Selective Zirconium Dioxide-Based Enrichment ofPhosphorylated Peptides for Mass SpectrometricAnalysis

Hye Kyong Kweon and Kristina Håkansson*

Department of Chemistry, University of Michigan, 930 North University Avenue, Ann Arbor, Michigan 48109-1055

Due to the dynamic nature and low stoichiometry ofprotein phosphorylation, enrichment of phosphorylatedpeptides from proteolytic mixtures is often necessary priorto their characterization by mass spectrometry. Severalphosphopeptide isolation strategies have been presentedin the literature, including immobilized metal ion affinitychromatography. However, that technique suffers frompoor selectivity and reproducibility. Recently, titaniumdioxide-based columns have been successfully employedfor phosphopeptide enrichment by several research groups.Here, we present, to our knowledge, the first demonstra-tion of the utility of zirconium dioxide microtips forphosphopeptide isolation prior to mass spectrometricanalysis. These microtips display similar overall perfor-mance as TiO2 microtips. However, more selective isola-tion of singly phosphorylated peptides was observed withZrO2 compared to TiO2 whereas TiO2 preferentially en-riched multiply phosphorylated peptides. Thus, these twochromatographic materials possess complementary prop-erties. For r- and â-casein, Glu-C digestion provided noevident advantage compared to trypsin digestion whencombined with TiO2 or ZrO2 phosphopeptide enrichment.

Reversible phosphorylation of proteins catalyzed by kinasesand phosphatases is recognized as a primary regulatory mecha-nism in eukaryotic cells.1,2 This mechanism controls a wide varietyof cellular events, including signal transduction, gene expression,metabolism, and cell growth, division, and differentiation. Toachieve detailed insights into phosphorylation-controlled cellularregulation, it is important to identify phosphorylated proteins anddetermine the precise sites of phosphorylation within thoseproteins as well as the phosphorylation residency at a certainmetabolic stage. However, phosphorylation profiling still remainsa challenge due to its low and dynamic stoichiometry.

Mass spectrometry (MS) has been widely applied as a powerfultool to characterize protein modifications, including phosphory-lation, due to its high sensitivity and capability of rapid sequencingby tandem mass spectrometric (MSn) techniques.3-8 However, the

commonly low occurrence of phosphorylation is still an issue inmass spectrometric analysis for localization of protein phospho-rylation sites. Thus, prior isolation and enrichment of phospho-peptides from a proteolytic peptide mixture (resulting from anenzymatic digest of a protein) is often required. This procedureserves to eliminate interferences and to enhance the signal fromphosphopeptides, which often exhibit signal suppression in MS.Commonly used enrichment strategies include immobilized metalion affinity chromatography (IMAC)9-11 incorporating Fe3+, Ga3+,or other metal ions (including Zr4+; however, that approach ismarkedly different from the use of zirconium oxide, which ispresented here), immunoprecipitation with phosphoprotein-specific antibodies,12,13 and the addition of an affinity tag tophosphorylated amino acids through chemical reactions.14 Ofthese methods, IMAC is the most widely used, both on-line andoff-line.11,15-17 However, nonspecific binding of nonphosphorylatedacidic peptides and the complexity of factors affecting phospho-peptide binding and release often result in low specificity andsensitivity for target phosphopeptides. Recently, highly specificphosphopeptide isolation has been demonstrated with titaniumdioxide columns in both off-line18 and on-line19,20 liquid chroma-

* To whom correspondence should be addressed. E-mail: kicki@umich.edu.Phone: (734) 615-0570. Fax: (734) 647-4865.(1) Graves, J. D.; Krebs, E. G. Pharmacol. Ther. 1999, 82, 111-121.(2) Hunter, T. Cell 2000, 100, 113-127.(3) McLachlin, D. T.; Chait, B. T. Curr. Opin. Chem. Biol. 2001, 5, 591-602.

(4) Mann, M.; Ong, S.-E.; Gronborg, M.; Steen, H.; Jensen, O. N.; Pandey, A.Trends Biotechnol. 2002, 20, 261-268.

(5) Schweppe, R. E.; Haydon, C. E.; Lewis, T. A.; Resing, K. A.; Ahn, N. G.Acc. Chem. Res. 2003, 36, 453-461.

(6) Chalmers, M. J.; Kolch, W.; Emmett, M. R.; Marshall, A. G. J. Chromatogr.,B 2004, 803, 111-120.

