Characterization of Protein Phosphorylation by Mass Spectrometry

The Ludwig Institute for Cancer Research, Cruciform Building, Gower Street, London WC1E ... fore, is essential in many proteomic studies.1,2 Mass spec...
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Anal. Chem. 2003, 75, 3232-3243

Characterization of Protein Phosphorylation by Mass Spectrometry Using Immobilized Metal Ion Affinity Chromatography with On-Resin β-Elimination and Michael Addition Andrew J. Thompson,† Sarah R. Hart,†,‡ Clemens Franz,†,‡ Karin Barnouin,†,‡ Anne Ridley,†,‡ and Rainer Cramer*,†,‡

The Ludwig Institute for Cancer Research, Cruciform Building, Gower Street, London WC1E 6BT, United Kingdom, Courtauld Building, 91 Riding House Street, London W1W 7BS, United Kingdom, and Department of Biochemistry and Molecular Biology, University College London, Darwin Building, Gower Street, London WC1E 6BT, United Kingdom

A protocol combining immobilized metal ion affinity chromatography and β-elimination with concurrent Michael addition has been developed for enhanced analysis of protein phosphorylation. Immobilized metal ion affinity chromatography was initially used to enrich for phosphorylated peptides. β-Elimination, with or without concurrent Michael addition, was then subsequently used to simultaneously elute and derivatize phosphopeptides bound to the chromatography resin. Derivatization of the phosphate facilitated the precise determination of phosphorylation sites by MALDI-PSD/LIFT tandem mass spectrometry, avoiding complications due to ion suppression and phosphate lability in mass spectrometric analysis of phosphopeptides. Complementary use of immobilized metal ion affinity chromatography and β-elimination with concurrent Michael addition in this manner circumvented several inherent disadvantages of the individual methods. In particular, (i) the protocol discriminated O-linked glycosylated peptides from phosphopeptides prior to β-elimination/Michael addition and (ii) the elution of peptides from the chromatography resin as derivatized phosphopeptides distinguished them from unphosphorylated species that were also retained. The chemical derivatization of phosphopeptides greatly increased the information obtained during peptide sequencing by mass spectrometry. The combined protocol enabled the detection and sequencing of phosphopeptides from protein digests at low femtomole concentrations of initial sample and was employed to identify novel phosphorylation sites on the cell adhesion protein p120 catenin and the glycoprotein fetuin. Characterization of protein phosphorylation sites is critical to the understanding of protein function and regulation and, therefore, is essential in many proteomic studies.1,2 Mass spectrometry * Corresponding author address: The Ludwig Institute for Cancer Research, Mass Spectrometry and Bioanalytical Chemistry Laboratories, Cruciform Building, Gower Street, London WC1E 6BT, United Kingdom. Phone: +44 20 7679 6683. Fax: +44 20 7679 6687. E-mail:[email protected]. † The Ludwig Institute for Cancer Research. ‡ University College London.

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(MS) is a key tool in proteomics for the identification and characterization of proteins and their modifications, typically involving 2D gel electrophoresis and enzymatic digestion, culminating in peptide mass fingerprinting or sequencing by tandem mass spectrometry (MS/MS).3,4 However, the mass spectrometric identification specifically of phosphorylation sites is hindered by several factors, including (i) the typically substoichiometric population of phosphorylated, as compared to unphosphorylated, species; (ii) the intrinsic ionization suppression in positive ion mode by the anionic phosphate functionality; and (iii) the lability of the phosphate group under mass spectrometric conditions, resulting in facile neutral losses of 98 and 80 Da during both MS and MS/MS analysis. Methods of phosphopeptide enrichment and separation prior to analysis can significantly improve mass spectrometric characterization of phosphorylation sites.5 Immobilized metal ion affinity chromatography (IMAC) using iron(III) or gallium(III) is one such method.6,7 IMAC employs a stationary phase with a binding affinity for phosphates capable of enriching phosphorylated peptides from a profusion of unphosphorylated species. Although IMAC significantly deconvolutes complex mixtures, some unphosphorylated peptides are also retained. In particular, peptides rich in acidic residues are frequently observed.7-10 Methylation of peptides in methanolic hydrochloric acid prior to IMAC subfractionation reportedly increases the specificity of the method for phosphopeptides by esterifying the carboxylic acid functionalities.8 The (1) Hunter, T. Cell 2000, 100, 113-127. (2) Cohen, P. Trends Biochem. Sci. 2000, 25, 596-601. (3) Yates, J. R., III J. Mass Spectrom. 1998, 33, 1-19. (4) Naaby-Hansen, S.; Waterfield, M. D.; Cramer, R. Trends Pharmacol. Sci. 2001, 22, 376-384. (5) Gronborg, M.; Kristiansen, T. Z.; Stensballe, A.; Andersen, J. S.; Ohara, O.; Mann, M.; Jensen, O. N.; Pandey, A. Mol. Cell. Proteomics 2002, 1, 517527. (6) Andersson, L.; Porath, J. Anal. Biochem. 1986, 154, 250-254. (7) Posewitz, M. C.; Tempst, P. Anal. Chem. 1999, 71, 2883-2892. (8) 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. (9) Muszynska, G.; Dobrowolska, G.; Medin, A.; Ekman, P.; Porath, J. O. J. Chromatogr. 1992, 604, 19-28. (10) Schlosser, A.; Bodem, J.; Bossemeyer, D.; Grummt, I.; Lehmann, W. D. Proteomics 2002, 2, 911-918. 10.1021/ac034134h CCC: $25.00

