Gygi, S. P. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 6940-6945. (11) Goshe, M. B.; Conrads, T. P.; Panisko, E. A.; Angell, N. H.; Veenstra, T. D.; Smith, R. D. Anal. Chem. 2001, 73, 2578-2586. (12) Beausoleil, S. A.; Jedrychowski, M.; Schwartz, D.; Elias, J. E.; Villen, J.; Li, J.; Cohn, M. A.; Cantley, L. C.; Gygi, S. P. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 12130-12135. (13) Blagoev, B.; Ong, S. E.; Kratchmarova, I.; Mann, M. Nat. Biotechnol. 2004, 22, 1139-1145. (14) MacCoss, M. J.; McDonald, W. H.; Saraf, A.; Sadygov, R.; Clark, J. M.; Tasto, J. J.; Gould, K. L.; Wolters, D.; Washburn, M.; Weiss, A.; Clark, J. I.; Yates, J. R. 3rd Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 7900-7905. (15) Olsen, J. V.; Blagoev, B.; Gnad, F.; Macek, B.; Kumar, C.; Mortensen, P.; Mann, M. Cell 2006, 127, 635-648. (16) Li, X.; Gerber, S. A.; Rudner, A. D.; Beausoleil, S. A.; Haas, W.; Villen, J.; Elias, J. E.; Gygi, S. P. J. Proteome Res. 2007, 6, 1190-1197. (17) Steen, H.; Jebanathirajah, J. A.; Rush, J.; Morrice, N.; Kirschner, M. W. Mol. Cell. Proteomics 2006, 5, 172-181. (18) Liu, S.; Zhang, C.; Campbell, J. L.; Zhang, H.; Yeung, K. K.; Han, V. K.; Lajoie, G. A. Rapid. Commun. Mass Spectrom. 2005, 19, 2747-2756.

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peptides and the coverage of phosphorylation sites.11,14,19-26 Most of these techniques employ metal-chelate affinity chromatography or ion-exchange chromatography to selectively enrich the substoichiometric phosphorylated peptides or phosphoproteins.1,19,24,25 Chemical modification type approaches have also been introduced. For example, phosphorylated peptides/proteins can be chemically modified to remove the negatively charged state of the phosphate moiety in order to increase ionization efficiencies. A second approach involves chemically changing phosphorylated amino acids into lysine homologues to allow distinct traces to be made after trypsin digestion.11,21-23,27 Other approaches using additives such as phosphoric acid or EDTA have also been developed; these techniques are designed to minimize unwanted interactions between phosphorylated peptides and reversed-phase column material or metal ions in an LC system.18,28 Among the mass spectrometric techniques that have been developed, only a few of them have focused on the improved detection of multiphosphorylated peptides in LC-MS analysis.18,28,29 The ionization behavior of multiphosphorylated peptides is reported to be quite different from that of mono- or diphosphorylated peptides.18,28,29 For example, trypsin digestion of R- and β-casein generates multiphosphorylated peptides (3, 4, and 5 phosphorylations) in addition to mono- or diphosphorylated peptides. Some of these multiphosphorylated peptides exhibit significantly decreased ion intensities compared to the less phosphorylated peptides by conventional RPLC-MS/MS analysis, and a significantly larger amount of enzymatic digests is required in order to detect them.18,28,29 We have recently determined that sub-stoichiometric yields of multiphosphorylated bovine β-casein peptides were not due to inefficient trypsin digestion near the multiphosphorylation sites.29 The tryptic digestion pattern of bovine caseins is relatively simple; therefore, the ionization of multiphosphorylated peptides might be governed by three previously reported causes for the unsuccessful mass spectrometric identification of phosphorylation sites: (1) their decreased retention on reversed-phase column material due to increased hydrophilicity, (2) selectively suppressed ionization by more ionizable coeluting peptides, and (3) lower ionization efficiency than unmodified counterparts.17 One way of investigating the effects of multiple phosphorylation on the ionization behavior of phosphopeptides would be to use synthetic phosphopeptides; however, it is difficult to obtain synthetic phosphopeptides with more than three modification sites by peptide synthesis. For this reason, researchers have used bovine R- and β-casein tryptic digest (19) Andersson, L.; Porath, J. Anal. Biochem. 1986, 154, 250-254. (20) 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. (21) Knight, Z. A.; Schilling, B.; Row, R. H.; Kenski, D. M.; Gibson, B. W.; Shokat, K. M. Nat. Biotechnol. 2003, 21, 1047-1054. (22) Li, W.; Boykins, R. A.; Backlund, P. S.; Wang, G.; Chen, H. C. Anal. Chem. 2002, 74, 5701-5710. (23) McLachlin, D. T.; Chait, B. T. Anal. Chem. 2003, 75, 6826-6836. (24) Oda, Y.; Nagasu, T.; Chait, B. T. Nat. Biotechnol. 2001, 19, 379-382. (25) Pinkse, M. W.; Uitto, P. M.; Hilhorst, M. J.; Ooms, B.; Heck, A. J. Anal. Chem. 2004, 76, 3935-3943. (26) Zhou, H.; Watts, J. D.; Aebersold, R. Nat. Biotechnol. 2001, 19, 375-378. (27) Vosseller, K.; Hansen, K. C.; Chalkley, R. J.; Trinidad, J. C.; Wells, L.; Hart, G. W.; Burlingame, A. L. Proteomics 2005, 5, 388-398. (28) Kim, J.; Camp, D. G., 2nd; Smith, R. D. J. Mass Spectrom. 2004, 39, 208215. (29) Kim, S.; Choi, H.; Park, Z. Y. Mol. Cells 2007, 23, 340-348.

