Mapping of Phosphorylation Sites of Gel-Isolated Proteins by

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Anal. Chem. 1999, 71, 235-242

Mapping of Phosphorylation Sites of Gel-Isolated Proteins by Nanoelectrospray Tandem Mass Spectrometry: Potentials and Limitations Gitte Neubauer and Matthias Mann*

European Molecular Biology Laboratory, Meyerhofstrasse 1, Postfach 102209, 69012 Heidelberg, Germany

Precursor ion scans have proven to be extremely useful for the characterization of unseparated peptide mixtures. In conjunction with the nanoelectrospray source, precursor ion scans provide a sensitive tool for the detection of posttranslationally modified peptides and have been used to determine phosphorylation sites of proteins digested in solution. In this report, we extend our previous work to the determination of protein phosphorylation sites of gel-isolated proteins. The in-gel digestion procedure developed in our laboratory for protein microsequencing was found to be suitable for phosphorylation mapping as well. The risk of losing hydrophilic peptides in the desalting step was decreased by using column packing material designed for the purification of oligonucleotides and by adjusting the pH conditions to the needs of phosphopeptide analysis. With this method, the tryptic phosphopeptides of β-casein were detected after in-gel digestion at a sensitivity of 250 fmol of protein applied to the gel. The phosphorylation sites of two other proteins, Src-∆U and Op18, have similarly been mapped. Subpicomole to low-picomole amounts of protein starting material are needed in general, although we and others have reported attomole sensitivity for the detection of model phosphopeptides using precursor ion scans. This indicates that the success in determining phosphorylation sites depends crucially on the digestion, extraction, and detection efficiency for individual phosphopeptides. Many cellular functions are controlled by reversible protein phosphorylation.1-4 In budding yeast, 2% of all genes encode conventional kinases5senzymes which catalyze the attachment of phosphate groups to target proteinssreflecting the importance of this regulatory mechanism. The knowledge of the location of the phosphorylation site in the protein sequence provides insight into the mechanism of regulation and allows informed speculations to be made about the kinases and phosphatases involved.6,7 * Corresponding author. New address: Odense University, Campusvej 55, DK-5230 Odense M, Denmark. E-mail: [email protected]. (1) Krebs, E. G. Trends Biol. Sci. 1994, 19, 439. (2) Hunter, T. Cell 1995, 80, 225-236. (3) Sun, H.; Tonks, N. K. Trends Biol. Sci. 994, 19, 480-485. (4) Faux, M. C.; Scott, J. D. Trends Biol. Sci. 1996, 21, 312-315. (5) Hunter, T.; Plowman, G. D. Trends Biol. Sci. 1997, 22, 18-22. (6) Kennelly, P. J.; Krebs, E. G. J. Biol. Chem. 1991, 266, 15555-15558. (7) Songyang, Z.; Cantley, L. C. Trends Biol. Sci. 1995, 20, 470-475. 10.1021/ac9804902 CCC: $18.00 Published on Web 12/01/1998

© 1998 American Chemical Society

Mass spectrometry is a rapid, sensitive, and nearly universal tool for the determination of posttranslational modifications. In the case of protein phosphorylation sites, several different approaches have been pursued in the past few years, using mass spectrometric methods either alone (see below) or in combination with other methods, such as Edman degradation.8-12 Several researchers reported the determination of phosphorylation sites through the isolation of individual peptides by HPLC, followed by mass spectrometric sequencing of the fractions off-line.13,14 Phosphopeptides in this case were not specifically detected but rather identified by their 80-Da mass difference compared to peptides expected from the sequence. A more specific identification of phosphopeptides utilizes the specificity of phosphatases: MALDI peptide mapping before and after phosphatase treatment detects phosphopeptides by the mass shift of 80 Da due to the removal of a phosphate moiety.15-17 Combining the enzyme specificity of protein tyrosine phosphatase with electrospray mass spectrometry (ESMS), Amankwa et al. demonstrated detection of tyrosine phosphorylated peptides by using an on-line reactor (with immobilized phosphatase) coupled to either capillary electrophoresis electrospray mass spectrometry (CE/ESMS) or liquid chromatography electrospray mass spectrometry (LC/ESMS).18 The 80-Da difference and the difference in retention times between the enzyme-treated and untreated sample identifies the masses of the phosphopeptides. Another on-line-ES approach utilizes the specific binding of phosphopeptides to an iron-chelating column (8) Palczewski, K.; Buczylko, J.; Hooser, P. v.; Carr, S. A.; Huddleston, M.; Crabb, J. W. J. Biol. Chem. 1992, 267, 18991-18998. (9) Taniguchi, H.; Suzuki, M.; Manenti, S.; Titani, K. J. Biol. Chem. 1994, 269, 22481-22484. (10) Lombardo, C. R.; Consler, T. G.; Kassel, D. B. Biochemistry 1995, 34, 16456-16466. (11) Gold, M. R.; Yungwirth, T.; Sutherland, C. L.; Ingham, R. J.; Vianzon, D.; Chiu, R.; Oostveen, I. v.; Morrison, H. D.; Aebersold, R. Electrophoresis 1994, 15, 441-453. (12) Palm, L.; Andersen, J.; Rahbek-Nielsen, H.; Hansen, T. S.; Kristiansen, K.; Hojrup, P. J. Biol. Chem. 1995, 270, 6000-6005. (13) Resing, K. A.; Johnson, R. S.; Walsh, K. A. Biochemistry 1995, 34, 94779487. (14) Ladner, R. D.; Carr, S. A.; Huddleston, M. J.; McNulty, D. E.; Caradonna, S. J. J. Biol. Chem. 1996, 271, 7752-7757. (15) Liao, P.-C.; Leykam, J.; Andrews, P. C.; Gage, D. A.; Allison, J. Anal. Biochem. 1994, 219, 9-20. (16) Yip, T.-T.; Hutchens, T. W. FEBS Lett. 1992, 308, 149-153. (17) 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. (18) Amankwa, L. N.; Harder, K.; Jirik, F.; Aebersold, R. Protein Sci. 1995, 4, 113-125.