(7) Cantin, G. T.; Yates, J. R. J. Chromatogr., A 2004, 1053, 7-14.(8) Meng, F.; Forbes, A. J.; Miller, L. M.; Kelleher, N. L. Mass Spectrom. Rev.

2005, 24, 57-77.(9) Neville, D. C.; Rozanas, C. R.; Price, E. M.; Gruis, D. B.; Verkman, A. S.;

Townsend, R. R. Protein Sci. 1997, 6, 2436-2445.(10) Posewitz, M. C.; Tempst, P. Anal. Chem. 1999, 71, 2883-2892.(11) Nuhse, T. S.; Stensballe, A.; Jensen, O. N.; Peck, S. C. Mol. Cell. Proteomics

2003, 2, 1234-1243.(12) Pandey, A.; Podtelejnikov, A. V.; Blagoev, B.; Bustelo, X. R.; Mann, M.;

Lodish, H. F. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 179-184.(13) Ficarro, S. B.; Chertihin, O.; Westbrook, V. A.; White, F.; Jayes, F.; Kalab,

P.; Marto, J. A.; Shabanowitz, J.; Herr, J. C.; Hunt, D. F.; Visconti, P. E. J.Biol. Chem. 2003, 278, 11579-11589.

(14) McLachlin, D. T.; Chait, B. T. Anal. Chem. 2003, 75, 6826-6836.(15) Stensballe, A.; Andersen, S.; Jensen, O. N. Proteomics 2001, 1, 207-222.(16) Haydon, C. E.; Eyers, P. A.; Aveline-Wolf, L. D.; Resing, K. A.; Maller, J. L.;

Ahn, N. G. Mol. Cell. Proteomics 2003, 2, 1055-1067.(17) Garcia, B. A.; Shabanowitz, J.; Hunt, D. F. Methods 2005, 35, 256-264.(18) Sano, A.; Nakamura, H. Anal. Sci. 2004, 20, 861-864.(19) Pinkse, M. W.; Uitto, P. M.; Hilhorst, M. J.; Ooms, B.; Heck, A. J. R. Anal.

Chem. 2004, 76, 3935-3943.(20) Larsen, M. R.; Thingholm, T. E.; Jensen, O. N.; Roepstorff, P.; Jorgensen,

T. J. D. Mol. Cell. Proteomics 2005, 4, 873-886.

Anal. Chem. 2006, 78, 1743-1749

10.1021/ac0522355 CCC: $33.50 © 2006 American Chemical Society Analytical Chemistry, Vol. 78, No. 6, March 15, 2006 1743Published on Web 02/07/2006

tography/MS and matrix-assisted laser desorption/ionization-MS.20 This approach is based on specific chemisorption ofphosphate groups on the surface of titanium oxide and was shownto result in less nonspecific binding than IMAC. In addition,column preparation is more straightforward, eliminating the metalion chelating and washing steps required for preparation of IMACcolumns. An alternative strategy involving magnetic Fe3O4/TiO2

core/shell nanoparticles as affinity probes for phosphopeptideshas also been recently demonstrated.21 Finally, utilization of ametal hydroxide, Al(OH)3, for phosphopeptide and phosphoproteinbinding also proved effective and more selective than commercialphosphoprotein enrichment kits.22 Here, we present enrichmentof phosphorylated peptides on the surface of zirconium dioxideloaded in a microtip. ZrO2 has drawn attention as chromatographiccolumn material due to its physical (e.g., toward extremes of pH)and thermal stability and its unique surface chemistry.23-25

However, to our knowledge, its use for phosphopeptide isolationhas not previously been reported in the literature. We investigatedthe performance of these microtips for trypsin and Glu-C pro-teolytic digests of R- and â-casein and compared ZrO2 phospho-peptide binding specificity and recovery to TiO2-based enrichment.Mass spectrometric phosphopeptide analysis was performed withelectrospray ionization (ESI) in both negative and positive ionmodes with a Fourier transform ion cyclotron resonance (FT-ICR)instrument.