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peptides as model phosphopeptides with multiple phosphorylation sites. Although such multiphosphorylated peptides may not be predominant in LC-MS analyses, the presence of phosphopeptides with more than three modifications has often been observed in actual proteome samples. Indeed, a recent study by Gygi et al. identified more than 900 multiphosphorylated peptides from a large-scale phosphorylation analysis of primary animal tissue sample (liver).2 In this study, we demonstrate the significantly improved detection of multiphosphorylated peptides of bovine R- and β-casein by RPLC-MS/MS analysis under low pH conditions. We reasoned that lowering the pH of the elution solvents during RPLC separation could decrease the negatively charged phosphopeptide species (deprotonated form) and thereby increase the degree of protonation or the ionization efficiency of multiphosphorylated peptides. We also investigated whether the increased hydrophilicity or negative (-) solution charge state resulting from multiple phosphorylation affects peptide retention on reversed-phase column material. To validate the utility of low pH phosphopeptide analysis in actual proteome samples, we analyzed a phosphopeptide enriched sample of mouse brain tissue lysates and compared the number of phosphopeptides identified and the number of modification sites per peptide under two different pH conditions. METHODS Bovine β-casein, bovine R-casein, phosphoric acid, formic acid, ammonium bicarbonate, ammonium hydroxide, calcium chloride, dithiothreitol (DTT), trifluoroacetic acid (TFA), and iodoacetamide (IAA) were purchased from Sigma. Acetonitrile, methanol, and HPLC grade water were all purchased from Fisher Science. Sequencing grade modified trypsin was obtained from Promega. Aqua C18 (particle size 5 µm) reversed-phase column material was purchased from Phenomenex. Titanium oxide (Titansphere, particle size 5 µm) was purchased from GL Sciences(GL Sciences Inc., Tokyo, Japan). In-Solution Enzymatic Digestion of r- and β-Casein. R-Casein. Bovine R-casein that had been dissolved in 50 mM NH4HCO3 (pH 8.0) was reduced by adding 5 mM DTT at room temperature for 1 h and alkylated with 25 mM IAA at room temperature for 30 min in the dark. In-solution digestion was performed using sequencing grade modified trypsin in 50 mM NH4HCO3 (pH 8.0) at a 1:50 (w/w) trypsin-to-protein ratio for 16 h at 37 °C. The digested peptides were adjusted with 5% formic acid (v/v) to quench the enzyme activity. β-Casein. Bovine β-casein in 5 mM CaCl2/100 mM Tris buffer (pH 8.0) was digested using sequencing grade modified trypsin in 5 mM CaCl2/50 mM NH4HCO3 (pH 8.0) at a 1:50 (w/w) trypsinto-protein ratio for 12-14 h at 37 °C. Bovine β-casein does not have a disulfide bond, and thus, reduction and alkylation processes were not carried out. The digested peptides were adjusted with 5% formic acid (v/v) to quench the enzyme activity and were immediately stored at -80 °C. Mouse Brain Tissue Collection. At 8 weeks of age, ICR male mice were obtained from Samtako Bio Korea and were sacrificed by vertebral fracture without anesthesia. The whole brain was immediately dissected and washed with ice-cold 1× PBS. The samples were immediately frozen in liquid nitrogen and transferred to a -80 °C freezer.