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(immobilized metal affinity column, IMAC). The IMAC column is either coupled directly to ESMS19 or coupled via a microLC column.20,21 An elegant mass spectrometric method for the selective detection of phosphopeptides in digest mixtures by LCESMS was reported by Carr’s group22 and later also by Ding et al.:23 analogous to their earlier work on the detection of glycopeptides,24 this method takes advantage of the generation of a specific diagnostic fragment ion for a given modification (m/z 79, PO3- for phosphopeptides) upon collision-induced dissociation (CID) in the orifice or skimmer region of the mass spectrometer.22 Later, Carr’s group applied LC/MS/MS to identify fractions containing phosphopeptides (by single-ion monitoring of m/z 79) and then used nanoelectrospay to confirm by precursor ion scans (see below) the presence of the phosphopeptides and, in some cases, to sequence the peptides by product ion scans.25 The loss of a diagnostic fragment in MALDI ion trap MS has also been used for the identification of phosphopeptides in unseparated digest mixtures.26 In this case, a difference of 98 ( 1 Da between two peaks is indicative of a phosphopeptide, which is due to the loss of either H3PO4 (∆ ) -98 Da) and/or the consecutive losses of HPO3 and NH3/H2O (∆ ) -97/98 Da). A tandem MALDI ion trap MS experiment can then be used for verification of the phosphopeptide identification.26 Recently, we combined the excellent selectivity of the precursor ion scan for PO3- with the high sensitivity provided by the nanoelectrospray source27,28 for the detection of phosphopeptides from unseparated digest mixtures.29-31 We29 and later others32 reported low femtomole or even attomole detection sensitivities when this method was applied to model phosphopeptides. The high selectivity and sensitivity of this method suggests its value for the determination of protein phosphorylation sites of proteins isolated from biological sources. Since gel electrophoresis is the most common method for the separation of proteins in biochemistry, it is desirable to combine gel electrophoresis with such a sensitive detection method. Previously, several groups have been able to determine phosphorylation sites of gel-isolated proteins by mass spectrometry either after digestion in gel12,33,34 or after transfer to a blot.17,35 (19) Nuwaysir, L. M.; Stults, J. T. J. Am. Soc. Mass Spectrom. 1993, 4, 662-669. (20) Russo, P.; Falchetto, R.; Hendrickson, R.; Smith, G.; Shabanowitz, J.; Hunt, D. Proceedings of the 44th ASMS Conference, Portland, OR, May 12-16, 1996; p 1084. (21) Watts, J. D.; Affolter, M.; Krebs, D. L.; Wange, R. L.; Samelson, L. E.; Aebersold, R. J. Biol. Chem. 1994, 269, 29520-29529. (22) Huddleston, M. J.; Annan, R. S.; Bean, M. T.; Carr, S. A. J. Am. Soc. Mass Spectrom. 1993, 4, 710-717. (23) Ding, J.; Burkhart, W.; Kassel, D. B. Rapid Commun. Mass Spectrom. 1994, 8, 94-98. (24) Carr, S. A.; Huddleston, M. J.; Bean, M. F. Protein Sci. 1993, 2, 183-196. (25) Verma, R.; Annan, R.; Huddleston, M.; Carr, S.; Reynard, G.; Deshaies, R. Science 1997, 278, 455-460. (26) Qin, J.; Chait, B. Anal. Chem. 1997, 69, 4002-4009. (27) Wilm, M. S.; Mann, M. Int. J. Mass Spectrom. Ion Processes 1994, 136, 167180. (28) Wilm, M.; Mann, M. Anal. Chem. 1996, 66, 1-8. (29) Wilm, M.; Neubauer, G.; Mann, M. Anal. Chem. 1996, 68, 527-533. (30) Weijland, A.; Neubauer, G.; Courtneidge, S. A.; Mann, M.; Wierenga, R. K.; Superti-Furga, G. Eur. J. Biochem. 1996, 240, 756-764. (31) Weijland, A.; Williams, J. C.; Neubauer, G.; Courtneidge, S. A.; Wierenga, R. K.; Superti-Furga, G. Proc. Natl. Ac. Sci. U.S.A. 1997, 94, 3590-3595. (32) Carr, S. A.; Huddleston, M. J.; Annan, R. S. Anal. Biochem. 1996, 239, 180192. (33) Betts, J. C.; Blackstock, W. P.; Ward, M. A.; Anderton, B. H. J. Biol. Chem. 1997, 272, 12922-12927.