EXPERIMENTAL SECTIONReagents and Sample Preparation. R-Casein and â-casein

from bovine milk (Sigma, St. Louis, MO) were prepared in 25mM ammonium bicarbonate (Fisher Scientific, Fair Lawn, NJ)buffer to a concentration of 25 µM. Trypsin (Promega, Madison,WI) digestion was performed for 15 h at 37 °C at an enzyme/substrate ratio of 1:50. Glu-C (Roche, Penzberg, Germany)digestion also proceeded for 15 h but at 25 °C at an enzyme/substrate ratio of 1:100. Microtips filled with ZrO2 and TiO2 (25or 50 µg) were either provided as a gift or purchased from Glygen(Columbia, MD) and used without further modification. Foroptimization of the phosphopeptide enrichment conditions, bindingsolutions of different pH were prepared by adding variousconcentrations (see below) of formic acid (Acros Organics, FairLawn, NJ) or ammonium bicarbonate to HPLC grade water(Fisher). Washing solutions consisted of pure water (pH 6.5), 250mM ammonium acetate (pH 6.9), 100 mM ammonium bicarbonate(pH 8.0), or 75 mM ammonium hydroxide (pH 10.5). Finally, 0.5%piperidine (pH 11.5) or 0.25 or 0.5% ammonium hydroxide (pH10.8 and 11, respectively) were used for eluting bound phospho-peptides. Peptide mixtures from the enzymatic digests werefractionated into 1-100-pmol aliquots, dried down in a vacuumconcentrator (Eppendorf, Hamberg, Germany), reconstituted inbinding solution, and loaded onto microtips that had beenequilibrated with the same binding solution. Unbound peptides

were removed with washing solution, and bound peptides wereeluted with the high-pH elution solutions. Eluted peptide sampleswere dried down and reconstituted in 2-propanol/acetonitrile/water (1:1:2) with 0.25% piperidine for negative ion mode and inacetonitrile/water (1:1) with 0.1% formic acid for positive ion modeESI-FT-ICR MS analysis.

Mass Spectrometry and Data Analysis. Mass measurementswere performed with a 7-T hybrid quadrupole (Q)-FT-ICR massspectrometer (Bruker Daltonics, Billerica, MA) equipped with anApollo electrospray ion source. The ESI flow rate was 50 µL/h,and nebulization was assisted by N2 gas. Peptide ions wereexternally accumulated for 0.5 s in a hexapole prior to beingtransferred to the ICR cell and captured by gated trapping. Theexternal accumulation and transfer events were looped twice priorto excitation and detection. Spectra were acquired with XMASS(version 6.1, Bruker Daltonics) from m/z 250 to 2500 with 512kdata points and summed over four or eight scans. Data analysiswas performed with the MIDAS analysis software.26 Data wereHanning apodized and zero filled once prior to fast Fouriertransformation and magnitude calculation. Lists of expectedproteolytic peptide masses were generated with the Peptide Masstool on the Expasy web site (www.us.expasy.org) using accessionnumbers P02662 and P02663 for the two forms (S1 and S2) ofR-casein and P02666 for â-casein. Another tool on the Expasy website, Compute pI, was used to compute pI values of certainpeptides. ZrO2 and TiO2 phosphopeptide binding selectivity wasdetermined by dividing the sum of the abundances of all phos-phopeptide isotopomers with the sum of all observed isotopomersin the same spectrum before and after phosphopeptide enrich-ment. Only peaks with a signal-to-noise (S/N) ratio above threewere included in the analysis. The noise level of a spectrum wasdetermined from the root-mean-square signal in a region devoidof peptide peaks. The Peptide Mass tool automatically considersphosphorylation when that modification is present in the database.However, we identified additional phosphopeptides by allowingdifferent numbers of phosphate groups. Those assignments wereconfirmed by collision-activated dissociation in the externalhexapole with argon as collision gas. Neutral loss of 98 Da(corresponding to phosphoric acid) was used as a marker ion.

RESULTS AND DISCUSSIONOptimization of Zirconium Dioxide Phosphopeptide En-

richment Conditions. Zirconium oxide is known to have am-photeric properties; that is, it can react either as a Lewis acid orbase depending on the pH of the reaction solution. This propertyis a result of unsatisfied valencies of both oxygen and zirconiumatoms in the surface layer.23 In acidic solution, ZrO2 behaves as aLewis acid with positively charged zirconium atoms, therebydisplaying anion-exchange properties.27 For example, high bindingaffinity for polyoxy anions, including phosphate, borate, carboxy-late, and sulfate, has been demonstrated.28 The binding constantof phosphate ions is markedly higher than for other Lewisbases,29,30 suggesting that high binding selectivity of phosphory-(21) Chen, C. T.; Chen, Y. C. Anal. Chem. 2005, 77, 5912-5919.

(22) Wolschin, F.; Wienkoop, S.; Weckwerth, W. Proteomics 2005, 5, 4389-4397.

(23) Nawrocki, J.; Rigney, J.; McCormick, A.; Carr, P. W. J. Chromatogr., A 1993,657, 229-282.