Protein Extraction and Enzymatic Digestions of Mouse Brain. After the whole mouse brain was ground using a mortar and pestle, the powder was collected in Eppendorf tubes that had been previously weighed, and the final weight of the tissue was calculated. To make protein extracts, the powder was dissolved in an appropriate volume of extraction buffer containing 50 mM Tris-HCl (pH 8.0), 8 M urea, and 1 mM DTT. The sample was incubated with rotation at 4 °C for 2 h and sonicated five times at 30% amplitude for 3 s on ice. In order to remove insoluble tissue debris from the suspensions, the samples were centrifuged at 100 000g for 1 h. The supernatant was transferred into separate tubes, and the protein concentration was determined using a BCA assay kit (Bio-Rad). After reduction (5 mM DTT, 1 h) and alkylation (25 mM IAA, 30 min in the dark), endoproteinase Lys-C (Roche) was added at a 1:100 enzyme/substrate ratio and digestion was allowed to proceed at 37 °C for 14 h. After 4 × dilution with 100 mM Tris (pH 8.0), lysate proteins were further digested with sequencing grade trypsin at a 1:50 enzyme/substrate ratio in the presence of calcium chloride (2 mM), at 37 °C, overnight. Isolation of Phosphopeptides Using Titansphere. Bodenmiller’s method was employed to enrich phosphopeptides using Titansphere.30 A 250-µm-i.d., 360-µm-o.d. fused-silica capillary was filled with 6-7 cm of TiO2 using a MicroFilter. Before initial use, the column was washed twice with 100 µL of 0.3 M NH4HCO3 (pH 10.5) and equilibrated with 80% CH3CN, 0.1% TFA. Samples in 80% CH3CN, 2.5% TFA were loaded on to the column at a flow rate of 1 µL/min, and then the column was washed twice with 80% CH3CN, 0.1% TFA, and finally twice with 0.1% TFA. Elution was performed with 0.3 M NH4HCO3 (pH 10.5). Eluted peptides were dried and reconstituted in 0.1% TFA before LC-MS/MS analysis RPLC Analysis under Normal pH Buffer Conditions and Low pH Buffer Conditions. Normal pH Buffer Conditions. For normal pH mobile-phase solutions, buffer A (5% CH3CN and 0.1% formic acid) and buffer B (80% CH3CN and 0.1% formic acid) were used. Low pH Buffer Conditions. For low pH mobile-phase solutions, buffer A (5% CH3CN and 3% formic acid) and buffer B (80% CH3CN and 3% formic acid) were used. Peptides were loaded onto a fused-silica capillary column (100-µm i.d., 360-µm o.d.) containing 7.5 cm of Aqua C18 reversed-phase column material (Phenomenex). The following gradient method was used for the separation of peptide mixtures over a period of 85 min: 0-10% B in 1 min; 10-60% B in 50 min; 60-100% B in 10 min; then 100% B hold for 15 min. Peptides were eluted at a flow rate of 250 nL/min provided across a flow splitter by the HPLC pumps. Mass Spectrometry. Analyses of peptide samples were performed using an Agilent 1100 Series HPLC pump (Agilent Technologies) coupled with a quadrupole ion trap mass spectrometer (LCQ Deca XP Plus, Thermo Finnigan, San Jose, CA) or a linear quadrupole ion trap mass spectrometer (LTQ, Thermo Finnigan) using an in-house-built nano ESI interface. In order to identify the eluting peptides, the ion trap mass spectrometer was operated in a data-dependent MS/MS mode (m/z 400-1400), in which a full MS scan was followed by 10 MS/MS scans (LTQ) or (30) Bodenmiller, B.; Mueller, L. N.; Mueller, M.; Domon, B.; Aebersold, R. Nat. Methods 2007, 4, 231-237.