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Betts et al. have used an additional IMAC step after in-gel digestion to specifically enrich the phosphopeptides before they were detected by precursor ion scans.33 However, the amounts of protein required for these different methods were usually at least tens of picomoles (applied to the gel), an amount which is generally not available for determining in vivo phosphorylation sites. During the preparation of this manuscript, Zhang et al. also reported on the issue of mapping phosphorylation sites of gelisolated proteins in the femtomole range.36 For the identification of phosphopeptides, the researchers employ comparative MALDI peptide mapping before and after phosphatase treatment of the protein digest. The phosphorylation site was then localized by an LC/MS/MS experiment using an ion trap instrument. Here we extend our previous work of mapping phosphorylation sites using precursor ion scans on unseparated digest mixtures to gel-isolated phosphoproteins. In extension of the rapid and sensitive sequencing of gel-isolated proteins by nanoelectrospray,37,38 a rapid screen for phosphorylation sites in the same experiment would be highly desirable. We investigate here whether the in-gel digestion, peptide extraction, and desalting procedures developed in our laboratory for microsequencing28,39 are compatible with phosphorylation mapping of low picomole or subpicomole amounts of proteins applied to the gel. In addition to developing methods optimized for the detection of phophopeptides derived from in-gel digests, we discuss limitations of this and other methods for the mass spectrometric detection of phosphorylation sites. MATERIALS AND METHODS Protein Samples and Digestion in Solution. Chicken Src∆U (a 51-kDa protein) was expressed in Schizosaccharomyces pombe and purified as previously described.31 This construct lacks the N-terminal domain of the c-Src protein, starting with a methionine residue followed by Ala 81. The protein was purified using two Q-Sepharose columns. One of the fractions was applied to a one-dimensional gel and stained with Coomassie Brilliant Blue (Serva, Heidelberg). The amount applied to the gel was estimated to be approximately 10 pmol. Op18, a 17-kDa protein, was in vitro phosphorylated by cdc2 kinase. Briefly, 1 µL of Op18 (5 mg/mL) in PBS was incubated with 5 µL of BRB80 (80 mM K-Pipes (pH 6.8), 1 mM MgCl2), 1 µL of 50 mM MgCl2, 2 µL of cdc2 kinase (in 80 mM β-glycerophosphate, 5 mM MgCl2, 150 mM KCl, 0.5% NP40, 1 mM DTT, protease inhibitors), and 1 µL of ATP (10 mM). The kinase assay was carried out for 20 min at 20 °C. It was stopped by heating for 1 min to 95 °C. The supernatant (containing approximately 10 pmol of protein) was applied to a 2D polyacrylamide gel and stained with Coomassie Brilliant Blue. (34) Szczepanowska, J.; Zhang, X.; Herring, C. J.; Qin, J.; Korn, E. D.; Brzeska, H. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 8503-8508. (35) Zhang, W.; Czernik, A. J.; Yungwirth, T.; Aebersold, R.; Chait, B. T. Protein Sci. 1994, 3, 677-686. (36) Zhang, X.; Herring, C.; Romano, P.; Szczepanowska, J.; Brzeska, H.; Hinnebusch, A.; Qin, J. Anal. Chem. 1998, 70, 2050-2059. (37) Wilm, M.; Shevchenko, A.; Houthaeve, T.; Breit, S.; Schweigerer, L.; Fotsis, T.; Mann, M. Nature 1996, 379, 466-469. (38) Shevchenko, A.; Jensen, O. N.; Podtelejnikov, A. V.; Sagliocco, F.; Wilm, M.; Vorm, O.; Mortensen, P.; Shevchenko, A.; Boucherie, H.; Mann, M. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 14440-14445. (39) Shevchenko, A.; Wilm, M.; Vorm, O.; Mann, M. Anal. Chem. 1996, 68, 850-858.

β-Casein was applied to a one-dimensional gel in differing amounts (1, 0.5, 0.25 and 0.1 pmol) and stained with either silver39 or Coomassie Brilliant Blue. The concentration of the stock solution was determined by amino acid analysis. A stock solution of tryptic digest of β-casein (Sigma) was prepared by digesting intact β-casein (1 µM in 0.1 M ammonium bicarbonate buffer) with unmodified trypsin (sequencing grade, Boehringer Mannheim) at an enzyme:substrate ratio of 1:50 at 37 °C overnight. In-Gel Digestion. All solvents used in this procedure and for mass spectrometry were HPLC grade (LabScan, Dublin, Ireland). The SDS-polyacrylamide gels were stained with either Coomassie Brilliant Blue or silver as previously described.39 The protein bands or spots were excised, washed, and digested with trypsin (Boehringer Mannheim, sequencing grade) and the resulting peptides extracted as described before,39 unless otherwise noted. Digests with Endoprotease Lys-C (Boehringer Mannheim, sequencing grade) were performed at twice the enzyme concentration compared to the tryptic digests (25 ng/µL instead of 12.5 ng/µL). The resulting peptide mixture was dried in a vacuum centrifuge and stored at -20 °C until use. Desalting of the Peptide Mixtures. “Desalting columns” were prepared and operated essentially as previously described,28 but different sorbent, washing, and elution conditions were used. About 100 nL of Poros oligoR3 sorbent (Perseptive Biosystems, Framingham, MA) was placed in a pulled glass capillary and equilibrated with 0.5% formic acid. The dried peptide mixture was reconstituted with 1 µL of 80% formic acid, which was quickly diluted with water to 10 µL. This solution was applied to the oligoR3 desalting column and washed with 3 × 3-4 µL of 0.5% formic acid. After all the solvent was washed through the column, the peptides were eluted stepwise into spraying capillaries: the first elution was done with 20% methanol, the second with 50% methanol, and the third with 50% methanol and 5% ammonia, unless described otherwise. Each eluate was analyzed individually. Mass Spectrometry. All experiments were carried out as previously described28,29 on an API III triple-quadrupole instrument (PE-Sciex, Ontario, Canada) with a nanoelectrospray source installed. Q1 scans were acquired with 0.1-Da step width and a dwell time of 1 ms, with unit resolution. In MS/MS mode, the step width was 0.2 Da and the dwell time 3 ms. The resolution was set to allow transmission of a mass window of 3-4 Da. The collision gas used was argon at a collision gas thickness of (2.83.0) × 1014 molecules/cm2, and the collision energy setting for precursor ion scans was 50 V (difference between R0 and R2 potentials on the Sciex API III instrument). For product ion scans, the collision energy was adjusted for each experiment individually. RESULTS AND DISCUSSION In this study, we investigated the usefulness of precursor ion scans in combination with the nanoelectrospray source for the sensitive determination of phosphorylation sites of gel-isolated proteins. The in-gel digestion, extraction, and desalting procedures developed for conventional microsequencing by nanoelectrospray MS/MS28,39 were examined for their compatibility with phosphorylation mapping using a precursor ion scan of m/z 79 in negative ion mode (Figure 1). A combined approach of protein identification and mapping of at least the most abundant phos-