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methylation procedure is specific and high-yielding, and no byproducts have been reported, despite the harsh reaction conditions. When fully optimized and characterized, methylation promises to be a significant complementary step to improve IMAC selectivity of phosphopeptides. However, tyrosine, histidine, and tryptophan residues also contribute to IMAC binding of unphosphorylated peptides,9-11 which may require an additional strategy to address. IMAC also shows pronounced differential recovery of phosphopeptides, as seen in the poor recovery of multiply phosphorylated species.7,12,13 In addition to its dependence on loading, washing, and elution conditions, the recovery of some phosphopeptides can be reportedly improved through substitution of iron(III) with gallium(III),7 although arguably at the cost of reduced retention.13,14 These and other factors, including the initial binding affinity, make IMAC a complex technique that requires further development and optimization in order to establish it as a simple but powerful affinity purification method for selective phosphopeptide enrichment. Elution of phosphorylated peptides from IMAC resin should be improved by removing the IMAC-bound phosphate(s) directly from the phosphopeptide, releasing the peptide into solution. One possibility is to use phosphatases to elute the phosphopeptides, but enzymatic dephosphorylation will leave the site of phosphorylation indistinguishable from other serine, threonine, or tyrosine residues. Another approach is the direct β-elimination of phosphopeptides from the IMAC resin. β-Elimination can occur at phosphoserine and phosphothreonine residues, and their respective conversions to dehydroalanine and dehydro-2-aminobutyric acid enables the precise determination of the phosphorylation site. Alkaline-mediated β-elimination forms the basis for a chemical modification strategy to address both the intrinsic ionization suppression of phosphorylated peptides and the lability of the phosphate moiety. Furthermore, this strategy provides a basis for specific chemical tagging of phosphorylation sites (Figure 1). β-Elimination coupled to Michael addition can be used to derivatize phosphopeptides substitutively at the phosphorylated residue15,16 and has recently been employed to derivatize phosphopeptides with biotin linkers to enable selective phosphopeptide isolation with streptavidin.17,18 The phosphoprotein isotope-coded affinity tag (PhIAT) approach extends this procedure by using an isotopecoded ethanedithiol linker to quantitate levels of protein phosphorylation19 in a fashion similar to isotope-coded affinity tag (ICAT) quantitation of protein expression.20 Simpler methods employ derivatizing the dehydrated products with ethanethiol to (11) Neville, D. C. A.; Rozanas, C. R.; Price, E. M.; Gruis, D. B.; Verkman, A. S.; Townsend, R. R. Protein Sci. 1997, 6, 2436-2445. (12) Hart, S. R.; Waterfield, M. D.; Burlingame, A. L.; Cramer, R. J. Am. Soc. Mass Spectrom. 2002, 13, 1042-1051. (13) Zhou, W.; Merrick, B. A.; Khaledi, M. G.; Tomer, K. B. J. Am. Soc. Mass Spectrom. 2000, 11, 273-282. (14) Stensballe, A.; Andersen, S.; Jensen, O. N. Proteomics 2001, 1, 207-222. (15) Clarke, R. C.; Dijkstra, J. Int. J. Biochem. 1980, 11, 577-585. (16) Meyer, H. E.; Hoffmann-Posorske, E.; Korte, H.; Heilmeyer, L. M., Jr. FEBS Lett. 1986, 204, 61-66. (17) Adamczyk, M.; Gebler, J. C.; Wu, J. Rapid Commun. Mass Spectrom. 2001, 15, 1481-1488. (18) Oda, Y.; Nagasu, T.; Chait, B. T. Nat. Biotechnol. 2001, 19, 379-382. (19) Goshe, M. B.; Conrads, T. P.; Panisko, E. A.; Angell, N. H.; Veenstra, T. D.; Smith, R. D. Anal. Chem. 2001, 73, 2578-2586. (20) Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R. Nat. Biotechnol. 1999, 17, 994-99.