(24) Nawrocki, J.; Dunlap, C.; McCormick, A.; Carr, P. W. J. Chromatogr., A2004, 1028, 1-30.

(25) Hoth, D. C.; Rivera, J. G.; Colon, L. A. J. Chromatogr., A 2005, 1079, 392-396.

(26) Senko, M. W.; Canterbury, J. D.; Guan, S.; Marshall, A. G. Rapid Commun.Mass Spectrom. 1996, 10, 1839-1844.

(27) Amplett, C. B. Inorganic Ion Exchangers; Elsevier: Amsterdam, TheNetherlands, 1964.

(28) Kraus, K. A.; Philips, H. O.; Carlson, T. A.; Johnson, J. S. Proceedings ofthe Second International Conference on Peaceful Uses of Atomic Energy,Geneva, Switzerland, 1958; 3.

1744 Analytical Chemistry, Vol. 78, No. 6, March 15, 2006

lated peptides over nonphosphorylated acidic peptides should beachievable with ZrO2 upon careful selection of the pH. Figure 1ashows a negative mode ESI FT-ICR mass spectrum of a 100 pmolnonenriched tryptic digest of R-casein. Identified phosphopeptidesare labeled with numbers from 1 to 12 and included in Table 1.Only the most abundant phosphopeptides are labeled in thespectrum. Spectra obtained following ZrO2 enrichment of phos-phopeptides from the same digest at different binding solutionpH values are shown in Figure 1b-e (100 pmol/spectrum). Allthese enrichment experiments involved water washing and elutionin 0.5% piperidine. These data show that it is critical to use low-pH solutions to achieve high phosphopeptide binding selectivity.At pH 2 (Figure 1b), only one nonphosphorylated peptide (doublydeprotonated EPMIGVNQELAYFYPELFR, corresponding to aminoacid residues 148-166 in the S1 form of R-casein) is detected withS/N > 3. This peptide contains three acidic glutamate residuesand has a pI of 4.25. The data obtained at pH 3 (Figure 1c) do notdiffer drastically from those obtained at pH 2. However, alreadyat this slightly higher pH, an increased abundance of nonphos-

phorylated peptides is seen with the peptide mentioned abovebeing detected both in its doubly and triply deprotonated forms.In addition, a singly deprotonated peptide with one glutamateresidue (VNELSK, corresponding to amino acid residues 52-57in the S1 form of R-casein with a pI of 5.97) is observed. However,phosphopeptides dominate the mass spectrum. Binding in water(Figure 1d) still provides phosphopeptide enrichment comparedto the spectrum obtained without the use of ZrO2 tips (Figure1a). However, the selectivity is further compromised. At higherpH (Figure 1e) for which the Lewis acid property of ZrO2

diminishes, phosphopeptides are no longer the dominant speciesand a very limited amount of peptides binds to the tip.

In chromatographic applications of zirconia, both the pH andthe ionic strength of the mobile phase can influence retentionproperties due to a mixed-mode ligand- and ion-exchange analytebinding mechanism.23 We did not control the ionic strength inour pH-dependence experiments; however, the carboxylate ionsoriginating from the formic acid added to lower the pH would beexpected to decrease phosphopeptide binding due to competitionfor ZrO2 acidic sites. Thus, because we observed increased ratherthan decreased binding at lower pH, we believe the pH is far moreimportant than ionic strength under our experimental conditions.

To achieve maximum recovery of bound phosphopeptides, wealso optimized the washing and elution conditions and found thatpure water was the best washing solution. We also found that apH as high as 10.8-11.5 was needed for optimum elutionperformance. The highest phosphopeptide recovery was achievedwith 0.5% piperidine (pH 11.5) and that elution condition was usedin all experiments presented below.

ZrO2 and TiO2 Phosphopeptide Enrichment from TrypticDigests of Phosphoproteins. To establish how ZrO2 phospho-peptide enrichment compares to TiO2 enrichment, we applied theprotocol established above to both ZrO2 and TiO2 microtips (50µg). Panels a-c in Figure 2 show this comparison for a trypticdigest of R-casein. In our hands, either technique is more selectivethan IMAC, similar to the results obtained previously by Larsenet al.20 However, we found that the relative abundance of singlyversus multiply phosphorylated peptides varies between ZrO2 andTiO2 with singly phosphorylated peptides being enriched to ahigher extent with ZrO2: For the R-casein tryptic digest, the mostabundant signal following ZrO2 enrichment (Figure 2b) originatesfrom the 2- charge state of the singly phosphorylated peptideYKVPQLEIVPNSAEER, corresponding to amino acid residues119-134 in the S1 form of R-casein (peptide 7 in Table 1) followedby the 2- charge state of the doubly phosphorylated peptideDIGSESTEDQAMEDIK, corresponding to amino acid residues58-73 in the S1 form of R-casein (peptide 6 in Table 1). Abundantsignal (31% relative abundance) is also observed from the 3-