Figure 1. Strategy for phosphorylation mapping of gel-separated proteins. The procedure is, in principle, identical to the sample preparation for microsequencing by nanoelectrospray MS/MS. Boxed in gray are the steps which determine the success of the phosphopeptide analysis: the digestion efficiency, the loss which could occur during the desalting step, and the detection efficiency of the phosphopeptides by nanoelectrospray MS.

phorylation sites of the identified protein in the same experiment would be highly desirable, considering the enormous biological significance of protein phosphorylation. The success of a phosphorylation mapping experiment after in-gel digestion dependssapart from the amount of phosphoproteinson several steps in the sample preparation, as illustrated in Figure 1. In the digestion step, the phosphopeptides have to be generated with high efficiency at a suitable size for mass spectrometric analysis. The extraction of the peptides created should not pose an additional problem, since phosphopeptides are more hydrophillic in comparison to their nonphosphorylated counterparts and, therefore, should be extracted even more readily. The peptides then have to be retained on the sorbent used in the desalting step. The detection of the phosphopeptides by precursor ion scans is dependent on the pH of the spraying solution and on the size of the peptides. β-Casein was used as phosphoprotein for most model experiments. It is a well-characterized 24-kDa protein with five phosphorylation sites, of which four are found in the tryptic peptide T1+2 (residues 1-25, with phosphorylation sites at Ser 17, 18, 19 and 22; MW 3123) and the remaining one in peptide T6 (residues 33-48, phosphorylated at serine 35; MW 2062). Digestion with Lys-C also produces two phosphopeptides; L1 (residues 1-28, MW 3478) and L4 (residues 33-48, MW 2062; identical to T6). In-Gel Digestion Procedure. Both Coomassie- and silverstained gels were processed and analyzed in the course of this study. At the same amount of protein applied to the gel (1-10 pmol β-casein), there was no noticeable difference in chemical noise or sensitivity observed for the detection of phosphopeptides in precursor ion scan mode. Therefore, both staining methods were used, depending on the amount of material applied to the gel. By comparing different washing, digestion, and extraction procedures, it was found that the in-gel digestion procedure we use routinely in our laboratory for sample preparation for microsequencing by nanoelectrospray MS/MS28,39 is suitable for phosphopeptide analysis. Reduction and carboxymethylation, basic extraction, or a combination of basic and acidic extraction did not influence the chemical background or the detection sensitivity significantly. An increase in background in the lowm/z area of the precursor ion scan for the phosphopeptide reporter ion (m/z 79) was observed only when the washing procedure before in-gel digestion was altered to several changes of 50% acetonitrile (data not shown). Digestion Efficiency. In-gel digestion with Lys-C was found to be more efficient for the recovery of the phosphopeptides of β-casein than trypsin (as was also noted by R. Annan, personal Analytical Chemistry, Vol. 71, No. 1, January 1, 1999

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Figure 2. Capillary holder for desalting of peptides after in-gel digestion. (A) Schematic drawing of the holder. The column capillary is filled with Poros perfusion material. During loading and washing of the peptide mixture, an R2 column can be aligned with an R3 column; the smaller or more hydrophilic peptides which are washed through the R2 column are then trapped by the more hydrophobic R3 column. For elution, both columns are aligned separately with the spraying capillary of the nanoelectrospray source.

communication). Interestingly, it seemed that both peptides could be recovered and detected at a higher sensitivity after Lys-C digestion compared with trypsin. For the larger peptide, carrying four phosphate groups, the Lys-C cleavage site is farther away from the phosphorylated residues and might, therefore, be cleaved more efficiently. At 250 fmol of β-casein applied to the gel, the tryptic phosphopeptide T6 is still detected with an adequate signal-to-noise ratio, while the T1+2 peptide can hardly be distinguished from the noise (data not shown). Digestion of 250 fmol of β-casein in-gel with Lys-C yielded good signal-to-noise ratios for both peptides (Figure 3). When only 100 fmol of β-casein was applied to the gel and then digested with Lys-C, only the smaller peptide (L4) could be detected with a good signal-to-noise ratio. The detection of the larger peptide is probably obscured by strong salt adduct formation and the presence of more charge states; i.e., the ion current for this peptide is split over many peaks, and therefore the signal intensity for each one is lower. After digestion of 100 fmol of β-casein with trypsin, none of the phosphopeptides were detected. Since trypsin is advantageous for microsequencing by tandem mass spectrometry (there are rarely internal arginines and there is a positive charge at the C-terminus), this would still be the first choice for detection and sequencing of phosphopeptides after ingel digestion. Depending on the protein sequence, different enzymes might be preferable, as shown here for the case of Lys-C and β-casein. Desalting of the Peptides after In-Gel Digestion. When we started to investigate the analysis of phosphorylation sites of gel-separated proteins at low picomole levels, one of the first problems encountered was the loss of phosphopeptides in the desalting step. This problem can be related to two effects. Small tryptic phosphopeptides can be very hydrophilic because of their extra charge and can, therefore, fail to be retained on the reversedphase material used in peptide purification. Large phosphospeptides can be hydrophobic and fail to be desorbed from the column material. A tryptic digest of β-casein in solution was used for evaluating the peptide desalting procedure. For microsequencing of gel-isolated proteins, desalting usually employs Poros R2 238 Analytical Chemistry, Vol. 71, No. 1, January 1, 1999

