Characterization of Protein Phosphorylation by Mass Spectrometry Using Immobilized Metal Ion...

Characterization of Protein Phosphorylation byMass Spectrometry Using Immobilized Metal IonAffinity Chromatography with On-Resinâ-Elimination and Michael Addition

Andrew J. Thompson,† Sarah R. Hart,†,‡ Clemens Franz,†,‡ Karin Barnouin,†,‡ Anne Ridley,†,‡ andRainer 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 andMolecular Biology, University College London, Darwin Building, Gower Street, London WC1E 6BT, United Kingdom

A protocol combining immobilized metal ion affinitychromatography and â-elimination with concurrent Michaeladdition has been developed for enhanced analysis ofprotein phosphorylation. Immobilized metal ion affinitychromatography was initially used to enrich for phosphor-ylated peptides. â-Elimination, with or without concurrentMichael addition, was then subsequently used to simul-taneously elute and derivatize phosphopeptides bound tothe chromatography resin. Derivatization of the phosphatefacilitated the precise determination of phosphorylationsites by MALDI-PSD/LIFT tandem mass spectrometry,avoiding complications due to ion suppression and phos-phate lability in mass spectrometric analysis of phospho-peptides. Complementary use of immobilized metal ionaffinity chromatography and â-elimination with concurrentMichael addition in this manner circumvented severalinherent disadvantages of the individual methods. Inparticular, (i) the protocol discriminated O-linked glycos-ylated peptides from phosphopeptides prior to â-elimina-tion/Michael addition and (ii) the elution of peptides fromthe chromatography resin as derivatized phosphopeptidesdistinguished them from unphosphorylated species thatwere also retained. The chemical derivatization of phos-phopeptides greatly increased the information obtainedduring peptide sequencing by mass spectrometry. Thecombined protocol enabled the detection and sequencingof phosphopeptides from protein digests at low femtomoleconcentrations of initial sample and was employed toidentify novel phosphorylation sites on the cell adhesionprotein p120 catenin and the glycoprotein fetuin.

Characterization of protein phosphorylation sites is critical tothe understanding of protein function and regulation and, there-fore, is essential in many proteomic studies.1,2 Mass spectrometry

(MS) is a key tool in proteomics for the identification andcharacterization of proteins and their modifications, typicallyinvolving 2D gel electrophoresis and enzymatic digestion, culmi-nating in peptide mass fingerprinting or sequencing by tandemmass spectrometry (MS/MS).3,4 However, the mass spectrometricidentification specifically of phosphorylation sites is hindered byseveral factors, including (i) the typically substoichiometricpopulation of phosphorylated, as compared to unphosphorylated,species; (ii) the intrinsic ionization suppression in positive ionmode by the anionic phosphate functionality; and (iii) the labilityof the phosphate group under mass spectrometric conditions,resulting in facile neutral losses of 98 and 80 Da during both MSand MS/MS analysis.

Methods of phosphopeptide enrichment and separation priorto analysis can significantly improve mass spectrometric charac-terization of phosphorylation sites.5 Immobilized metal ion affinitychromatography (IMAC) using iron(III) or gallium(III) is one suchmethod.6,7 IMAC employs a stationary phase with a binding affinityfor phosphates capable of enriching phosphorylated peptides froma profusion of unphosphorylated species. Although IMAC signifi-cantly deconvolutes complex mixtures, some unphosphorylatedpeptides are also retained. In particular, peptides rich in acidicresidues are frequently observed.7-10 Methylation of peptides inmethanolic hydrochloric acid prior to IMAC subfractionationreportedly increases the specificity of the method for phospho-peptides by esterifying the carboxylic acid functionalities.8 The

* Corresponding author address: The Ludwig Institute for Cancer Research,Mass Spectrometry and Bioanalytical Chemistry Laboratories, Cruciform Build-ing, Gower Street, London WC1E 6BT, United Kingdom. Phone: +44 20 76796683. Fax: +44 20 7679 6687. E-mail:[email protected].

† The Ludwig Institute for Cancer Research.‡ University College London.

(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, 517-527.

(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.

Anal. Chem. 2003, 75, 3232-3243

3232 Analytical Chemistry, Vol. 75, No. 13, July 1, 2003 10.1021/ac034134h CCC: $25.00 © 2003 American Chemical SocietyPublished on Web 05/14/2003

methylation procedure is specific and high-yielding, and nobyproducts have been reported, despite the harsh reactionconditions. When fully optimized and characterized, methylationpromises to be a significant complementary step to improve IMACselectivity of phosphopeptides. However, tyrosine, histidine, andtryptophan residues also contribute to IMAC binding of unphos-phorylated peptides,9-11 which may require an additional strategyto address.

IMAC also shows pronounced differential recovery of phos-phopeptides, as seen in the poor recovery of multiply phosphor-ylated species.7,12,13 In addition to its dependence on loading,washing, and elution conditions, the recovery of some phospho-peptides can be reportedly improved through substitution ofiron(III) with gallium(III),7 although arguably at the cost ofreduced retention.13,14 These and other factors, including the initialbinding affinity, make IMAC a complex technique that requiresfurther development and optimization in order to establish it as asimple but powerful affinity purification method for selectivephosphopeptide enrichment.

Elution of phosphorylated peptides from IMAC resin shouldbe improved by removing the IMAC-bound phosphate(s) directlyfrom the phosphopeptide, releasing the peptide into solution. Onepossibility is to use phosphatases to elute the phosphopeptides,but enzymatic dephosphorylation will leave the site of phosphor-ylation indistinguishable from other serine, threonine, or tyrosineresidues. Another approach is the direct â-elimination of phos-phopeptides from the IMAC resin. â-Elimination can occur atphosphoserine and phosphothreonine residues, and their respec-tive conversions to dehydroalanine and dehydro-2-aminobutyricacid enables the precise determination of the phosphorylation site.

Alkaline-mediated â-elimination forms the basis for a chemicalmodification strategy to address both the intrinsic ionizationsuppression of phosphorylated peptides and the lability of thephosphate moiety. Furthermore, this strategy provides a basis forspecific chemical tagging of phosphorylation sites (Figure 1).â-Elimination coupled to Michael addition can be used to derivatizephosphopeptides substitutively at the phosphorylated residue15,16

and has recently been employed to derivatize phosphopeptideswith biotin linkers to enable selective phosphopeptide isolationwith streptavidin.17,18 The phosphoprotein isotope-coded affinitytag (PhIAT) approach extends this procedure by using an isotope-coded ethanedithiol linker to quantitate levels of protein phos-phorylation19 in a fashion similar to isotope-coded affinity tag(ICAT) quantitation of protein expression.20 Simpler methodsemploy derivatizing the dehydrated products with ethanethiol to

facilitate positive ion formation and tryptic enzymatic digestion21

or with dual thiol mixtures to identify phosphorylated peptides ina single experiment by the observed mass difference.22 However,â-elimination and Michael addition also occur at cysteine andglycosylated serine and threonine residues. Although cysteinescan be stabilized to the elimination reaction by alkylation withiodoacetamide or similar reagents, O-linked carbohydrates remainproblematic, rendering the method unspecific for phosphorylationsites alone.