Figure 1. A general schematic for β-elimination and Michael addition for phosphoserine (X ) H)- and phosphothreonine (X ) CH3)containing peptides. Treatment with base liberates the free phosphate generating dehydroalanine or dehydro-2-aminobutyric acid for phosphoserine and phosphothreonine, respectively. Dehydrated amino acids are excellent targets for nucleophilic attack. Thiols with designed tags can further derivatize phosphorylation sites with a variety of physicochemical properties.

facilitate positive ion formation and tryptic enzymatic digestion21 or with dual thiol mixtures to identify phosphorylated peptides in a single experiment by the observed mass difference.22 However, β-elimination and Michael addition also occur at cysteine and glycosylated serine and threonine residues. Although cysteines can be stabilized to the elimination reaction by alkylation with iodoacetamide or similar reagents, O-linked carbohydrates remain problematic, rendering the method unspecific for phosphorylation sites alone. (21) Jaffe, H.; Veeranna; Pant, H. C. Biochemistry 1998, 37, 16211-16224. (22) Molloy, M. P.; Andrews, P. C. Anal. Chem. 2001, 73, 5387-5394.

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Both the IMAC and β-elimination strategies suffer individual disadvantages that can be overcome by their complementary application. Directly eluting peptides from IMAC resin by β-elimination potentially improves the recovery of phosphopeptides, particularly multiply phosphorylated species, and preserves the phosphorylation site information. In combination with concurrent Michael addition, phosphorylated residues can be further derivatized with a variety of purposely designed tags. Derivatization distinguishes phosphopeptides from other peptides that adhere to IMAC resin by the resultant mass shift or by other discriminative physicochemical properties. The chemical modification also circumvents ionization suppression and neutral loss of phosphate that typically occurs with positive ion mass spectrometry. Furthermore, IMAC screening prior to β-elimination improves the selectivity of the elimination reaction for phosphopeptides by subfractionating the phosphopeptides from O-linked glycopeptides. Although phosphotyrosine does not undergo β-elimination, a combined IMAC/β-elimination protocol can enable the unambiguous determination of phosphorylated serine and threonine residues and still provide the usual benefits of IMAC enrichment for phosphotyrosine containing peptides. EXPERIMENTAL SECTION Reagents. Nickel(II)-loaded nitrilotriacetic acid (NTA) immobilized on sepharose beads was purchased from Qiagen (Hilden, Germany). HPLC grade water was purchased from Rathburn (Walkerburn, U.K.). For enzymatic digestions, modified porcine trypsin was purchased from Promega (Madison, WI). Glycosylated, phosphorylated, and unmodified synthetic peptides (YSPTgSPSK, YSPTpSPSK, and YSPTSPSK, respectively) corresponding to the C-terminal repeat domain (CTD) of RNA polymerase II, were kindly provided by Professor Gerald W. Hart (Johns Hopkins University School of Medicine, Baltimore, MD). 2,5-Dihydroxybenzoic acid (DHB) was purchased from Bruker Daltonik (Bremen, Germany). All other materials were purchased from Sigma-Aldrich (Poole, U.K.) unless stated otherwise. Generation of GST-p120ctn Expression Vector. A mammalian glutathione-(S)-transferase-p120 catenin fusion protein (GST-p120ctn) expression vector incorporating murine p120 isoform 1A, pEF-Bos GST-p120ctn, was made using the pEF-Bos vector. The GST sequence from pGEX-2T (incorporating the polylinker) was amplified by PCR and ligated with XbaI-digested pEF-Bos. Using the pEGFP-p120ctn plasmid23 as a template and primers 5′-CAA-GGA-TCC-ATG-GAC-GAC-TCA-GAG-GTG-3′ and 5′-GATT-GGT-CAC-CCG-GGA-CAG-ACT-G-3′, the p120ctn N-terminus was amplified by PCR, inserting a BamHI restriction site upstream of the first p120ctn start codon. pEGFP-p120ctn was kindly provided by K. Burridge (Department of Cell Biology and Anatomy and the Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC). The PCR product was subjected to a BstEII/BamHI double digest. In addition, the pEGFP-p120 plasmid23 was sequentially digested with BstEII and SmaI. The resulting 2.1 kb fragment was ligated in tandem with the N-terminal BstEII/BamHI fragment into the pEF-Bos-GST vector using the polylinker BamHI and SmaI sites. GST-p120ctn Fusion Protein Purification. Cos-7 cells were maintained at 10% carbon dioxidein DMEM, supplemented with (23) Noren, N. K.; Liu, B. P.; Burridge, K.; Kreft, B. J. Cell Biol. 2000, 150, 567-579.