Figure 3. Sensitivity of phosphorylation mapping of β-casein after one-dimensional SDS-PAGE. Here, 250 fmol of intact β-casein was applied to the gel. The silver-stained band was excised and in-gel digested with Lys-C. The peptides were stepwise eluted from the desalting oligoR3 column. (A) Elution with 50% methanol. Only the smaller L4 (residues 33-48) peptide was detected, even when ammonia was added to the spraying solution (data not shown). (B) Elution from the same desalting column with 50% methanol/5% ammonia. The strongly basic pH is crucial for elution of this large phosphopeptide from the oligo R3 column (see Table 1).

perfusion material.37 The desalting column is equilibrated, and peptides are washed with 5% methanol/5% formic acid solution. The peptides are then eluted in one step with 0.5-1 µL of 50% methanol/5% formic acid.28,39 Based on this method, different column materials and different solvents for loading, washing, and elution were tested. To avoid differing detection efficiencies due to different pH conditions of the analyte solution, two precursor ion scans were acquired for each experiment, the first one directly with the eluate from the column, and the second after addition of approximately 0.2 µL of ammonia (25%) to the spraying needle. Since the sensitivity of electrospray ionization for phosphopeptides depends strongly on the pH of the spraying solution (being best at basic pH29,32 and see below), the acid was omitted for the elution from the column. In a first experiment, the whole desalting procedure was carried out as described above, but the acid was omitted in all steps. After desalting of 1 pmol of the solution digest of β-casein, the smaller phosphopeptide (T6, 2062 Da; sequence F Q SP E E Q Q Q T E D E L Q D K) was not detected by a precursor ion scan for m/z 79 in negative ion mode, while the larger phosphopeptide (T1+2, 3123 Da; sequence R E L E E L N V P G E I V E SP L SP SP SP E E S I T R) could be detected, but only after addition

Table 1. Detection of Phosphopeptides of β-Casein by Parent Ion Scans of m/z 79 after Stepwise Elution from an OligoR3 Columna elution, percentage of methanol

loading

step 1, 20%

step 2, 50%

step 3, 80%

step 4, 50% + 5% NH4OH

5% methanol H2O 0.5% formic acid

-b nac -

T6

-

T1+2 T1+2 T1+2

a After each elution, a parent ion scan of m/z 79 was acquired first in neutral conditions and then in basic conditions. When elution is done in only one step with 50% methanol and 5% ammonia, both peptides are detected. b -, not detected in the eluate. c Data not available.

of 0.2 µL of ammonia to the spraying needle. In contrast, when the digest mixture of β-casein was diluted twice with 50% methanol and analyzed without micropurification, both peptides were observed with strong signal intensities after the pH was adjusted to basic conditions. This demonstrates that the smaller phosphopeptide was lost in the desalting stage. Effect of pH on the Retention and Elution of Phosphopeptides. A more hydrophobic chromatographic material (Poros oligoR3) was tested for its ability to retain the phosphopeptides of β-casein. The oligoR3 perfusion material was originally designed for purification of oligonucleotides and is more hydrophobic than the R2 material. However, using neutral conditions for loading washing and elution, the T6 peptide of β-casein was still lost during the desalting procedure. It was possible to retain both phosphopeptides of the tryptic β-casein digest only when the peptide mixture was loaded in acidic conditions (0.5% formic acid; see Table 1). At low pH, the acidic groups of the peptides (including the phosphate groups) are protonated and, therefore, more hydrophobic, i.e., more efficiently retained on the desalting column. The elution of the phosphopeptides from the oligoR3 desalting column was found to be strongly dependent on the pH: while the smaller, T6 peptide was detected in precursor ion scan mode after elution with 50% methanol in neutral conditions, the larger phosphopeptide could only be eluted from the column with 50% methanol in basic conditions (5% ammonia). In a stepwise elution series with 5%, 20%, 50%, and 80% methanol and finally with 50% methanol/5% ammonia, the phosphopeptides selectively elute at 50% methanol and 50% methanol/5% ammonia, respectively (Table 1). In general, it was found that the smaller and/or more hydrophillic peptides are retained by this method while also the larger, more hydrophobic ones (like the 4-kDa peptide of the Src∆U sample (see below)) can be eluted efficiently in basic pH. Double-Column Alignment. Since the oligoR3 perfusion material is more hydrophobic than R2, it is now used routinely in our laboratory for the desalting of phosphopeptides. It has also proven to be very useful generally for peptides which are too small or too hydrophilic in acidic conditions to be retained by a PorosR2 desalting column. A combination of the two columns is used in our laboratory for the sample preparation of peptide mixtures after in-gel digestion for protein microsequencing by tandem mass spectrometry. Two column capillaries are aligned in the capillary holder (Figure 2), the upper one filled with R2 and the lower one with R3 sorbent. During the loading and the washing steps, the