(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. MassSpectrom. 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.(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.

Figure 1. A general schematic for â-elimination and Michael additionfor phosphoserine (X ) H)- and phosphothreonine (X ) CH3)-containing peptides. Treatment with base liberates the free phosphategenerating dehydroalanine or dehydro-2-aminobutyric acid for phos-phoserine and phosphothreonine, respectively. Dehydrated aminoacids are excellent targets for nucleophilic attack. Thiols with designedtags can further derivatize phosphorylation sites with a variety ofphysicochemical properties.

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Both the IMAC and â-elimination strategies suffer individualdisadvantages that can be overcome by their complementaryapplication. Directly eluting peptides from IMAC resin by â-elim-ination potentially improves the recovery of phosphopeptides,particularly multiply phosphorylated species, and preserves thephosphorylation site information. In combination with concurrentMichael addition, phosphorylated residues can be further deriva-tized with a variety of purposely designed tags. Derivatizationdistinguishes phosphopeptides from other peptides that adhereto IMAC resin by the resultant mass shift or by other discrimina-tive physicochemical properties. The chemical modification alsocircumvents ionization suppression and neutral loss of phosphatethat typically occurs with positive ion mass spectrometry. Fur-thermore, IMAC screening prior to â-elimination improves theselectivity of the elimination reaction for phosphopeptides bysubfractionating the phosphopeptides from O-linked glycopeptides.Although phosphotyrosine does not undergo â-elimination, acombined IMAC/â-elimination protocol can enable the unambigu-ous determination of phosphorylated serine and threonine resi-dues and still provide the usual benefits of IMAC enrichment forphosphotyrosine containing peptides.

EXPERIMENTAL SECTIONReagents. Nickel(II)-loaded nitrilotriacetic acid (NTA) im-

mobilized on sepharose beads was purchased from Qiagen(Hilden, Germany). HPLC grade water was purchased fromRathburn (Walkerburn, U.K.). For enzymatic digestions, modifiedporcine trypsin was purchased from Promega (Madison, WI).Glycosylated, phosphorylated, and unmodified synthetic peptides(YSPTgSPSK, YSPTpSPSK, and YSPTSPSK, respectively) corre-sponding to the C-terminal repeat domain (CTD) of RNA poly-merase 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 BrukerDaltonik (Bremen, Germany). All other materials were purchasedfrom Sigma-Aldrich (Poole, U.K.) unless stated otherwise.

Generation of GST-p120ctn Expression Vector. A mam-malian glutathione-(S)-transferase-p120 catenin fusion protein(GST-p120ctn) expression vector incorporating murine p120isoform 1A, pEF-Bos GST-p120ctn, was made using the pEF-Bosvector. The GST sequence from pGEX-2T (incorporating thepolylinker) was amplified by PCR and ligated with XbaI-digestedpEF-Bos. Using the pEGFP-p120ctn plasmid23 as a template andprimers 5′-CAA-GGA-TCC-ATG-GAC-GAC-TCA-GAG-GTG-3′ and5′-GATT-GGT-CAC-CCG-GGA-CAG-ACT-G-3′, the p120ctn N-ter-minus was amplified by PCR, inserting a BamHI restriction siteupstream of the first p120ctn start codon. pEGFP-p120ctn waskindly provided by K. Burridge (Department of Cell Biology andAnatomy and the Lineberger Comprehensive Cancer Center,University of North Carolina, Chapel Hill, NC). The PCR productwas subjected to a BstEII/BamHI double digest. In addition, thepEGFP-p120 plasmid23 was sequentially digested with BstEII andSmaI. The resulting 2.1 kb fragment was ligated in tandem withthe N-terminal BstEII/BamHI fragment into the pEF-Bos-GSTvector using the polylinker BamHI and SmaI sites.

GST-p120ctn Fusion Protein Purification. Cos-7 cells weremaintained at 10% carbon dioxidein DMEM, supplemented with

10% fetal bovine serum, 100 units/mL penicillin, and 100 µg/mLstreptomycin and split 1:2 the day before transfection. The pEF-Bos GST-p120ctn plasmid (10 µg) was electroporated into 2 ×107 Cos-7 cells in electroporation buffer (120 mM potassiumchloride, 10 mM potassium phosphate pH 7.6, 2 mM magnesiumchloride, 25 mM Hepes pH 7.6, 0.5% Ficoll 400) using a BioradGenepulser at 250 V, 960 mF. After expression for 24 h, the cellswere incubated with the serine/threonine phosphatase inhibitorCalyculin A (100 nM) (Sigma, Poole, U.K.) for 30 min andsubsequently lysed in CHAPS lysis buffer (1% CHAPS, 150 mMsodium chloride, 10 mM Tris pH 7.5, 1 mM ethylenediamine-tetraacetic acid (EDTA), 1% phosphatase inhibitor cocktail II(Sigma, Poole, U.K.), and 0.1 tablet/mL Mini EDTA-free proteaseinhibitor cocktail (Roche Molecular Biochemicals, Lewes, U.K.).The lysate was centrifuged for 10 min at 13 000 rpm, and thesupernatant was incubated with 20 µL of glutathione-sepharose4B beads (Amersham Biosciences, Little Chalfont, U.K.) for 1 h.The beads were removed, and the lysate was incubated with anadditional 20 µL of beads for 1.5 h. Beads were combined andwashed three times with CHAPS lysis buffer, two times with ahigh-salt buffer (0.5 M lithium chloride, 10 mM Tris pH7.5), andtwo 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 priorto enzymatic digestion according to the protocol employed byAdamczyk et al.17 with minor modifications. Briefly, performic acidwas 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 performicacid mixture (40 µL) was added to a solution of fetuin in water(10 pmol/µL, 10 µL), and the resulting solution was incubated at2 °C for 1 h. After protein oxidation, the solvent was removedunder a stream of nitrogen. The oxidized protein was redissolvedin ammonium bicarbonate (25 mM, 50 µL) and digested overnightwith trypsin at 37 °C. R-Casein was digested overnight with trypsinat 37 °C without prior performic acid oxidation.

GST-p120ctn fusion protein on glutathione sepharose beadswas washed with water (3 × 20 µL) and resuspended inammonium bicarbonate (25 mM, 50 µL). The bound protein wasreduced on-bead with dithiothreitol (DTI) (1 M, 0.5 µL) at 37 °Cfor 1 h and alkylated with iodoacetamide (IAM) (1 M, 2.5 µL) atroom temperature for 1 h. The excess IAM was then quenchedwith 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, 30µL), and digested overnight with trypsin at 37 °C.