3 MS/MS scans (LCQ). The temperature of the heated capillary was 200 °C, and 2.3 kV of electrospray voltage was applied via a gold electrode. The dynamic exclusion function of the Xcalibur software was employed to minimize redundant MS/MS data acquisition for identical peptides. Normalized collision energy of 35% was used to generate MS/MS spectra. Mass Spectrometric Data Analysis. MS/MS spectra that were obtained in the LC-MS/MS analyses were searched against an in-house protein database containing the bovine R- and β-casein sequence or a composite protein database containing the mouse IPI protein database (v.3.28) and its reversed complement using TurboSequest or Sequest Sorcerer Solo (SageN). The differential modification search option for phosphorylation modification (+80 on Ser, Thr, Tyr) and oxidation (+16 on Met) was considered in the search, and the maximum number of modifications that were allowed per peptide was seven (the maximum number of modification per type was 5). Scaffold (Proteomesoftware, version 1.6) was used to filter the search results, and the following Xcorr values were applied to fully tryptic peptides based on the charge state: 2.0 for singly charged peptides, 2.0 for doubly charged peptides, 3.0 for triply charged peptides, and 0.0 for ∆ Cn. Somewhat less stringent criteria were used in the Sequest score filtering process since phosphopeptides tend to show low Xcorr scores due to extensive neutral loss of phosphate group. A false positive rate of these filtering criteria was estimated to be over 10%, thus manual validation of all MS/MS spectra assigned as phosphopeptides was followed to confirm the search results and to remove incorrect assignments. The presence of neutral loss of phosphoric acid (M + H - 98 for singly charged, M + 2H - 49 Da for doubly charged, and M + 3H - 32.6 for triply charged peptides) was used first as a way of confirming Ser- and Thr-phosphorylated peptides and major fragment ions (b and y series ions, N-Terminal side fragment ions of Pro-containing peptides, neutral loss ion of oxidized Met-containing peptides) of the phosphorylated peptide sequences were then manually assigned and checked for their intensities. The requirement of neutral loss of phosphoric acid was not applied to Tyr phosphorylated peptides. Manual validations appeared to be necessary in the phosphopeptide analysis because the number of MS/MS spectra removed after manual validations was always higher than those calculated from estimated false positive rates. For example, 26% of filtered MS/MS spectra were actually removed in a 12.5% false positive rate phosphopeptide search case (10 reverse sequence phosphopeptide matches out of 159 total phosphopeptide matches). Construction of Selected Ion Chromatograms. XCalibur v.2.0 was used to construct selected ion chromatograms of phosphopeptides. Peptide ions in a range of m/z 400-1400 were scanned, and mass window was set at 500 ppm. A peak smoothing feature using Gaussian function was employed prior to the peak integration process. The peak area of each peptide was calculated with the built-in peak area integration feature of Xcalibur v.2.0. Each peak in the selected ion chromatogram was manually confirmed by the presence of corresponding tandem mass spectra and residual peaks resulting from the background noises in the MS scan were removed for clarity. RESULTS Previously, various mass spectrometric phosphopeptide analysis techniques have been tested using one of the model phosAnalytical Chemistry, Vol. 80, No. 8, April 15, 2008