Figure 4. Application of the “double-column” technique. The peptide mixture obtained from in-gel digestion of a protein is applied to the R2 desalting column, which is aligned with the oligoR3 column during the loading and washing steps. Marked with asterisks are those ions which were fragmented and the sequence tags which were used to identify RPS4X (SwissProt P12750). (A) Precursor ion scan of m/z 86 for the detection of peptides in the R2 eluate. The most intense peaks correspond to the doubly charged ion of a peptide from RPS4X (sequence L S N I F V I G K, m/z 496.2) and to autodigestion products of trypsin, as indicated. (B) Precursor ion scan of m/z 86 of the eluate from the oligoR3 column. The trypsin autodigestion products which are prominent in the R2 eluate are hardly visible here, indicating that those peptides are retained well by the R2 column. The most intense peak in the spectrum corresponds to a doubly charged peptide from RPS4X (sequence I G V I T N R, m/z 387.2), which was not retained at all by the R2 column.

peptides which are too small or too hydrophobic to be retained by the R2 sorbent are trapped by the oligoR3 column. The peptides of each of the two columns can be eluted directly into a spraying capillary. As can be seen in Figure 4, the elution pattern from the two columns is strikingly different: while the larger peptides are eluted from the R2 column, some of the smaller ones would be lost by a single-step purification (for example, peptides at m/z 387 and 417 in Figure 4). We also found that digest mixtures can be stored at +4 °C for at least some hours adsorbed to a R2 or R3 column without any apparent loss of peptides. Application of This Method to Tyrosine Phosphorylation. Once the procedure was established with β-casein as a model protein, it was applied to a tyrosine phosphorylated protein, chicken Src-∆U, a protein overexpressed in the yeast S. pombe in structural studies of Src.30,31 This construct lacks the N-terminal unique domain of c-Src (starting with a methionine residue Analytical Chemistry, Vol. 71, No. 1, January 1, 1999

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followed by Ala 81 of the c-Src sequence). Src-∆U was purified over two Q-Sepharose columns as previously described.31 The fraction applied to the gel and analyzed here is heterogeneous in its phosphorylation. After in-gel digestion with trypsin, the resulting peptides were extracted and desalted using the oligoR3 sorbent in a capillary, as described above for β-casein. The peptides were eluted stepwise with 20% and 50% methanol and finally with 50% methanol/5% ammonia. Precursor ion scans of m/z 79 in negative mode were performed for each fraction. For the two neutral fractions, the mode was switched to positive after the phosphopeptides were detected and the phosphorylation sites localized by a product ion scan (data not shown). The pH was then raised by addition of approximately 0.2 µL of ammonia (5%), and the mode was switched back to negative for an additional precursor ion scan. For the first fraction (eluted in 20% methanol), one phosphopeptide (1303.2 Da) was detected in neutral pH, and the phosphorylation site was mapped to Tyr 416 (sequence L410 I E D N E YP T A R419) by a product ion scan in positive ion mode (data not shown). After the mode was switched back to negative and ammonia was added, a second precursor ion scan for m/z 79 was acquired, and a second, less abundant peptide was also detected (1686.7 Da) which fits in its mass only to a peptide containing the same phosphorylation site and one uncleaved tryptic site (sequence L410 I E D N E YP T A R Q G A K423, see Figure 5A). In the second eluate (50% methanol), three phosphopeptides were detected at neutral pH (1303.3, 1314.0, and 1799.3 Da). The 1303.3-Da peptide had already been sequenced in the first eluate and the phosphorylation site determined to be Tyr 416. The two other peptides were sequenced in positive ion mode and the phosphorylation sites determined (data not shown). Again, both peptides contain the same phosphorylation site at Tyr 436, the larger one containing an uncleaved tryptic site (1799.3 Da; sequence F424 P I K W T A P E A A L YP G R438). After both peptides were sequenced, the mode was switched back to negative, and after the pH was raised, a precursor ion scan was again acquired (Figure 5B). No additional peptides were detected. The final elution with 50% methanol/5% ammonia showed as the predominant peptide species a 4062.6-Da peptide, which corresponds to the very C-terminus of the protein containing the phosphorylated Tyr 527 residue (Figure 5 C). We also observed a minor phosphopeptide with mass 2654.0 Da, which could be assigned to a tryptic peptide (residues 322-342) carrying one phosphate group. Apart from the phosphorylation site contained in the 2654.0Da peptide, all other phosphorylation sites determined in this experiment had previously been observed in different fractions of Src-∆U after an additional Q-Sepharose column separation31 or when different constructs of c-Src expressed in S. pombe were analyzed.30 However, in previous experiments, the digestion was carried out in solution, and possibly for that reason no uncleaved tryptic peptides were observed. Importantly, none of the posphorylation sites determined after in solution digest were missed by our optimized in-gel method. This demonstrates the reliability of this method. So far, we have not had any proof for a missed phosphorylation site. Interestingly, also the very large phosphopeptide (4063 Da) was detected with good sensitivity, indicating that, in this case, the size the of phosphopeptide generated by 240 Analytical Chemistry, Vol. 71, No. 1, January 1, 1999