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10% fetal bovine serum, 100 units/mL penicillin, and 100 µg/mL streptomycin and split 1:2 the day before transfection. The pEFBos GST-p120ctn plasmid (10 µg) was electroporated into 2 × 107 Cos-7 cells in electroporation buffer (120 mM potassium chloride, 10 mM potassium phosphate pH 7.6, 2 mM magnesium chloride, 25 mM Hepes pH 7.6, 0.5% Ficoll 400) using a Biorad Genepulser at 250 V, 960 mF. After expression for 24 h, the cells were incubated with the serine/threonine phosphatase inhibitor Calyculin A (100 nM) (Sigma, Poole, U.K.) for 30 min and subsequently lysed in CHAPS lysis buffer (1% CHAPS, 150 mM sodium chloride, 10 mM Tris pH 7.5, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% phosphatase inhibitor cocktail II (Sigma, Poole, U.K.), and 0.1 tablet/mL Mini EDTA-free protease inhibitor cocktail (Roche Molecular Biochemicals, Lewes, U.K.). The lysate was centrifuged for 10 min at 13 000 rpm, and the supernatant was incubated with 20 µL of glutathione-sepharose 4B beads (Amersham Biosciences, Little Chalfont, U.K.) for 1 h. The beads were removed, and the lysate was incubated with an additional 20 µL of beads for 1.5 h. Beads were combined and washed three times with CHAPS lysis buffer, two times with a high-salt buffer (0.5 M lithium chloride, 10 mM Tris pH7.5), and two times with 1 mM Tris pH 8.0. After removal of excess buffer, the beads were stored at -40 °C. Enzymatic Digestion. Fetuin glycoprotein was oxidized prior to enzymatic digestion according to the protocol employed by Adamczyk et al.17 with minor modifications. Briefly, performic acid was prepared freshly before use by standing a mixture of 30% aqueous hydrogen peroxide and 98% formic acid in a ratio of 5:95 (v/v) at room temperature for 30 min. An aliquot of the performic acid mixture (40 µL) was added to a solution of fetuin in water (10 pmol/µL, 10 µL), and the resulting solution was incubated at 2 °C for 1 h. After protein oxidation, the solvent was removed under a stream of nitrogen. The oxidized protein was redissolved in ammonium bicarbonate (25 mM, 50 µL) and digested overnight with trypsin at 37 °C. R-Casein was digested overnight with trypsin at 37 °C without prior performic acid oxidation. GST-p120ctn fusion protein on glutathione sepharose beads was washed with water (3 × 20 µL) and resuspended in ammonium bicarbonate (25 mM, 50 µL). The bound protein was reduced on-bead with dithiothreitol (DTI) (1 M, 0.5 µL) at 37 °C for 1 h and alkylated with iodoacetamide (IAM) (1 M, 2.5 µL) at room temperature for 1 h. The excess IAM was then quenched with additional DTT (1 M, 1.0 µL). The supernatant was removed, and the beads were washed with ammonium bicarbonate (25 mM, 2 × 20 µL), resuspended in ammonium bicarbonate (25 mM, 30 µL), and digested overnight with trypsin at 37 °C. Immobilized Metal Ion Affinity Chromatography. Iron(III)NTA-IMAC beads were prepared in bulk as previously described with minor modifications.12 Briefly, nickel(II)-NTA sepharose beads were washed with water (3 volumes), and bound Ni(II) was removed with EDTA (100 mM, 3 volumes). The stripped beads were then washed with water (3 volumes) and charged with iron(III) using a solution of 100 mM iron(III) chloride in 100 mM acetic acid. The iron(III)-loaded beads were finally washed with acetic acid (100 mM, 3 volumes) and stored at 4 °C as a suspension (1:1, volume of beads/volume of acetic acid).