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Table 1. Comparisons of Tandem Mass Spectrometry Database Search Score of Bovine β-Casein Mono- and Tetraphosphorylated Peptides with Various Loading Amounts under Normal (0.1% Formic Acid) and Low pH (3% Formic Acid) Elution Solvent Conditions (Instrument: LCQ Deca XP) tandem mass spectrometric database search score (SEQUEST Xcorr) monophosphorylated peptide FQpSEEQQQTEDELQDK bovine β-casein

tetraphosphorylated peptide RELEELNVPGEIVEpSLpSpSpSEESITR

2+

3+

3+

9.6 pmol 1 pmol 100 fmol 50 fmol

4.5 4.6 5.0 5.3

Low pH Condition 3.6 -

5 4.8 4.7 3.9

9.6 pmol 1 pmol 100 fmol

4.4 4.9 4.3

Normal pH Condition -

4.7 nd nd

phoproteins, bovine R- and β-casein, to measure detection limits. In this study, we compared two different pH conditions (0.1% formic acid and 3% formic acid containing elution solvents) for RPLC-MS/MS analysis to identify any changes in the level of detection of phosphorylated β-casein peptides. We varied the amounts of trypsin-digested β-casein that were loaded, and the tandem mass spectrometric database search score was monitored for phosphorylated peptides (mono- and tetraphosphorylated) under two different pH conditions.(Table 1) As previously reported, the detection limit of β-casein tetraphosphorylated peptide under normal pH conditions (conventional method) was ∼10 pmol. Changes in the pH condition from 2.9 to 1.7 significantly improved the detection limit of the multiphosphorylated peptide. Indeed,

the tetraphosphorylated peptide was detected when only 50 fmol was loaded under low pH conditions, which is 200 times less than the amount of peptide that is necessary for detection under normal pH conditions. The detection limit of monophosphorylated peptide was also slightly enhanced under low pH conditions. pH condition lower than pH 1.7 were tested using higher concentrations of formic acid; however, under these conditions, the detection limit of β-casein phosphopeptides was not improved.(data not shown) Figure 1 shows a base peak chromatogram of 50 fmol of bovine β-casein tryptic digests that were obtained by RPLC-MS/MS analysis under low pH conditions (inset) and a manually assigned tandem mass spectrum of β-casein tetraphosphorylated peptide. Characteristic neutral losses of phosphoric acid from fragment ions and the parent ion were evident in the MS/MS spectra. Since the improved detection limit of β-casein tetraphosphorylated peptide in low pH conditions can be interpreted as increased ionization efficiencies, the ion intensities of phosphorylated and unmodified peptides under two different pH conditions were examined. Instead of a direct ion intensity comparison, selected ion chromatograms of individual peptide ions were constructed for the elution profiles and the peak areas were calculated.(Table 2) Tryptic digest peptides of R-casein, a major contaminant of β-casein, were also analyzed and included in the comparison of ionization efficiencies. While tetraphosphorylated β-casein peptides exhibited greater peak areas in the low pH analysis, nearly all of the monophosphorylated and unmodified β-casein peptides showed similar or greater peak areas in the normal pH analysis. In order to explain the improved ionization efficiencies of β-casein tetraphosphorylated peptides under low pH conditions, elution profiles of β-casein tryptic digest peptides were compared using selected ion chromatograms. Interestingly, tetraphosphorylated peptides under normal pH conditions showed an unusual elution profile compared to tetraphosphorylated peptides under low pH conditions.(Figure 2) The elution of