charge state of the singly phosphorylated peptide 7 as well asminor signal from its quadruply deprotonated form. In addition,a shorter version of peptide 7, corresponding to no missed trypsincleavage sites (amino acid residues 121-134 in the S1 form ofR-casein, peptide 4 in Table 1), is detected following ZrO2

enrichment. By contrast, the doubly phosphorylated peptide 6 isthe most abundant species following TiO2 enrichment (Figure 2c),and the abundance for both the 2- and 3- charge states of thesingly phosphorylated peptide 7 is lower in that spectrum (53 and12%, respectively) compared to the spectrum obtained following

(29) Blackwell, J. A.; Carr, P. W. J. Chromatogr., A 1991, 549, 43-57.(30) Blackwell, J. A.; Carr, P. W. J. Chromatogr., A 1991, 549, 59-75.

Figure 1. Negative mode ESI FT-ICR mass spectra (8 scans) from100 pmol of a tryptic digest of R-casein obtained prior to ZrO2

phosphopeptide enrichment (a) and following enrichment at variousbinding pH values (b-e). Phosphopeptides are labeled with numbersthat are identified in Table 1. Nonphosphorylated peptides observedfollowing enrichment are labeled with their corresponding amino acidresidue numbers and R-casein isoform. *, noise.

Analytical Chemistry, Vol. 78, No. 6, March 15, 2006 1745

ZrO2 enrichment (74 and 31%, respectively). In addition, the 4-

charge state of peptide 7 is not detected following TiO2 enrich-ment, nor is the singly phosphorylated peptide 4. These enrich-ments were performed from 100-pmol aliquots of the same trypticdigest. Thus, variations in the digestion conditions cannot explainthe absence of peptide 4. We believe these results constitute animportant finding because a plentitude of biological regulatorymechanisms involve a single phosphorylation event and our resultsimply that ZrO2 phosphopeptide enrichment would be preferredover other isolation strategies in the characterization of such cases.We do not believe the observed behavior is a result of irreversiblebinding of multiply phosphorylated peptides to ZrO2 because wecould still detect such phosphopeptides in the left-over solutionof tryptic peptides. Also, we do not believe the differentiationbetween singly and multiply phosphorylated peptides is a resultof our specific enrichment protocol because changing the condi-tions did not result in varied ratios of, for example, peptides 6and 7 for R-casein (Figure 1).

The finding that ZrO2 more selectively isolates singly phos-phorylated peptides as compared to TiO2 was confirmed with atryptic digest of â-casein; see Figure 2d-f. Again, the mostabundant peak following ZrO2 enrichment originates from a singlyphosphorylated peptide (the 2- charge state of FQSEEQQQT-EDELQDK, corresponding to amino acid residues 48-63 inâ-casein (peptide 13 in Table 1) whereas its abundance followingTiO2 enrichment is comparable to the quadruply phosphorylatedpeptide RELEELNVPGEIVESLSSSEESITR (peptide 17, triplydeprotonated). The latter peptide displays similar S/N ratios

following both techniques, again indicating that its lower relativeyield following ZrO2 enrichment is not due to poorer recoveryfrom the column.

The observed difference in phosphopeptide binding selectivitybetween zirconia and titania may be related to their differentsurface chemistry: First, zirconia is a stronger Lewis acid thantitania,31 which could contribute to an enhanced binding of singlyphosphorylated peptides. Second, the coordination numbers ofZr and Ti are different in the crystalline forms typically presentin chromatographic materials, six for Ti and seven for Zr,31 whichcould result in different binding properties for singly and multiplyphosphorylated peptides. However, because the surface propertiesof zirconia and titania are not well understood,24 these suggestionsare rather speculative at this point.