IMAC was performed as previously described.12,13 Briefly, synthetic peptides or tryptic digests were typically added as a 1-µL solution (1 pmol to 50 fmol/µL) directly to 1 µL of iron(III)-NTA bead suspension and allowed to stand at room temperature for 15 min with occasional agitation. The supernatant was removed using gel loading pipet tips (Eppendorf AG, Hamburg, Germany), and the beads were washed with acetic acid (3 × 10 µL, 100 mM) then water (3 × 10 µL). Bound peptides were eluted using either 30% ammonium hydroxide (2 µL, 5 min) or ammonium phosphate (2 µL, 100 mM, 5 min) or by β-elimination with or without Michael addition. On-Resin β-Elimination and Michael Addition. Barium hydroxide (100 mM, 1 µL) for β-elimination, or barium hydroxide (100 mM, 1 µL) and 2-aminoethanethiol (50 mM, 0.4 µL) for β-elimination with concurrent Michael addition, was added directly to peptide-loaded iron(III)-NTA-IMAC beads. The suspension was incubated at 37 °C for 1 h, and the reaction was quenched with ammonium sulfate (100 mM, 1 µL). The barium sulfate precipitate was pelleted by centrifugation (13 000 rpm, 1 min), and the supernatant was analyzed by mass spectrometry. Matrix-Assisted Laser Desorption Ionization Mass Spectrometry. All samples were prepared with 2,5-dihydroxybenzoic acid (DHB) matrix on 600-800-µm-diameter Anchorchip targets (Bruker Daltonik, Bremen, Germany), except for samples for PSD/LIFT analysis, which were prepared with R-cyano-4-hydroxycinnamic acid (CHCA) as matrix. The DHB matrix solution was prepared by diluting a saturated aqueous solution 1 in 4 with water for use with the 800-µm Anchorchip targets. A 1-µL aliquot of this solution was added to 1 µL of the analyte solution and subsequently air-dried. In the case of aqueous ammonia eluents from IMAC, the analyte solution was spotted first and partially air-dried before adding the matrix solution to avoid ammonia interference with DHB crystallization. The CHCA matrix solution was prepared as a saturated solution in acetone/0.1% trifluoroacetic acid (TFA) in a ratio of 97:3 (v/v) and applied as a thin layer to 600-µm Anchorchip target spots. A 2-µL aliquot of 5% aqueous TFA was deposited on the thin layer, 2 µL of analyte solution was added, and the sample was incorporated for 5 min. The solvent was removed, and the thin layer was washed with 2% TFA. Mass spectra were recorded on a Bruker Ultraflex TOF/TOF (Bruker Daltonik, Bremen, Germany) in positive ion reflectron mode. The instrument was calibrated immediately prior to the experiments using the commercial Sequazyme Peptide Mass Standards Kit solution CalMix2 (PE Biosystems, Foster City, CA). Mass spectra typically constituted 100 shots, except for PSD/LIFT spectra, for which 800 shots were typically summed. Data were processed using the Bruker XTOF software v5.1.5 supplied with the Ultraflex TOF/TOF instrument. RESULTS Discrimination of O-Linked Glycopeptides from Phosphopeptides by IMAC. IMAC was applied to a mixture of synthetic phosphorylated, glycosylated, and unmodified peptides to assess the effectiveness of the procedure at separating glycopeptides from phosphopeptides. Unmodified and O-glycosylated peptides were not retained on iron(III)-IMAC resin, and elution of resin-captured peptides with ammonium hydroxide yielded only the phosphopeptides (Figure 2). Fractionation in this manner

Figure 2. MALDI-TOF mass spectra of (A) a mixture of phosphorylated, O-glycosylated, and unmodified CTD peptides; (B) the supernatant from iron(III)-IMAC of the CTD peptide mixture; and (C) CTD peptides eluted from iron(III)-IMAC resin with 5% ammonium hydroxide. Labels represent unmodified peptide (CTD: YSPTSPSK), O-glycosylated peptide (G-CTD: YSPTgSPSK), phosphorylated peptide (P-CTD: YSPTpSPSK), doubly phosphorylated peptide (P2-CTD), and phosphorylated peptide with a serine deletion (P-CTD - Ser).