Immobilized Metal Ion Affinity Chromatography. Iron(III)-NTA-IMAC beads were prepared in bulk as previously describedwith minor modifications.12 Briefly, nickel(II)-NTA sepharosebeads were washed with water (3 volumes), and bound Ni(II) wasremoved with EDTA (100 mM, 3 volumes). The stripped beadswere then washed with water (3 volumes) and charged withiron(III) using a solution of 100 mM iron(III) chloride in 100 mMacetic acid. The iron(III)-loaded beads were finally washed withacetic acid (100 mM, 3 volumes) and stored at 4 °C as asuspension (1:1, volume of beads/volume of acetic acid).

(23) Noren, N. K.; Liu, B. P.; Burridge, K.; Kreft, B. J. Cell Biol. 2000, 150,567-579.

3234 Analytical Chemistry, Vol. 75, No. 13, July 1, 2003

IMAC was performed as previously described.12,13 Briefly,synthetic peptides or tryptic digests were typically added as a 1-µLsolution (1 pmol to 50 fmol/µL) directly to 1 µL of iron(III)-NTAbead suspension and allowed to stand at room temperature for15 min with occasional agitation. The supernatant was removedusing 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 either30% ammonium hydroxide (2 µL, 5 min) or ammonium phosphate(2 µL, 100 mM, 5 min) or by â-elimination with or without Michaeladdition.

On-Resin â-Elimination and Michael Addition. Bariumhydroxide (100 mM, 1 µL) for â-elimination, or barium hydroxide(100 mM, 1 µL) and 2-aminoethanethiol (50 mM, 0.4 µL) forâ-elimination with concurrent Michael addition, was added directlyto peptide-loaded iron(III)-NTA-IMAC beads. The suspension wasincubated at 37 °C for 1 h, and the reaction was quenched withammonium sulfate (100 mM, 1 µL). The barium sulfate precipitatewas pelleted by centrifugation (13 000 rpm, 1 min), and thesupernatant was analyzed by mass spectrometry.

Matrix-Assisted Laser Desorption Ionization Mass Spec-trometry. All samples were prepared with 2,5-dihydroxybenzoicacid (DHB) matrix on 600-800-µm-diameter Anchorchip targets(Bruker Daltonik, Bremen, Germany), except for samples forPSD/LIFT analysis, which were prepared with R-cyano-4-hydroxy-cinnamic acid (CHCA) as matrix. The DHB matrix solution wasprepared by diluting a saturated aqueous solution 1 in 4 with waterfor use with the 800-µm Anchorchip targets. A 1-µL aliquot of thissolution was added to 1 µL of the analyte solution and subse-quently air-dried. In the case of aqueous ammonia eluents fromIMAC, the analyte solution was spotted first and partially air-driedbefore adding the matrix solution to avoid ammonia interferencewith DHB crystallization. The CHCA matrix solution was preparedas 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-µmAnchorchip target spots. A 2-µL aliquot of 5% aqueous TFA wasdeposited on the thin layer, 2 µL of analyte solution was added,and the sample was incorporated for 5 min. The solvent wasremoved, and the thin layer was washed with 2% TFA. Massspectra were recorded on a Bruker Ultraflex TOF/TOF (BrukerDaltonik, Bremen, Germany) in positive ion reflectron mode. Theinstrument was calibrated immediately prior to the experimentsusing the commercial Sequazyme Peptide Mass Standards Kitsolution CalMix2 (PE Biosystems, Foster City, CA). Mass spectratypically constituted 100 shots, except for PSD/LIFT spectra, forwhich 800 shots were typically summed. Data were processedusing the Bruker XTOF software v5.1.5 supplied with the UltraflexTOF/TOF instrument.

RESULTSDiscrimination of O-Linked Glycopeptides from Phos-

phopeptides by IMAC. IMAC was applied to a mixture ofsynthetic phosphorylated, glycosylated, and unmodified peptidesto assess the effectiveness of the procedure at separating glyco-peptides from phosphopeptides. Unmodified and O-glycosylatedpeptides were not retained on iron(III)-IMAC resin, and elutionof resin-captured peptides with ammonium hydroxide yielded onlythe phosphopeptides (Figure 2). Fractionation in this manner

enabled subsequent â-elimination and derivatization of phospho-peptides, but precluded derivatization of glycopeptides.

Conditions for â-Elimination and Michael Addition. Recentpublications employing alkali-mediated â-elimination frequentlyutilize sodium hydroxide as the â-elimination reagent and requirea 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.

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


purification of subpicomole concentrations of samples often resultsin significant sample losses and ideally should be avoided. In thisprotocol, barium hydroxide was employed as the base, sincebarium(II) can be removed after the reaction by precipitation withsulfate or carbonate, abrogating the need for chromatographiccleanup. In preliminary experiments, barium(II) was rapidlyprecipitated from solution as barium carbonate after exposure toa carbon dioxide atmosphere or as barium sulfate by quenchingwith ammonium sulfate. Although precipitation under carbondioxide atmosphere would be preferable, since no salts are addeddirectly to the solution and no sample dilution occurs, ammoniumsulfate was found to be more effective for barium(II) precipitationand was adopted as the method of choice.

Barium(II) is also reported to catalyze specifically the â-elim-ination of phosphate from phosphoserine and phosphothreonine.26

The barium(II) cation has a high affinity for phosphate, and insolution, it rapidly neutralizes the anionic phosphate charges,facilitating the approach of electron-rich nucleophiles. Bariumphosphate ion-pairing is also postulated to increase the acidity ofthe CR proton of phosphoserine and promote the â-eliminationreaction.26 Therefore, short reaction times were possible withoutusing elevated temperatures typically used for phosphopeptideâ-elimination.17,19,21,25,27 Additionally, barium(II)-catalyzed phos-phate elimination can proceed at lower concentrations of base,avoiding the potential alkaline-hydrolysis of peptide bonds. Simi-larly, the use of aprotic solvent mixtures often employed to inhibithydrolysis15,16,19,21,27 was avoided.

The product from â-elimination of phosphoserine and phos-phothreonine is generally stable to mass spectrometric analysis.28

However, unwanted reactions during storage, elimination of theambiguity with dehydrated peptide species, and the ease ofsecondary derivatization using Michael addition advocate forfurther modification of the â-eliminated phosphopeptides. Sub-sequent derivatization with Michael addition can create a stableor functionally enhanced product with an unambiguously uniqueresidue mass. Michael addition can be performed concurrentlywith â-elimination, adding no extra sample preparation or purifica-tion steps to the protocol. Thiol nucleophiles are frequentlyemployed for the Michael addition of â-eliminated peptides, butthe most commonly used are thioalkanes that are poorly solublein water and exude an offensive stench. 2-Aminoethanethiol wasselected for use in the presented protocol because of its excellentsolubility in water and absence of offensive odor. Protonation ofthe amine in water was also reasoned to assist Michael additionby potentially facilitating deprotonation of the thiol and activatingit for nucleophilic attack.