Figure 1. MS/MS spectrum of tetraphosphorylated peptide obtained from a loading amount of 50 fmol of bovine β-casein tryptic digests in the RPLC-MS/MS analysis. Fragment ions were manually assigned with a mass tolerance of (0.5 Da. Inset shows the base peak ion chromatogram obtained in the LC-MS/MS analysis and the retention time of the β-casein tetraphosphorylated peptide. 3010

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Table 2. Comparisons of Integrated Peak Areas (× 106) of Phosphopeptides and Unmodified Peptides under Low pH and Normal pH Conditionsa low pH

normal pH

176.9 (53.99) 22.0 (3.20) 2.3 (0.27)

537.0 (18.27) 63.0 (10.42) 9.5 (0.96)

435.6 (124.17) 48.4 (5.24) 2.2 (0.96) 0.59 (0.033)

55.1 (16.98) 1.6 (0.37) not detected not detected

17.2 (8.51) 2.5 (0.30) 0.25 (0.042)

33.7 (4.88) 3.4 (0.39) 0.45 (0.063)

25.0 (6.15) 1.6 (3.20) 0.16 (0.001)

60.3 (10.91) 3.5 (1.50) not detected

Phosphopeptide FQpSEEQQQTEDELQDK 9.6 pmol 1 pmol 100 fmol RELEELNVPGEIVEpSLpSpSpSEESITR 9.6 pmol 1 pmol 100 fmol 50 fmol VPQLEIVPNpSAEER (alpha casein S1) 9.6 pmol 1 pmol 100 fmol TVDMEpSTEVFTK (alpha casein S2) 9.6 pmol 1 pmol 100 fmol

Non-Phosphopeptide MHQPHQPLPPTVMFPPQSVLSLSQSK+3 9.6 pmol 15.4 (2.70) 1 pmol 1.8 (0.46) 100 fmol 0.14 (0.003) DMPIQAFLLYQEPVLGPVR+2 9.6 pmol 50.2 (2.74) 1 pmol 3.2 (0.87) 100 fmol 0.24 (0.007) HQGLPQEVLNENLLR+2 (R-casein S1) 9.6 pmol 16.3 (13.91) 1 pmol 2.5 (0.25) 100 fmol 0.12 (0.019) FPQYLQYLYQGPIVLNPWDQVK+2 (R-casein S2) 9.6 pmol 15.8 (3.77) 1 pmol 1.2 (0.32)

65.1 (3.06) 2.0 (1.10) Not detected 100.6 (9.42) 4.7 (1.55) 0.57 (0.144) 23.9 (2.37) 2.5 (0.70) 0.64 (0.176) 21.0 (3.02) 1.1 (0.33)

a Standard deviation is shown in parentheses. [Three replicate experiments on LTQ MS)

tetraphosphorylated peptide under normal pH conditions was extended over a broad range of retention time, which is very different from the conventional peptide elution that was observed in the RPLC separation. Tetraphosphorylated peptide under low pH conditions and other tryptic digest peptides from β-casein all showed typical peptide elution profiles. The elution profiles of monophosphorylated β-casein peptides under normal and low pH conditions are shown in Figure 3 for comparison purposes. The tendency for the improved ionization efficiency of multiphosphorylated peptides under low pH conditions was also confirmed in a separate RPLC-MS/MS analysis of bovine R-casein, another model phosphoprotein.(Table 3) A tryptic digestion of bovine R-casein generates phosphorylated peptides of various modification degrees: two monophosphorylated peptides, two diphosphorylated peptides, one triphosphorylated peptide, two tetraphosphorylated peptides, and one pentaphosphorylated peptide. Various amounts of bovine R-casein tryptic digests were analyzed under normal and low pH conditions. The tandem mass spectrometric database search score was monitored for identified R-casein phosphopeptides. We identified a total of six phosphopeptides (out of eight phosphopeptides) from the tryptic digestion of R-casein; phosphopeptides containing more than two phosphorylation sites exhibited better ionization efficiencies in low pH conditions. When the elution profiles of R-casein tetraphosphorylated peptides were compared in the two pH conditions, only NANEEEYSIGpSpSpSEEpSAEVATEEVK exhibited extended elution under normal pH conditions.(Figure 4)