enabled subsequent β-elimination and derivatization of phosphopeptides, but precluded derivatization of glycopeptides. Conditions for β-Elimination and Michael Addition. Recent publications employing alkali-mediated β-elimination frequently utilize sodium hydroxide as the β-elimination reagent and require a subsequent desalting step.18,19,24,25 However, chromatographic (24) Li, W.; Boykins, R. A.; Backlund, P. S.; Wang, G.; Chen, H.-C. Anal. Chem. 2002, 74, 5701-5710. (25) Steen, H.; Mann, M. J. Am. Soc. Mass Spectrom. 2002, 13, 996-1003.

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purification of subpicomole concentrations of samples often results in significant sample losses and ideally should be avoided. In this protocol, barium hydroxide was employed as the base, since barium(II) can be removed after the reaction by precipitation with sulfate or carbonate, abrogating the need for chromatographic cleanup. In preliminary experiments, barium(II) was rapidly precipitated from solution as barium carbonate after exposure to a carbon dioxide atmosphere or as barium sulfate by quenching with ammonium sulfate. Although precipitation under carbon dioxide atmosphere would be preferable, since no salts are added directly to the solution and no sample dilution occurs, ammonium sulfate was found to be more effective for barium(II) precipitation and was adopted as the method of choice. Barium(II) is also reported to catalyze specifically the β-elimination of phosphate from phosphoserine and phosphothreonine.26 The barium(II) cation has a high affinity for phosphate, and in solution, it rapidly neutralizes the anionic phosphate charges, facilitating the approach of electron-rich nucleophiles. Barium phosphate ion-pairing is also postulated to increase the acidity of the CR proton of phosphoserine and promote the β-elimination reaction.26 Therefore, short reaction times were possible without using elevated temperatures typically used for phosphopeptide β-elimination.17,19,21,25,27 Additionally, barium(II)-catalyzed phosphate elimination can proceed at lower concentrations of base, avoiding the potential alkaline-hydrolysis of peptide bonds. Similarly, the use of aprotic solvent mixtures often employed to inhibit hydrolysis15,16,19,21,27 was avoided. The product from β-elimination of phosphoserine and phosphothreonine is generally stable to mass spectrometric analysis.28 However, unwanted reactions during storage, elimination of the ambiguity with dehydrated peptide species, and the ease of secondary derivatization using Michael addition advocate for further modification of the β-eliminated phosphopeptides. Subsequent derivatization with Michael addition can create a stable or functionally enhanced product with an unambiguously unique residue mass. Michael addition can be performed concurrently with β-elimination, adding no extra sample preparation or purification steps to the protocol. Thiol nucleophiles are frequently employed for the Michael addition of β-eliminated peptides, but the most commonly used are thioalkanes that are poorly soluble in water and exude an offensive stench. 2-Aminoethanethiol was selected for use in the presented protocol because of its excellent solubility in water and absence of offensive odor. Protonation of the amine in water was also reasoned to assist Michael addition by potentially facilitating deprotonation of the thiol and activating it for nucleophilic attack. On-Resin β-Elimination of IMAC-Bound Phosphopeptides. Procedures for high-sensitivity proteomic analyses should ideally minimize sample transfers and preparation steps in order to maximize the overall sample recovery. Consistent with this tenet, IMAC isolation of phosphopeptides can be combined directly with β-elimination of the phosphorylation sites, with and without concurrent Michael addition, in a single-vessel process with no sample transfer prior to mass spectrometric analysis. R-Casein phosphoprotein was employed as a model protein in this (26) Byford, M. F. Biochem. J. 1991, 280, 261-265. (27) Fadden, P.; Haystead, T. A. Anal. Biochem. 1995, 225, 81-88. (28) Weil, H. P.; Becksickinger, A. G.; Metzger, J.; Stevanovic, S.; Jung, G.; Josten, M.; Sahl, H. G. Eur. J. Biochem. 1990, 194, 217-223.