On-Resin â-Elimination of IMAC-Bound Phosphopep-tides. Procedures for high-sensitivity proteomic analyses shouldideally minimize sample transfers and preparation steps in orderto maximize the overall sample recovery. Consistent with thistenet, IMAC isolation of phosphopeptides can be combineddirectly with â-elimination of the phosphorylation sites, with andwithout concurrent Michael addition, in a single-vessel processwith no sample transfer prior to mass spectrometric analysis.R-Casein phosphoprotein was employed as a model protein in this

study, since its phosphorylation sites are well characterized. IMACwas performed on a tryptic digest of R-casein to isolate thephosphopeptides from the peptide mixture. After appropriatewashes, the resin-bound phosphopeptides were simultaneouslyeluted and derivatized by â-elimination using barium hydroxide.Following the reaction, barium(II) was precipitated from themixture with ammonium sulfate and the supernatant analyzed bymass spectrometry without further cleanup. R-Casein phospho-peptides were efficiently isolated from the digest mixture andeluted by â-elimination using this procedure (Figure 3). Forconsistency and to ensure maximal elution and â-elimination, thereaction was typically allowed to proceed for 1 h, although elutionand â-elimination of these peptides was easily observable after a5-min reaction time.

Elution of the phosphopeptides using â-elimination reliablyreleased multiply phosphorylated peptides from the chromatog-raphy resin (Figure 3). For R-casein, the quintuply phosphorylatedtryptic peptide (P75) corresponding to residues 74-94 (S1 variant)was consistently eluted by â-elimination for 500 fmol of trypticdigest initially loaded on IMAC resin and was observed in mostexperiments using only 100 fmol of digest. In contrast, consistentdetection of the quintuply phosphorylated peptide using am-monium phosphate required total IMAC loading amounts of 20pmol of digest, applying 1 pmol on-target (data not shown andHart et al.12).

After barium precipitation, the supernatant was suitable foranalysis without further purification in both DHB and CHCA on600-800 µm Anchorchip targets. Use of CHCA enabled theprecise determination of phosphorylation sites by PSD/LIFT for50 fmol of digest applied to the IMAC/â-elimination protocol(Figure 4). In contrast to conventional IMAC elution usingammonium phosphate (Figure 4A), derivatization of the phos-phorylation site greatly improved the sequence informationobtained by precluding neutral loss of the phosphate as theprimary pathway of fragmentation (Figure 4B, C). In both casesof derivatization with and without Michael addition, significantlymore fragment ions were detected, both in number of differentions and in ion intensities. The additional information enabledfurther assignment of y-type and b-type peptide fragment ions,greatly increasing the confidence of sequence identification.

On-Resin â-Elimination with Concurrent Michael Additionof IMAC-Bound Phosphopeptides. Michael addition can beperformed simultaneously with â-elimination of phosphopeptidesboth in solution and on iron(III)-IMAC resin. R-Casein trypticphosphopeptides bound to IMAC resin were eluted and derivatizedefficiently by â-elimination with concurrent Michael addition usingbarium hydroxide and 2-aminoethanethiol. In general the additionof thiol to the â-eliminated residue occurred extremely rapidly innear-quantitative yields as determined from the mass spectra(Figure 3C). However, for the quintuply phosphorylated R-caseinpeptide P75, peak splitting was observed corresponding to Michaeladdition at four of five and five of five â-eliminated phosphorylationsites. Although the reaction efficiency was 90-95% complete, thepeak splitting effectively halved the sensitivity of the method todetect the quintuply phosphorylated peptide. This hyperphospho-rylated peptide represents an extreme case, and given thegenerally lower occurrence of multiple phosphorylations in closeproximity within the primary sequence, the majority of phospho-

(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.


peptides will not suffer an appreciable loss of sensitivity fromincomplete Michael addition.

Michael addition of 2-aminoethanethiol to â-eliminated residuesafforded a derivatized residue with a side chain that remainedintact during PSD/LIFT experiments as well as during storage.The unique mass for the derivatized residue enabled the precisedetermination of phosphorylation sites from 50 fmol of R-caseinapplied to the combined IMAC-â-elimination procedure (Figure4C.).

Identification of Phosphorylation Sites on Bovine FetuinGlycoprotein. Fetuin is a heavily glycosylated mammalian plasmaprotein belonging to the cystatin superfamily.29 The human, ratand bovine forms are recognized to be serine-phosphorylated30-35

in addition to being extensively N- and O-glycosylated.36-39

Although several phosphorylation sites of human and rat fetuinhave been determined, phosphorylation of bovine fetuin is not aswell characterized. To illustrate the effectiveness of the IMAC-â-elimination protocol for the exclusive derivatization of phospho-rylated but not O-glycosylated serine and threonine, the methodwas applied to bovine fetuin. Three phosphopeptides were identi-fied corresponding to residues 313-333 (HTFSGVASVESSS-GEAFHVGK) being singly, doubly, and triply phosphorylated(Figure 5). PSD/LIFT of these peptides unambiguously identifiedthe phosphorylation sites as serines 320, 323, and 325. Noglycosylated peptides were isolated during this procedure. Thereported phosphorylation sites are, to the best of the authors’knowledge, novel assignments.

Identification of Phosphorylation Sites on GST-p120ctnFusion Protein. To assess the efficacy of the combined protocolto analyze protein obtained from mammalian cell culture, the

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260, 15965-15971.(34) Pedersen, K. J. Phys. Colloid Chem. 1947, 51, 164-171.(35) Deutsch, H. J. Biol. Chem. 1954, 208, 669-678.

(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.

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) â-elimination and (D) â-elimination with concurrentMichael addition. â-Elimination results in a -98 Da mass shift per eliminated phosphate. â-Elimination with Michael addition using2-aminoethanethiol (Mw 77) results in a -21 Da mass shift per derivatized phosphoamino acid. Peptide P75 readily underwent N-terminalpyroglutamization, evident by the loss of 17 Da. Michael addition of this quintuply phosphorylated peptide was estimated to be 90-95% completefrom 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 ofderivatization. Labels correspond to P11, YKVPQLEIVPNpSAEER, residues 119-134 of RS1-casein; P22, DIGpSEpSTEDQAMEDIK, residues58-73 of RS1-casein; P31, VPQLEIVPNpSAEER, residues 121-134 of RS1-casein; P41, (K)TVDMEpSTEVFTK(K), residues 152(3)-164(5) ofRS1-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 trypticpeptides that are not phosphorylated are labeled with their first and last amino acids and corresponding residue numbers. Matrix-related peaksare labeled “M”. A fragment peptide corresponding to YKVPQLEIVPN, labeled “+”, was also observed after â-elimination. For clarity, onlyphosphopeptides are labeled in the pre-IMAC digest spectrum.