Figure 5. Analysis of a tyrosine phosphorylated protein (Src-∆U) after in-gel digestion with trypsin. The precursor ion scans of all three fractions were acquired in basic conditions. (A) Precursor ion scan of m/z 79 of the peptide mixture eluted with 20% methanol from an oligoR3 desalting column. Two phosphopeptides were observed after addition of ammonia to the spraying needle; both contain the same phosphorylation site, Tyr 416. (B) Phosphopeptide detection in the 50% methanol eluate from the same desalting column. Three phosphopeptides are detected. The phosphorylation sites of the 1314.0- and the 1799.3-Da peptides were both localized to Tyr 436. (C) Elution with 50% methanol/5% ammonia. The major phosphopeptide detected is the C-terminal peptide phosphorylated at Tyr 527 (4062.6 Da; sequence K501 D P E E R P T F E Y L Q A F L E D Y F T S T E P Q YP Q P G E N L533). A minor phosphopeptide was detected at 2654.0 Da, which could correspond to the tryptic peptide L322 V Q L Y A V V S E E P I Y I V T E Y M S K343, with one phosphate group attached either to a serine, threonine, or tyrosine residue.

in-gel digestion did not hinder efficient extraction. The greater obstacle for the detection of large phosphopeptides is a diminished detection efficiency by strong sodium adduct formation and a broad distribution of charge states, which is particularly strong for the L1 (and T1+2) peptide of β-casein (see above). Thus, the signal is, in this case, distributed over 5 (charge states) × 5 (sodium adducts at each charge state) channels (see Figure 3). The strong salt adduct formation can be reduced by methylating the acidic residues on the whole peptide mixture after digestion. However, this involves additional sample handling and, consequently, additional sample loss. Mapping Phosphorylation Sites after 2D Gel Electrophoresis. Using 2D gel electrophoresis for the separation of proteins has the advantage that, in principle, all phospho forms of a protein are separated. However, the loss of protein is higher

Figure 6. Phosphopeptide analysis of Op18. (A) 2D gel of Op18 after in vitro phosphorylation with cdc2 kinase. Two of the spots (Op18.1 and Op18.2) were cut out and the phosphorylation sites determined after in-gel digestion. B) Q1 scan of the peptide mixture of Op18.1 after elution from the R3 column with 50% methanol. Among other peptide ions, the doubly charged ions of the phosphopeptides are detected above the noise. (C) Precursor ion scan of m/z 79 of the same sample. The triply and doubly charged species of two phosphate containing peptides and their sodium adducts are observed. (D) Determination of the site of phosphorylation of the 1406-Da peptide. The mode was switched from negative to positive, and a product ion scan was acquired. The sequence of the peptide and the phosphorylation site could be determined by a clear Y′′ ion series (E29 S V P E F P L SP P P K40). (E) Determination of the site of modification of the 1479-Da peptide. A product ion scan again yielded a clear Y′′ ion series of the same peptide sequence, but carrying a phosphate containing modification instead of a phosphate at Ser 37. Marked with bullets are those peaks which belong to a Y′′ series with a phosphate group only (also in E). The predominant species are the Y′′ ions containing the modified phosphate group on Ser 37 (marked with asterisks). (F) Product ion scan of the doubly charged phosphorylated peptide (1406 Da) in the low-m/z range in negative ion mode. The two marker ions for phosphate are observed (m/z 79 and 97). (G) Product ion scan of the doubly charged modified peptide (1479 Da). Only one of the marker ions for phosphate (m/z 79, corresponding to PO3-) is observed, but not the ion for H2PO4-, indicating that the modification contains a phosphodiester bond. Instead, a fragment at m/z 170 is observed, corresponding to the phosphate plus modification.

for two-dimensional compared to one-dimensional gel electrophoresis. Analysis of in Vitro Phosphorylated Op18. In this experiment, the phosphorylation sites of Op18 were determined after in vitro phosphorylation with cdc2 kinase and separation of the different phosphorylation forms by two-dimensional SDS-PAGE (Figure 6 A). Op18 is a cytosolic phosphoprotein which is phosphorylated in response to a variety of extracellular signals (reviewed in ref 39). The spots were excised and individually digested in gel by Lys-C (in this case, Lys-C was chosen since the protein is very basic and trypsin might have created too small phosphopeptides). The resulting peptides were purified over an oligoR3 desalting capillary column, as described above. Only in the eluate with 50% methanol were two different phosphopeptides (1406.0 and 1479.2 Da) detected in precursor ion scan mode (Figure 6C). While the 1406.0-Da peptide corresponds in mass to a phosphorylated Lys-C peptide (residues 29-40), the 1479-

Da peptide could not be assigned to any phosphorylated peptide generated by digestion with Lys-C. The sequence of the 1406.0Da peptide was confirmed to be E29 S V P E F P L SP P P K,40 where the phosphorylation site could be localized to Ser 37 by a product ion scan of the doubly charged peptide ion (Figure 6D). Sequencing of the second peptide revealed the same sequence as for peptide 29-40, however, with a different modification on the site of phosphorylation: instead of the difference of 80 Da in the Y′′ ion series (the mass difference for a phosphogroup), a gap of 153 Da is observed, in addition to a weaker series of Y′′ ions corresponding to the “normal” phosphorylation on Ser 37 (Figure 6E). The mass difference of 153 Da is most likely due to a modification on the phosphate moiety, since a product ion scan in negative ion mode of the phosphorylated peptide (1406.0 Da) produced the two well-known fragments in the low-m/z region, (40) Sobel, A. Trends Biol. Sci. 1991, 16, 301-305.