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study, since its phosphorylation sites are well characterized. IMAC was performed on a tryptic digest of R-casein to isolate the phosphopeptides from the peptide mixture. After appropriate washes, the resin-bound phosphopeptides were simultaneously eluted and derivatized by β-elimination using barium hydroxide. Following the reaction, barium(II) was precipitated from the mixture with ammonium sulfate and the supernatant analyzed by mass spectrometry without further cleanup. R-Casein phosphopeptides were efficiently isolated from the digest mixture and eluted by β-elimination using this procedure (Figure 3). For consistency and to ensure maximal elution and β-elimination, the reaction was typically allowed to proceed for 1 h, although elution and β-elimination of these peptides was easily observable after a 5-min reaction time. Elution of the phosphopeptides using β-elimination reliably released multiply phosphorylated peptides from the chromatography resin (Figure 3). For R-casein, the quintuply phosphorylated tryptic peptide (P75) corresponding to residues 74-94 (S1 variant) was consistently eluted by β-elimination for 500 fmol of tryptic digest initially loaded on IMAC resin and was observed in most experiments using only 100 fmol of digest. In contrast, consistent detection of the quintuply phosphorylated peptide using ammonium phosphate required total IMAC loading amounts of 20 pmol of digest, applying 1 pmol on-target (data not shown and Hart et al.12). After barium precipitation, the supernatant was suitable for analysis without further purification in both DHB and CHCA on 600-800 µm Anchorchip targets. Use of CHCA enabled the precise determination of phosphorylation sites by PSD/LIFT for 50 fmol of digest applied to the IMAC/β-elimination protocol (Figure 4). In contrast to conventional IMAC elution using ammonium phosphate (Figure 4A), derivatization of the phosphorylation site greatly improved the sequence information obtained by precluding neutral loss of the phosphate as the primary pathway of fragmentation (Figure 4B, C). In both cases of derivatization with and without Michael addition, significantly more fragment ions were detected, both in number of different ions and in ion intensities. The additional information enabled further assignment of y-type and b-type peptide fragment ions, greatly increasing the confidence of sequence identification. On-Resin β-Elimination with Concurrent Michael Addition of IMAC-Bound Phosphopeptides. Michael addition can be performed simultaneously with β-elimination of phosphopeptides both in solution and on iron(III)-IMAC resin. R-Casein tryptic phosphopeptides bound to IMAC resin were eluted and derivatized efficiently by β-elimination with concurrent Michael addition using barium hydroxide and 2-aminoethanethiol. In general the addition of thiol to the β-eliminated residue occurred extremely rapidly in near-quantitative yields as determined from the mass spectra (Figure 3C). However, for the quintuply phosphorylated R-casein peptide P75, peak splitting was observed corresponding to Michael addition at four of five and five of five β-eliminated phosphorylation sites. Although the reaction efficiency was 90-95% complete, the peak splitting effectively halved the sensitivity of the method to detect the quintuply phosphorylated peptide. This hyperphosphorylated peptide represents an extreme case, and given the generally lower occurrence of multiple phosphorylations in close proximity within the primary sequence, the majority of phospho-

Figure 3. MALDI-TOF mass spectra of (A) the R-casein tryptic digest (500 fmol), and the iron(III)-IMAC eluents of the R-casein tryptic digest (500 fmol total loading on IMAC) obtained using (B) 100 mM ammonium phosphate, (C) β-elimination and (D) β-elimination with concurrent Michael addition. β-Elimination results in a -98 Da mass shift per eliminated phosphate. β-Elimination with Michael addition using 2-aminoethanethiol (Mw 77) results in a -21 Da mass shift per derivatized phosphoamino acid. Peptide P75 readily underwent N-terminal pyroglutamization, evident by the loss of 17 Da. Michael addition of this quintuply phosphorylated peptide was estimated to be 90-95% complete from the ratio of the quadruply (m/z 2539.11) and quintuply (m/z 2616.03) derivatized P75 peptide and the absence of a lower degree of derivatization. Labels correspond to P11, YKVPQLEIVPNpSAEER, residues 119-134 of RS1-casein; P22, DIGpSEpSTEDQAMEDIK, residues 58-73 of RS1-casein; P31, VPQLEIVPNpSAEER, residues 121-134 of RS1-casein; P41, (K)TVDMEpSTEVFTK(K), residues 152(3)-164(5) of RS1-casein; P52, EQLpSTpSEENSKK, residues 141-152 of RS2-casein; P61, TVDMESTEVFTK, residues 153-164 of RS2-casein; and P75, QMEAEpSIpSpSpSEEIVPNpSVEQK, residues 74-94 of RS1-casein. Subscript values denote the number of phosphorylations. R-Casein tryptic peptides that are not phosphorylated are labeled with their first and last amino acids and corresponding residue numbers. Matrix-related peaks are labeled “M”. A fragment peptide corresponding to YKVPQLEIVPN, labeled “+”, was also observed after β-elimination. For clarity, only phosphopeptides are labeled in the pre-IMAC digest spectrum.