method was applied to GST-p120ctn fusion protein overexpressedin and isolated from Cos-7 cells. Initially, 4 minor peaks (<3% basepeak intensity) out of 52 peaks corresponding to GST-p120ctn wereassigned as phosphopeptides from the tryptic digest massspectrum. Iron(III)-IMAC using ammonium phosphate elutionenabled the assignment of 19 phosphopeptides out of 34 peakscorresponding to GST-p120ctn. After iron(III)-IMAC with on-resinâ-elimination/Michael addition, only 11 phosphopeptides weredetected, but the derivatization facilitated peptide sequencing by

PSD/LIFT of the more intense peaks. Most of the phosphoryla-tions detected appeared to occur at serine residues, although afew threonine phosphorylations were also identified by the on-resin derivatization strategy on the basis of the mass measure-

Figure 4. MALDI-PSD/LIFT-TOF/TOF mass spectra of R-caseintryptic phosphopeptide P11 (YKVPQLEIVPNpSAEER) eluted fromiron(III)-IMAC (50 fmol total loading) with (A) ammonium phosphate,(B) â-elimination, and (C) â-elimination with concurrent Michaeladdition using 2-aminoethanethiol. Samples were prepared in CHCA.Neutral loss of phosphate was the major fragmentation pathway forthe intact phosphopeptide (ammonium phosphate elution). Derivati-zation by â-elimination, with or without Michael addition, precludedneutral loss of phosphate; significantly increased fragmentation withinthe peptide backbone; and discriminated the phosphorylation site asa unique residue mass (69 and 146 Da, respectively).

Figure 5. MALDI-TOF mass spectra of (A) the iron(III)-IMAC eluentof bovine fetuin tryptic digest (2 pmol loading on IMAC, 400 fmol ontarget) using ammonium phosphate and (B) the iron(III)-IMAC eluentof bovine fetuin tryptic digest (2 pmol loading on IMAC, 400 fmol ontarget) using on-resin â-elimination. The ions P11 and P12 correspondto single and double phosphorylations within residues 313-333(HTFSGVASVESSSGEAFHVGK). The ion P21 corresponds to asingle phosphorylation within residues 132-143 (CDSSPDSAEDVR).P11â, P12â, and P13â correspond to â-elimination derivatization ofresidues 313-333 at one, two, and three phosphorylation sites,respectively. Matrix-related contaminants are labeled “M”, metastabledecay is labeled “MSD”, and an identified contaminant present in theoriginal digest is labeled “C”. (C) MALDI-PSD/LIFT-TOF/TOF massspectrum of P13â identifying the three phosphorylation sites as serines320, 323, and 325 by their conversion to dehydroalanines. Becauseof the low abundance of the triply phosphorylated peptide, 5 pmol ofdigest was applied to the iron(III)-IMAC â-elimination protocol toensure reliable identification of the three sites.


ments before and after â-elimination. The characterization dataof two of the phosphopeptides, corresponding to residues 216-233 (HYEDGYPGGSDNYGSLSR) and residues 250-264 (APSRQD-VYGPQPQVR) of murine p120ctn isoform 1A, respectively, arepresented in Figure 6. PSD/LIFT of these peptides identified thephosphorylation sites as serine 230 and serine 252. The remainderof the data will be described elsewhere (manuscript in prepara-tion).

DISCUSSIONElution and Derivatization of Phosphopeptides on Iron-

(III)-IMAC Resin by â-Elimination and Michael Addition.

Combining â-elimination with IMAC circumvents several dis-advantages inherent to the individual protocols. â-Eliminationalone occurs at both glycosylated and phosphorylated serine andthreonine residues.40,41 Therefore, the method does not unambigu-ously identify the phosphorylation sites from complex unknownpeptide mixtures. This problem has not been addressed by currentstrategies that employ â-elimination derivatization of phospho-peptides without prior enzymatic deglycosylation of the sample.17,18,42

However, IMAC is a suitable technique to separate phosphopep-

(40) Whitaker, J. R.; Feeney, R. E. Adv. Exp. Med. Biol. 1977, 86B, 155-175.(41) Mega, T.; Nakamura, N.; Ikenaka, T. J. Biochem. (Tokyo) 1990, 107, 68-

72.

Figure 6. MALDI-TOF mass spectra of the iron(III)-IMAC eluent of the GST-p120ctn tryptic digest obtained using (A, B) ammonium phosphate,centered on the phosphorylated peptide ions at 1777.84 (p120ctn residues 250-264 APSRQDVYGPQPQVR) and 2053.83 (p120ctn residues216-233 HYEDGYPGGSDNYGSLSR), respectively; and (C, D) the eluent obtained using â-elimination with Michael addition. (E, F) MALDI-PSD/LIFT-TOF/TOF mass spectra of the derivatized peptides at 1756.6 and 2032.8, respectively, identifying serines 252 and 230 of p120ctnas phosphorylation sites. For (E), the derivatized serine within the series of internal ions that clearly characterize the derivatization site is labeled“S*”.


tides from glycopeptides in peptide mixtures, and its use prior toâ-elimination facilitates the specific derivatization of phosphopep-tides. Conversely, mass spectrometric-based identification ofphosphorylation sites using IMAC is also improved by â-elimina-tion. Iron(III)-IMAC does not exclusively retain phosphopeptidesbut also binds acidic-rich and other peptides.7-11 Elution ofiron(III)-IMAC-bound peptides using basic conditions ideal forâ-elimination specifically derivatizes serine and threonine phos-phorylated peptides at the phosphorylated residue, but does notmodify unphosphorylated peptides. The â-elimination of phosphatefrom phosphoserine and phosphothreonine generates dehydroala-nine and dehydro-2-aminobutyric acid, respectively, marking thesite of phosphorylation with a unique residue mass. Removal ofthe phosphate circumvents phosphate-associated ionization sup-pression of the peptide and, more importantly, avoids neutral lossof phosphate as the primary mode of fragmentation during PSD/LIFT. The information obtained during peptide fragmentation isgreatly increased, facilitating the precise determination of theoriginal phosphorylated residue.

â-Elimination of phosphopeptides is frequently performed usinghigh concentrations of base, elevated temperatures, and longreaction times. Under these conditions, peptide hydrolysis isincreasingly probable, and aprotic solvent mixtures, often includ-ing dimethyl sulfoxide (DMSO), are employed to minimizehydrolysis.15,16,19,21 The presence of involatile organic solvents andthe high concentration of base and salt, typically sodium hydrox-ide, necessitate chromatographic purification prior to MS analysis.The risk of sample degradation and sample loss by the requiredadditional purification step is undesirable, particularly for highsensitivity analyses. The reaction conditions for â-elimination ofphosphopeptides are significantly improved using barium hydrox-ide as base. Group II metal ions, including barium(II), catalyzethe â-elimination of phosphates from phosphoserine and phos-phothreonine.26 Barium hydroxide-mediated â-elimination pro-ceeds at much lower concentrations of base using mildertemperatures and shorter reaction times than sodium hydroxide,greatly reducing the probability of undesired peptide hydrolysis.26

The need for aprotic solvent mixtures is similarly made redundant.Conveniently, barium(II) can also be precipitated from solutionas barium sulfate, and the supernatant is immediately compatiblewith MS analysis using DHB and CHCA without chromatographiccleanup. Barium hydroxide is, therefore, an ideal reagent for high-sensitivity MS-based protocols, enabling rapid derivatization undermild conditions and avoiding sample-consuming purification steps.