In order to validate the use of low pH phosphopeptide analysis in an actual phosphoproteome analysis, a complex phosphopeptide mixture sample that was prepared from mouse brain whole tissue lysate was analyzed. A total of 50 µg of mouse brain protein was trypsinized to generate peptide mixtures, and then a one-step phosphopeptide enrichment procedure was performed using the phosphopeptide affinity column material, Titansphere (TiO2). In order to increase the phosphopeptide coverage of the sample, aliquots from the phosphopeptide-enriched sample were analyzed by RPLC-MS/MS at least twice under each pH condition, and the two best tandem mass spectrometric database search results of each pH condition were combined to make phosphopeptide/ protein lists. We applied simple and stringent criteria when completing the phosphopeptide lists in order to quickly compare the results between the two pH conditions. SEQUEST score-based filtering and manual validation was used. The SEQUEST score filtering was only applied to phosphopeptides with fully tryptic digest ends and the following Xcorr values for different charge state ions (singly charged, 1.8; doubly charged, 2.0; triply charged, 3.0; ∆ Cn, 0) were employed. Somewhat less stringent criteria were used in the filtering process since phosphorylated peptides usually exhibit lower Xcorr scores than unmodified counterparts. All the filtered MS/MS spectra were then manually validated with the requirements of having phosphate group neutral loss ions as the major ions (within 10% of base peak ion intensity) and matching major fragment ions (b or y series ions, N-terminal side preferential cleavage of Pro-containing case, and neural loss of oxidized Met containing case). The number of phosphopeptides/ proteins that were identified in the normal pH analysis was only 63/43; however, the number of phosphopeptides/proteins that were identified in the low pH analysis was 137/89.(Table 4) A complete list of the phosphopeptides that were identified in the two pH analyses is provided as Supporting Information.(SI; Table 1) More than 62% of the phosphopeptides that were identified in the normal pH analysis were redundantly identified in the low pH analysis. Most of the phosphopeptides that were identified contained only one modification site per peptide.(84.1% for normal pH and 84.7% for low pH analysis) The number of multiphosphorylated peptides with more than 2 modification sites was 10 (15.9% of total number of phosphopeptides) for normal pH analysis and 21 (15.3% of a total number of phosphopeptides) for low pH analysis. No more than three phosphorylation sites per peptide were observed in the normal pH analysis, but phosphopeptides with four or five modification sites were observed in the low pH analysis. DISCUSSION The mass spectrometric identification of multiphosphorylated peptides in positive ion mode has been a challenging task because the ionization efficiencies of these peptides are much lower than their unmodified counterparts.18,28,29 In this study, we have demonstrated that a simple pH change (from pH 2.9 to pH 1.7) in the elution solvents used in the RPLC-MS/MS analysis can effectively improve the detection of multiphosphorylated peptides of model phosphoproteins. Conventional RPLC separation uses a low concentration (0.1-0.4%) of formic acid to adjust the pH conditions of elution solvents; in this case, the pH ranges between 2.5 and 3. Most unmodified tryptic digest peptides maintain solution charge states of +2 at this pH range; however, the Analytical Chemistry, Vol. 80, No. 8, April 15, 2008

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Figure 2. Elution profiles of bovine β-casein tetraphosphorylated peptide under two different pH conditions. Three different loading amounts of bovine β-casein tryptic digests were analyzed by RPLC-MS/MS. (A) Selected ion chromatograms of m/z 1042.3 under low pH conditions, (B) Selected ion chromatograms of m/z 1042.3 under normal pH conditions.

solution charge state of the phosphorylated peptides is lower than +2 depending on the number of phosphorylation sites and basic amino acid residues.2 Some multiphosphorylated peptides with very low pI values (pI