ZrO2 and TiO2 Phosphopeptide Enrichment from Glu-CDigests of Phosphoproteins. Regnier and co-workers haverecently shown that the use of Glu-C rather than trypsin forphosphoprotein digestion prior to IMAC phosphopeptide enrich-ment significantly reduces nonspecific binding of acidic nonphos-phorylated peptides.32 This behavior is a result of the specificityof Glu-C, which can cleave a protein (depending on the digestionconditions) at the C-terminal side of acidic amino acid residues(Asp and Glu). Thus, in theory, Glu-C digestion results in onlyone acidic amino acid residue per peptide (not counting the

(31) Grun, M.; Kurganov, A. A.; Schacht, S.; Schuth, F.; Unger, K. K. J.Chromatogr., A 1996, 740, 1-9.

(32) Seeley, E. H.; Riggs, L. D.; Regnier, F. E. J. Chromatogr., A 2005, 817,81-88.

Table 1. List of Phosphopeptides Found in Negative Mode FT-ICR Mass Spectra from Proteolytic Digests of r- andâ-Casein without (w) Phosphopeptide Enrichment and Following ZrO2 and TiO2 Enrichment

no. peptide identity enzyme phosphorylationmonoisotopic

mass method

1 R-casein-S2:153-164 trypsin 1phos:158 1465.6047 w, Zr, Ti2 R-casein-S2:141-152 trypsin 2phos:144,146 1538.5902 w, Zr, Ti3 R-casein-S2:152-164(153-165) trypsin 1phos:158 1593.6997 w, Zr, Ti4 R-casein-S1:121-134 trypsin 1phos:130 1659.7868 w, Zr, Ti5 R-casein-S1:58-73 trypsin 1phos:61,63,68a 1846.7179 w, Zr, Ti6 R-casein-S1:58-73 trypsin 2phos:61,63,68a 1926.6842 w, Zr, Ti7 R-casein-S1:119-134 trypsin 1phos:130 1950.9451 w, Zr, Ti8 R-casein-S1:118-134 trypsin 1phos:130 2079.0401 w, Zr, Ti9 R-casein-S1:74-94 trypsin 1phos:79 2719.9055 w, Zr, Ti10 R-casein-S2:16-36 trypsin 4phos:23,24,25,31 2745.9923 Zr, Ti11 R-casein-S2:61-85 trypsin 4phos:71,71,73,76 3007.0221 Zr, Ti12 R-caseinS2:57-95 trypsin 4phos:71,72,73,76 4717.9274 w, Zr, Ti13 â-casein:48-63 trypsin 1phos:50 2060.8211 w, Zr, Ti14 â-casein:45-63 trypsin 1phos:50 2431.0430 Zr15 â-casein:17-40 trypsin 4phos:30,32,33,34 2965.1571 w, Zr, Ti16 â-casein:16-40 trypsin 3phos:30,32,33,34a 3041.5000 w, Zr, Ti17 â-casein:16-40 trypsin 4phos:30,32,33,34 3121.2582 w, Zr, Ti18 R-casein-S1:126-133 Glu-C 1phos:130 937.3793 w, Zr, Ti19 R-casein-S1:55-65 Glu-C 2phos:61,63 1324.4836 w20 R-casein-S1:126-140 Glu-C 1phos:130 1818.8335 w, Zr, Ti21 R-casein-S1:55-70 Glu-C 3phos:61,63,68 1978.6556 w22 R-casein-S1:77-92 Glu-C 5phos:79,81,82,83,90 2075.6103 w, Zr, Ti23 R-casein-S2:67-83 Glu-C 4phos:71,72,73,76 2093.6080 w24 R-casein-S2:16-33 Glu-C 4phos:23,24,25,31 2353.7863 w25 â-casein:47-52 Glu-C 1phos:50 846.3160 w, Zr, Ti26 â-casein:47-57 Glu-C 1phos:50 1460.5820 Zr, Ti27 â-casein:47-59 Glu-C 1phos:50 1704.6516 w, Zr, Ti28 â-casein:21-36 Glu-C 4phos:30,32,33,34 2007.6804 w, Zr, Ti29 â-casein:21-46 Glu-C 3phos:30,32,33,34a 3110.4225 w, Zr, Ti30 â-casein:21-46 Glu-C 4phos:30,32,33,34 3190.3888 w, Zr, Ti

a All possible (known) phosphorylation sites are listed.