â-Elimination confers significant benefits to IMAC and MSanalysis of phosphopeptides, but several complications maypotentially arise. Free cysteine can readily â-eliminate underalkaline conditions to form dehydroalanine. To prevent this, allcysteine residues must be fully protected. Frequently, proteinsare reduced with DTT and alkylated with IAM before MS analysis.Alkylation of cysteine reportedly prevents â-elimination at thisresidue,43 and in preliminary experiments for this work, proteinswere reduced and alkylated in this manner prior to iron(III)-IMACand on-resin â-elimination. Nonetheless, trace amounts of unalky-lated free cysteine-containing peptides persisted in the raw digest

(<5% by the ion signal intensities) and were observed to bind toiron(III)-IMAC resin, effectively being coenriched together withphosphopeptides (data not shown). These peptides were alsoeluted and derivatized along with phosphopeptides by â-elimina-tion, despite repeated thorough alkylation of protein digests withIAM, and were frequently seen as dominant ion peaks in the massspectra. The free cysteine-containing peptides were not observedto elute from iron(III)-IMAC with ammonium phosphate. There-fore, in these experiments, some free cysteine-containing trypticpeptides present in trace amounts bound to iron(III)-IMAC resinwith very high affinity consistent with previous publications44 andwere only eluted by â-elimination of the sulfhydryl moiety fromthe peptide. For example, for the most prominent free cysteine-containing peptide (AQFVPLPVSVSVEFAVAATDCIAK), the alky-lated form, which probably adhered to the resin via weak acidicinteractions, was observed to elute with ammonium phosphateand also under the conditions for â-elimination as a minor peakin both cases. However, during the latter elution, the same peptidesequence with a â-eliminated cysteine was the base peak in themass spectrum but was not observed during ammonium phos-phate elution. It has been reported that alkylation of free cysteineprevents â-elimination at the residue.43 Therefore, the observedâ-eliminated peptide must have been derived from retention andâ-elimination of the free cysteine-containing peptide and not ofthe alkylated peptide. The â-elimination of cysteine was readilyrecognizable as a -34 Da loss from the free cysteine-containingpeptide or as a -91 Da difference from carbamidomethylatedcysteine-containing peptides, and as such, was distinguishablefrom â-elimination of phosphoserine for known sequences. Alter-native protection of cysteine by oxidation to cysteic acid (+48 Da)using performic acid also prevented â-elimination of the sulf-hydryl17,45 and was significantly more complete than the commonlyused reduction/alkylation protocol (data not shown). Performicacid treatment is considerably harsher and oxidizes methionineto the sulfone (+32 Da) as well as tryptophan (+48 Da). However,these modifications can be included into search parameters tofacilitate database matching of mass spectra. To avoid derivatiza-tion of free cysteine-containing peptides on iron(III)-IMAC resin,performic acid pretreatment of proteins prior to enzymatic diges-tion was the preferred method to cleave disulfides and protectcysteines.

â-Elimination of a phosphopeptide generates a product identicalto dehydration of the unphosphorylated sequence. This can beconfusing if both the phosphorylated and unphosphorylated formsof a peptide are present in the initial sample and is furthercomplicated if the unphosphorylated peptide undergoes facile lossof water. Additionally, the double bond formed from eliminationof the phosphate, although stable during MS analysis, is anexcellent substrate for nucleophilic addition. In preliminaryâ-elimination time-course experiments over a 24-h period, 10-20% of the reaction products involved water addition across thedouble bond (data not shown). Although this was an excessivereaction time, the potential for unwanted nucleophilic additionexists, particularly if samples are held in storage. Undesiredaddition and possible confusion of â-eliminated phosphopeptideswith unphosphorylated dehydrated peptides can be avoided by

(42) Goshe, M. B.; Veenstra, T. D.; Panisko, E. A.; Conrads, T. P.; Angell, N. H.;Smith, R. D. Anal. Chem. 2002, 74, 607-616.

(43) Simpson, D. L.; Hranisavljevic, J.; Davidson, E. A. Biochem. 1972, 11, 1849-1856.

(44) Ramadan, N.; Porath, J. J. Chromatogr. 1985, 321, 93-104.(45) Meyer, H. E.; Hoffmann-Posorske, E.; Heilmeyer, L. M., Jr. Methods Enzymol.

1991, 201, 169-185.


further derivatization of the â-eliminated phosphopeptide byMichael addition.

Current methods employing Michael addition derivatizationof â-eliminated peptides frequently utilize thioalkane nucleo-philes.17-19,22 Although these are excellent reagents for Michaeladdition, they are also malodorous and generally poorly solublein water. The latter can significantly retard reaction times inaqueous media without additional solvent components to helpsolubilize the thioalkane. Recently, sodium sulfite has beenemployed to derivatize â-eliminated phosphoserine and phospho-threonine residues to cysteic acid and â-methylcysteic acid,respectively, for mass spectrometric analysis.24 The sodium sulfiteprocedure is similar to a previously reported method for deriva-tization of O-linked glycosylation.38 Sodium sulfite is water-solubleand odorless and is reported to perform Michael addition moreefficiently than thioalkanes.41 Unfortunately, the addition of excesssodium necessitates sample purification prior to MS analysis. Moresignificantly, the cysteic acid generated from derivatization ofâ-eliminated phosphoserine with sodium sulfite is identical tocysteic acid generated by performic acid oxidation of cysteine.This ambiguity renders the sulfite nucleophile incompatible withprotocols employing performic acid oxidation of cysteine-contain-ing proteins as presented in this paper.

To explore on-resin â-elimination and Michael addition of iron-(III)-IMAC-bound phosphopeptides, 2-aminoethanethiol was usedas the nucleophile. This reagent is odorless and very water soluble.The reaction therefore proceeded rapidly and efficiently in waterwithout the need for additional solvent components. Addition of2-aminoethanethiol to â-eliminated phosphopeptides generated aside-chain stable to PSD/LIFT, and the derivatized residue wasdistinguishable by its unique mass (146 Da for derivatized serineand 160 Da for derivatized threonine). Michael addition wasperformed concurrently with â-elimination directly on iron(III)-IMAC resin using low concentrations of 2-aminoethanethiol. Thenucleophile was fully compatible with MS analysis using DHBand CHCA, and no purification additional to barium precipitationwas required before spotting samples onto MALDI targets.