peptides will not suffer an appreciable loss of sensitivity from incomplete Michael addition. Michael addition of 2-aminoethanethiol to β-eliminated residues afforded a derivatized residue with a side chain that remained intact during PSD/LIFT experiments as well as during storage. The unique mass for the derivatized residue enabled the precise determination of phosphorylation sites from 50 fmol of R-casein applied to the combined IMAC-β-elimination procedure (Figure 4C.). Identification of Phosphorylation Sites on Bovine Fetuin Glycoprotein. Fetuin is a heavily glycosylated mammalian plasma protein belonging to the cystatin superfamily.29 The human, rat and bovine forms are recognized to be serine-phosphorylated30-35 (29) Brown, W. M.; Saunders: N. R.; Mollgard, K.; Dziegielewska, K. M. Bioessays 1992, 14, 749-755. (30) Haglund, A. C.; Ek, B.; Ek, P. Biochem. J. 2001, 357, 437-445. (31) Jahnen-Dechent, W.; Trindl, A.; Godovac-Zimmermann, J.; Muller-Esterl, W. Eur. J. Biochem. 1994, 226, 59-69. (32) Ohnishi, T.; Nakamura, O.; Ozawa, M.; Arakaki, N.; Muramatsu, T.; Daikuhara, Y. J. Bone Miner. Res. 1993, 8, 367-377. (33) Le Cam, A.; Magnaldo, I.; Le Cam, G.; Auberger, P. J. Biol. Chem. 1985, 260, 15965-15971. (34) Pedersen, K. J. Phys. Colloid Chem. 1947, 51, 164-171. (35) Deutsch, H. J. Biol. Chem. 1954, 208, 669-678.

in addition to being extensively N- and O-glycosylated.36-39 Although several phosphorylation sites of human and rat fetuin have been determined, phosphorylation of bovine fetuin is not as well characterized. To illustrate the effectiveness of the IMAC-βelimination protocol for the exclusive derivatization of phosphorylated but not O-glycosylated serine and threonine, the method was applied to bovine fetuin. Three phosphopeptides were identified corresponding to residues 313-333 (HTFSGVASVESSSGEAFHVGK) being singly, doubly, and triply phosphorylated (Figure 5). PSD/LIFT of these peptides unambiguously identified the phosphorylation sites as serines 320, 323, and 325. No glycosylated peptides were isolated during this procedure. The reported phosphorylation sites are, to the best of the authors’ knowledge, novel assignments. Identification of Phosphorylation Sites on GST-p120ctn Fusion Protein. To assess the efficacy of the combined protocol to analyze protein obtained from mammalian cell culture, the (36) Edge, A. S.; Spiro, R. G. J. Biol. Chem. 1987, 262, 16135-16141. (37) Hayase, T.; Rice, K. G.; Dziegielewska, K. M.; Kuhlenschmidt, M.; Reilly, T.; Lee, Y. C. Biochemistry 1992, 31, 4915-4921. (38) Spiro, R. G.; Bhoyroo, V. D. J. Biol. Chem. 1974, 249, 5704-5717. (39) Watzlawick, H.; Walsh, M. T.; Yoshioka, Y.; Schmid, K.; Brossmer, R. Biochemistry 1992, 31, 12198-12203.

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Figure 4. MALDI-PSD/LIFT-TOF/TOF mass spectra of R-casein tryptic phosphopeptide P11 (YKVPQLEIVPNpSAEER) eluted from iron(III)-IMAC (50 fmol total loading) with (A) ammonium phosphate, (B) β-elimination, and (C) β-elimination with concurrent Michael addition using 2-aminoethanethiol. Samples were prepared in CHCA. Neutral loss of phosphate was the major fragmentation pathway for the intact phosphopeptide (ammonium phosphate elution). Derivatization by β-elimination, with or without Michael addition, precluded neutral loss of phosphate; significantly increased fragmentation within the peptide backbone; and discriminated the phosphorylation site as a unique residue mass (69 and 146 Da, respectively).

method was applied to GST-p120ctn fusion protein overexpressed in and isolated from Cos-7 cells. Initially, 4 minor peaks (