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PO3- (m/z 79) and H2PO4- (m/z 97), while for the other modified peptide (1479.2 Da) only m/z 79 is observed, but not m/z 97 (Figure 6F and G, respectively). Instead, a fragment ion at m/z 170 is observed which could correspond to the intact modification, linked to the serine by a phosphodiester bond. Since the expected mass accuracy for this experiment is not expected to be lower than 0.5 Da, one can exclude that the modification is due to a glycerophosphate moiety, as was previously described for a pilin protein.41 The nature of the modification was not further investigated. The second, more acidic spot was analyzed in the same manner. The same peptides as for the first spot were found in the eluate with 50% methanol (1405.8 and 1479.2 Da); however, elution with 50% methanol/5% ammonia also yielded two major phosphopeptides, corresponding to a phosphorylated Lys-C fragment and a modified one, again with a mass difference of 153 Da compared to the unmodified peptide (1839.6 and 1912.8 Da, respectively, with the sequence R13A S G Q A F E L I L S P R S K,28 data not shown). The site of the modifications could not be localized in positive ion mode, probably because of low ionization efficiency due to the extremely basic pH of the spraying solution. Previously, the phosphorylation sites of Op18 were determined upon phorbol 12-myristate 13-acetate induction,17 where the same sites were identified but no other modifications were observed. CONCLUSION In this study, we investigated whether rapid protein identification of low levels of gel-isolated proteins can be combined with efficient screening of phosphorylation sites by nanoelectrospray MS/MS experiments. Using β-casein as a model, we demonstrate detection of both phosphopeptides when only 250 fmol of protein was applied to the gel. However, whether all phosphopeptides of any given phosphoprotein can be detected when subpiocomole or low-picomole amounts of protein are applied to a gel depends on the percentage of phosphorylation at each site and the digestion and detection efficiency of the particular phosphopeptides. For protein identification using nanoelectrospray MS/MS and the sequence tag approach, partial sequences of one or only a few peptides from anywhere in the sequence are required in order to unambiguously identify the protein in a database. For phosphorylation mapping, however, it is important to find one particular peptide. Even at a sequence coverage of 100%, one cannot exclude the possibility of missing one phosphorylation site which is occupied only to a small percentage. More than one digest with different enzymes might be necessary to generate with high efficiency a phosphopeptide in the right size range for detection by the precursor ion scan technique. Even though phosphopeptides as large as 4 kDa could be detected with good signal-tonoise ratio, the detection efficiency decreases with the number of charge states and the number of sodium adducts (as shown for the bigger peptide of β-casein). Which enzyme works best and how well a phosphopeptide can be detected depends, therefore, on the individual peptide/protein sequence and cannot always be predicted without some initial experiments. While attomole detection sensitivities for synthetic model peptides in solution are not difficult to obtain, the amount of protein required (41) Stimson, E.; Virji, M.; Barker, S.; Panico, M.; Blench, I.; Saunders: J.; Payne, G.; Moxon, E. R.; Dell, A.; Morris, H. R. Biochem. J. 1996, 316, 29-33.

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for phosphorylation mapping of gel-isolated proteins depends strongly on the individual protein. Possible drawbacks of any method operating with unseparated mixtures are two-fold. First, the dynamic range of the precursor ion scan of the mixture might not be sufficient to detect low-abundance phosphopeptides next to a number of highly abundant ones. In our experience, it is not a problem, however, to detect low occupancy of a phosphorylation site amidst high amounts of unphosphorylated peptides. In fact, in most cases the phosphopeptides are not detected in a normal Q1 scan. The second problem is overlaying peptides when sequencing of the phosphopeptide is required. This is particularly true for large proteins, i.e., over 100 kDa, with a high number of phosphorylation sites. While it is usually possible to assign fragmentation series of at least two peptides in the same MS/MS spectrum, a HPLC separation on- or off-line might be preferable to our approach of a rough fractionation of the peptide mixture only. However, any type of separation is costly in material. Furthermore, one advantage of our technique is that the pH can be optimized for the detection of the phosphopeptides in negative ion mode. Another advantage of our method compared to that, for example, of Zhang et al.34 is that there is sufficient time to find the charge state of the phosphopeptide which is best for fragmentation in positive mode and that the fragmentation parameters can be adjusted to each peptide individually. Furthermore, in the approach Zhang et al. use, an entire LC/MS/MS run needs to be performed in order to sequence one phosphopeptide only. Depending on the amount of material, in principle all phosphopeptides can be detected and sequenced in one experiment using the nanoelectrospray approach. In general, sequencing of a particular phosphopeptide can be a difficult task, and whether the phosphorylation site can be localized to one residue depends not only on the amount of material used but also on the sequence of the individual phosphopeptide. Our conclusion, therefore, is that high-throughput screening of phosphorylation sites is, at the moment, not feasible at the same speed and sensitivity as protein identification (at least with the methodology applied here). However, it is encouraging that, in model experiments, phosphorylation mapping was feasible when low-picomole to subpicomole amounts of protein were applied to the gel. These amounts should be sufficiently low to allow the determination of in vivo phosphorylation sites and are, in any case, at least an order of magnitude improvement over sensitivities reported in the literature for practical biochemical problem solving. Several projects are under way in our laboratory which involve the mapping of phosphorylation sites of gel-isolated proteins which were isolated after in vivo phosphorylation. ACKNOWLEDGMENT We thank the other members of the Protein and Peptide Group for fruitful discussions. Drs. O. N. Jensen and M. Wilm are gratefully acknowledged for critically reading the manuscript.

Received for review May 5, 1998. Accepted September 16, 1998. AC9804902