Secondary derivatization of â-eliminated phosphopeptides byMichael addition can greatly increase the flexibility of the protocol.Sulfur nucleophiles incorporating or bridging to fluorescent,27

isotopic,19 or affinity labels17,18 have been previously employed tomark â-eliminated substrates. These methods can facilitate selec-tive purification and quantitation of phosphopeptides and shouldbe compatible with the protocol for â-elimination and Michaeladdition on iron(III)-IMAC resin presented here. Recently, Michaeladdition with 2-dimethylaminoethanethiol to derivatize â-elimi-nated phosphoserine was also reported.25 Oxidation of the thio-ether-bridged Michael addition product to the sulfonyl-bridgedderivative generated a labile substituent effective as a precursorion for precursor ion scanning-based detection of phosphopeptides.The derivatized phosphoserine was then readily detected asdehydroalanine during MS/MS. In preliminary work for this study,thiocholine was trialed as a water-soluble nucleophile for Michaeladdition and was observed to undergo facile PSD during MALDIin DHB matrix. Thiocholine and 2-dimethylaminoethanethiol arestructurally analogous and would likely behave similarly duringon-resin â-elimination and Michael addition of phosphopeptideson iron(III)-IMAC resin. Consequently, the precursor ion-scanning

method is also likely to be compatible with the on-resin protocolpresented here.

A potential disadvantage of Michael addition is the formationof diastereoisomers during nucleophilic addition at the â-elimi-nated residue. These diastereoisomers have been reported toresolve independently during HPLC in some studies employingâ-elimination/Michael addition protocols.17,21,26,42 Although thisphenomenon was not problematic in this study, if LC/MS wereto be applied, then the sensitivity of detection of phosphorylationsites after derivatization would be reduced if the diastereoisomerseluted separately. Since LC/MS was not employed in this work,the general suitability of â-elimination/Michael addition protocolswith LC/MS was not assessed.

Identification of Novel Phosphorylation Sites on BovineFetuin and p120ctn. Treatment of a tryptic digest of the bovineglycoprotein fetuin with iron(III)-IMAC subfractionated phospho-peptides but excluded glycopeptides. Iron(III)-IMAC alone enabledthe detection of three phosphopeptides corresponding to a singlephosphorylation within residues 132-143 and single and doublephosphorylations within residues 313-333. The phosphorylationwithin residues 132-143 was identified to occur at serine 138 byPSD/LIFT on the phosphorylated peptide (data not shown),consistent with an earlier reported identification of the samephosphorylation site in human fetuin.30 Identification of the otherunderivatized phosphorylation sites by PSD/LIFT was not possiblewithout the presented derivatization strategy. Iron(III)-IMAC withon-resin â-elimination detected single, double, and triple phos-phorylations within residues 313-333. Derivatization by â-elimina-tion enabled the precise identification of the sites as serines 320,323, and 325 by PSD/LIFT. The peptide 132-143 and itsphosphorylated counterpart were not observed after iron(III)-IMAC and â-elimination. These peptides contain a cysteic acidresidue and were likely lost into the barium pellet duringammonium sulfate precipitation.

Serine phosphorylation of fetuin has been postulated to occurat SXE motifs, where X is glycine or alanine.30,31 Phosphorylationat serine 325, which is followed by glycine and glutamic acid,conforms with this consensus sequence. However, the phosphor-ylation at serine 320 indicates that substitution of glycine or alaninewith valine also satisfies the requirements for site recognition bythe kinase. Finally, phosphorylation of serine 325 may act as aglutamic acid mimic, subsequently enabling phosphorylation ofserine 323 within the sequence S323-S324-S325 if X can also besubstituted by serine. These data therefore suggest that neutralamino acids other than glycine or alanine should be included inthe consensus sequence.

The biological function of fetuin glycoprotein is unclear, butsome downstream effects are phosphorylation-dependent. Forexample, the phosphorylated form of rat fetuin, also known aspp63, inhibited receptor tyrosine kinase autophosphorylation46 anddecreased HGF-stimulated DNA synthesis in hepatocytes47 whereasthe unphosphorylated form did not. The triply phosphorylatedpeptide sequence identified by the iron(III)-IMAC-â-eliminationprotocol is essentially conserved across porcine, bovine, ovine,and murine species32,37 but is apparently absent from the human

(46) Auberger, P.; Falquerho, L.; Contreres, J. O.; Pages, G.; Le Cam, G.; Rossi,B.; Le Cam, A. Cell 1989, 58, 631-640.

(47) Ohnishi, T.; Nakamura, O.; Arakaki, N.; Daikuhara, Y. Eur. J. Biochem.1997, 243, 753-761.


form. Further studies are required to evaluate the functionalimplications of phosphorylation in this region.

To determine the ability of the protocol to detect phosphory-lation sites on protein derived from mammalian cells, the methodwas applied to GST-p120ctn fusion protein overexpressed in Cos-7cells and isolated by affinity chromatography on glutathione beads.A host of phosphorylated peptides were detected and derivatizedby the presented protocol, including singly, doubly, and triplyphosphorylated tryptic peptides of p120ctn. The majority of theseidentifications will be published in a forthcoming manuscript, butto demonstrate the efficacy of the protocol, the characterizationof two phosphorylation sites are presented in this report. Sub-fractionation of the GST-p120ctn tryptic digest by iron(III)-IMACenhanced the ion signals attributed to the phosphopeptides andclarified the spectra by eliminating overlapping ion signals fromnonphosphorylated species. MS analysis of the ammoniumphosphate eluent detected ions at m/z 1777.84 and 2053.79,corresponding to the p120ctn-tryptic peptides HYEDGYPGGSD-NYGSLSR and APSRQDVYGPQPQVR (residues 216-233 and250-264 of murine p120 isoform 1A, respectively) being singlyphosphorylated. Alternative elution of the phosphopeptides fromiron(III)-IMAC by â-elimination or by â-elimination with Michaeladdition demonstrated single derivatization within these sequencesby loss of 98 or 21 Da, respectively, confirming the phosphory-lation. Derivatization also enabled the identification of serine 230and serine 252 in these peptides as the sites of phosphorylationby PSD/LIFT.

p120ctn is a member of the catenin family of proteins thatincludes â-catenin, R-catenin, and plakoglobin. p120ctn bindsdirectly to the membrane-proximal region of E-cadherin and iscrucial for the ability of the E-cadherin complex to mediate cell-cell adhesion. Both tyrosine and serine/threonine phosphorylationof p120ctn have been proposed to regulate the adhesive functionof the E-cadherin complex.48-50 A number of tyrosine phosphor-ylation sites have been mapped on p120ctn recently,50,51 but thesites of serine/threonine phosphorylation remain unknown.Identification of sites of serine/threonine phosphorylation aspresented in this work should allow the effect of p120ctnphosphorylation on cadherin function to be studied in more detail.