1746 Analytical Chemistry, Vol. 78, No. 6, March 15, 2006

phosphopeptides), hence reducing nonspecific binding. We ex-plored the use of Glu-C digestion prior to ZrO2 and TiO2

enrichment; see Figure 3. For R-casein (Figure 3a-c), Glu-Cdigestion resulted in lower overall (i.e., with or without enrich-ment) phosphopeptide signal as compared to trypsin digestion(see Table 2). With TiO2, the enrichment factor (i.e., the increasein relative phosphopeptide signal) following Glu-C digestion wasabout the same (∼2.5-fold) as for the R-casein trypsin digestwhereas a higher enrichment factor (∼4-fold relative increase)was observed with ZrO2 for the Glu-C digest (compared to ∼2.5-fold relative increase for the trypsin digest). From repeatedexperiments, the error of the tabulated selectivity values wasestimated to be ∼8%. Again, one additional singly phosphorylatedpeptide (peptide 20 in Table 1) was detected following ZrO2

enrichment as compared to TiO2 enrichment. However, severalmultiply phosphorylated peptides (peptides 22-24 in Table 1)detected prior to TiO2 and ZrO2 treatment were not observedfollowing the use of metal oxide microtips, rendering trypsindigestion the preferred strategy for R-casein.

For â-casein, the enrichment factors following both TiO2 andZrO2 treatment were similar for the Glu-C and trypsin digests (seeTable 2). Additional evidence for the selective enrichment of singlyphosphorylated peptides with ZrO2 is evident from Figure 3e andf: Following both ZrO2 and TiO2 enrichment, the most abundantspecies correspond to the 2- charge state of singly phosphorylatedKFQSEEQQQTEDE (peptide 27 in Table 1). However, the relativeabundance is 64% following ZrO2 treatment and 44% following TiO2

treatment. Again, TiO2 shows higher selectivity than ZrO2 for a

multiply phosphorylated peptide (quadruply phosphorylated LN-VPGEIVESLSSSEE, peptide 28 in Table 1): The 2- charge stateof this peptide is detected at a 27% relative abundance followingTiO2 enrichment versus 15% for ZrO2. Also, its triply deprotonatedform is only observed following TiO2 enrichment.

Sensitivity of ZrO2 and TiO2 Microtip PhosphopeptideEnrichment. All spectra presented above were obtained followingenrichment of 100 pmol of a phosphoprotein digest employingmicrotips containing 50 µg of ZrO2 or TiO2. Table 3 shows theselectivity of these microtips with decreasing amounts of anR-casein tryptic digest (50 and 25 pmol, respectively). Again, theerror of these values was found to be ∼8%. It is clear that, forR-casein, the phosphopeptide selectivity is not compromised whenthe sample amount is decreased. However, because our massspectrometer had not been optimized for high sensitivity (e.g.,we did not use nanospray or an ion funnel for improved iontransmission), we obtained poor S/N values with lower than 25pmol of sample.

To allow utilization of the ZrO2 microtips for sample amountscompatible with proteomic applications, it will be necessary toscale down the size of these columns. An initial attempt to halfthe amount of metal oxide to 25 µg resulted in improved S/Nratios of phosphopeptides from a tryptic digest of 1 pmol ofR-casein (Figure 4). These experiments were performed with adigest different from the one used for the experiments shown inFigures 1 and 2. Thus, the relative abundance of the detectedphosphopeptides is somewhat different. However, again, singlyphosphorylated peptides (peptides 4 and 7) are more abundant

Figure 2. Negative mode ESI FT-ICR mass spectra (8 scans) from 100 pmol of tryptic digests of R- and â-casein obtained prior to phosphopeptideenrichment (a, d) and following ZrO2 (b, e) and TiO2 phosphopeptide enrichment (c, f). Phosphopeptides were bound in 3.3% formic acid (pH2), washed with water, and eluted in 0.5% piperidine (pH 11.5). Highly selective enrichment of singly phosphorylated peptides (7 and 13, seeTable 1) is observed following ZrO2 enrichment. Nonphosphorylated peptides observed following enrichment are labeled with their correspondingamino acid residue numbers and isoform (for R-casein). *, noise.

Analytical Chemistry, Vol. 78, No. 6, March 15, 2006 1747

than the doubly phosphorylated peptide 6. All three peptides aremore abundant following enrichment with the smaller as com-pared to the larger column. However, it should be noted that thephosphopeptide selectivity was compromised at 1 pmol, even withthe 25-µg tip: Several singly deprotonated nonphosphorylatedpeptides as well as some unidentified peaks were observed.Calculated selectivity values were 41% for the 25-µg tip and 36%for the 50-µg tip whereas values as high as 80% were obtained athigher sample amounts (Table 3).