Limitations of the On-Resin Derivatization Strategy.Iron(III)-IMAC for the enrichment of phosphopeptides is recog-nized to retain some unphosphorylated peptides in addition to thedesired phosphorylated sequences. Frequently, acidic peptides arethe primary culprits,7-10 although other sequences, includingcysteine, histidine, and tyrosine-rich peptides, have also beenreported to bind.9-11 In this work, â-elimination of phosphoserineand phosphothreonine was initially developed as a method tospecifically elute the phosphopeptides from the resin but leaveother nonphosphorylated peptides bound to the resin. Unfortu-nately, under the conditions required for effective â-elimination,hydroxide-mediated elution from iron(III)-IMAC also occurred.This rendered the elution method unspecific for phosphopeptides,although only phosphoserine and phosphothreonine were deriva-

tized. Despite this, the specific nature of the derivatization enabledthe unambiguous marking of serine and threonine phosphorylatedpeptides at the phosphorylated residue. These phosphopeptideswere easily distinguishable from unphosphorylated species by themass shift resulting from derivatization. Subsequent Michaeladdition at the â-elimination sites is also a powerful tool topotentially identify serine and threonine phosphorylation bydiscriminative features attributed to the Michael addition nucleo-phile.

Although phosphothreonine phosphorylations were identifiedfrom the mass measurements and mass shifts after â-elimination,the signal intensities after â-elimination were lower than expected,suggestive of incomplete derivatization. Underivatized phospho-peptides were generally not observed after â-elimination/Michaeladdition, probably because of trapping of the phosphopeptides inthe barium sulfate precipitate. This prevented semiquantitativeestimation of the extent of the derivatization from mass spectraldata. However, â-elimination of phosphothreonine is recognizedto proceed slower than for phosphoserine.26 The reaction of bothresidues also exhibits a degree of sequence dependence, notablypoor reactivity of phosphoserine/threonine preceding proline,17

as well as preceding glutamic or aspartic acid or the C-terminal.41

Although derivatization of phosphothreonine can be achieved,21,22,24,41

improving the â-elimination of this residue would increase theoverall reaction efficiency and, therefore, the sensitivity of detect-ing threonine phosphorylation.

Glycosylation did not appear to contribute to retention ofpeptides on iron(III)-IMAC resin. However, since the resin canalso retain nonphosphorylated peptides, it is conceivable that anO-linked glycopeptide rich in acidic or other residues recognizedto adhere to iron(III)-IMAC resin may also be retained, togetherwith phosphopeptides. Such a glycopeptide would likely bederivatized along with phosphopeptides and confuse the identifica-tion of phosphorylation sites. Although this was never observedto occur in any of the experiments conducted, and glycosylationmay, in fact, sterically hinder peptide binding to IMAC resin, thepossibility, however small, must be acknowledged. Phosphopep-tides can still be verified by comparison of the IMAC eluent fromâ-elimination with the eluent from ammonium phosphate or othertraditional elution reagents. Derivatization of the phosphate isrecognizable by the mass shift per phosphate present (-98 Dafor â-elimination, -21 Da for â-elimination and Michael additionwith 2-aminoethanethiol), whereas derivatization of O-glycosylatedpeptides generates a mass shift dependent on the extent ofglycosylation but distinguishable from phosphate derivatization.Methods that improve the selectivity of IMAC for phosphopep-tides, by minimizing or preventing the interaction of nonphos-phorylated peptides with the resin, would also address thepotential ambiguity of O-linked glycopeptides derivatizing identi-cally as phosphopeptides. This problem highlights the importanceof further refining the IMAC system to create a more definitivemethod for phosphopeptide isolation. Methyl esterification ofacidic residues has been employed to improve IMAC selectivityfor phosphopeptides and was very effective in facilitating thephosphoproteome analysis of whole cell lysate.8 However, furtherexperiments would be desirable to fully characterize the efficiencyand specificity of the methylation treatment and assess potentialpitfalls. The retention of histidine, tyrosine, and cysteine-containing

(48) Aono, S.; Nakagawa, S.; Reynolds, A. B.; Takeichi, M. J. Cell Biol. 1999,145, 551-562.

(49) Ohkubo, T.; Ozawa, M. J. Biol. Chem. 1999, 274, 21409-21415.(50) Ozawa, M.; Ohkubo, T. J. Cell Sci. 2001, 114, 503-512.(51) Mariner, D. J.; Anastasiadis, P.; Keilhack, H.; Bohmer, F. D.; Wang, J.;

Reynolds, A. B. J. Biol. Chem. 2001, 276, 28006-28013.


peptides to IMAC resin also needs to be addressed to improvethe specificity of the technique for phosphopeptides. However,solid-phase derivatization of phosphopeptides directly on IMACresin, as described here, provides a complementary method tospecifically mark phosphopeptides after IMAC subfractionationand enrichment. Although this does not improve the specificityof IMAC for phosphopeptides, it can enable their discriminationfrom other retained species and facilitate their mass spectrometricidentification.

CONCLUSIONA protocol combining IMAC and â-elimination strategies has

been developed to improve the identification of phosphorylationsites by mass spectrometry. The combination of these strategiescircumvents inherent disadvantages of the individual techniques.IMAC treatment of samples prior to â-elimination-based elutionand derivatization precludes the â-elimination of O-glycosylatedpeptides. â-Elimination-mediated elution of phosphopeptides im-proves the recovery of multiply phosphorylated species. Moreover,the derivatization distinguishes the phosphorylation site(s) andfacilitates mass spectrometric sequencing by avoiding phosphate-related ionization suppression of phosphopeptides and problemswith phosphate lability during both MS and MS/MS. Michaeladdition using 2-aminoethanethiol can be performed concurrentlywith â-elimination to further modify the phosphorylation sites. Theease of concurrent Michael addition potentially increases theflexibility of the protocol, enabling the labeling of phosphorylationsites with discriminative physicochemical properties attributed to

tailored nucleophiles. The presented protocol is designed with aminimum of sample preparation steps and sample transfers andavoids further chromatographic purification to maximize thesensitivity of the technique. To demonstrate the effective applica-tion of the protocol, the method was employed to identify novelphosphorylation sites on the bovine glycoprotein fetuin and thecell adhesion protein p120ctn. A triply phosphorylated sequencecorresponding to residues 313-333 of bovine fetuin was isolatedand derivatized, and the major phosphorylation site was identified.No glycosylation sites were detected in this analysis, affirmingthe ability of the protocol to promote the selective derivatizationand identification of phosphorylation sites only. A host of phos-phorylation sites were also detected on the cell adhesion proteinp120ctn derived from the Cos-7 mammalian cell line.

ACKNOWLEDGMENTThe authors thank Steve Corless and Malcolm Saxton (Ludwig

Institute for Cancer Research, University College London branch,London, U.K.) for their assistance in acquiring mass spectrometricdata. Clemens Franz was supported by a scholarship fromBoehringer-Ingelheim Fonds and Karin Barnouin was supportedby the Kay Kendall Leukaemia Fund. This work was supportedby the Ludwig Institute for Cancer Research.

Received for review February 11, 2003. Accepted April 10,2003.

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