ZrO2 Phosphopeptide Enrichment for Positive Ion ModeMass Spectrometry. All data discussed above were obtained innegative ion mode. Although negative ionization generally pro-vides higher sensitivity for phosphopeptides, many phosphopro-teomics experiments are performed in positive ion mode becausethe dissociation behavior of peptide cations is much better

understood than that of peptide anions.33,34 However, the majorfragmentation pathway of serine- and threonine-phosphorylatedpeptides in both positive and negative modes is loss of phosphoricacid rather than backbone cleavage,3,4,6 and thus, their sequencingcan be difficult. Phosphate loss is not observed to a large extentin electron capture dissociation (ECD) of phosphopeptides,35-37

and that technique, which requires positive ion mode operation,

(33) Ewing, N. P.; Cassady, C. J. J. Am. Soc. Mass Spectrom. 2001, 12, 105-116.

(34) Bowie, J. H.; Brinkworth, C. S.; Dua, S. Mass Spectrom. Rev. 2002, 21,87-107.

(35) Stensballe, A.; Norregaard-Jensen, O.; Olsen, J. V.; Haselmann, K. F.;Zubarev, R. A. Rapid Commun. Mass Spectrom. 2000, 14, 1793-1800.

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Figure 3. Negative mode ESI FT-ICR mass spectra (8 scans) from 100 pmol of Glu-C digests of R- and â-casein obtained prior to phosphopeptideenrichment (a, d) and following ZrO2 (b, e) and TiO2 phosphopeptide enrichment (c, f). Phosphopeptides were bound in 3.3% formic acid (pH2), washed with water, and eluted in 0.5% piperidine (pH 11.5). For R-casein, poorer phosphopeptide signal is observed as compared to trypsindigestion (Figure 2) whereas similar performance is seen for â-casein with selective enrichment of a singly phosphorylated peptide (27, seeTable 1) following ZrO2 treatment. Nonphosphorylated peptides observed following enrichment are labeled with their corresponding amino acidresidue numbers. *, noise.

Table 2. Selectivitya (%) of ZrO2 and TiO2 Microtips forPhosphopeptide Enrichment of Proteolytic Digests ofr- and â-Casein

trypsindigest ofR-casein

trypsindigest ofâ-casein

Glu-Cdigest ofR-casein

Glu-Cdigest ofâ -casein

without enrichment 27 22 8 22ZrO2 enrichment 67 61 31 62TiO2 enrichment 62 65 22 77

a Defined as relative phosphopeptide signal; see text.

Table 3. Selectivitya (%) of 50-µg ZrO2 and TiO2

Microtips for Phosphopeptide Enrichment of a TrypticDigest of r-casein as a Function of Sample Amount

100 pmol 50 pmol 25 pmol

without enrichment 27 29 29ZrO2 enrichment 67 85 83TiO2 enrichment 62 77 74

aDefined as relative phosphopeptide signal; see text.

1748 Analytical Chemistry, Vol. 78, No. 6, March 15, 2006

can therefore facilitate phosphopeptide sequencing and localizesites of phosphorylation. However, ECD also requires high signal-to-noise ratios of precursor ions, and thus, enrichment of phos-phopeptides may be crucial to its applicability in phosphopro-teomics.

Figure 5 shows positive ion mode ESI-FT-ICR mass spectraobtained prior to and following ZrO2 enrichment of a tryptic digestof R-casein. Only three singly phosphorylated peptides (peptides1, 4, and 7 in Table 1) are observed without enrichment (Figure5a) whereas one more charge state of peptide 7 and four additionalphosphopeptides are detected following ZrO2 enrichment (Figure5b). Three of these additional phosphopeptides (peptides 2, 3, and6 in Table 1), of which two are doubly phosphorylated, were alsoobserved in negative ion mode (Figure 2). However, the fourthadditional phosphopeptide (labeled 31 in Figure 5b) was identifiedas the 2+ charge state of the singly phosphorylated peptideNMAINPSKENLCSTFCK, corresponding to amino acid residues40-56 in the S2 form of R-casein. No peptides including thatregion of the protein were observed in negative ion mode. Thereare also several peaks (mainly singly charged) in Figure 5b thatcould not be identified as either phosphorylated or nonphospho-rylated peptides from R-casein; however, R-casein phosphopeptidesdominate the enriched spectrum. The relative phosphopeptidesignal increased a factor of 3 following enrichment.

CONCLUSIONSIn our hands, both zirconium oxide and titanium oxide

microtips provide highly selective phosphopeptide enrichmentfrom proteolytic peptide mixtures. However, the zirconium oxidecolumns possess a unique selectivity for singly phosphorylatedpeptides whereas titanium oxide selectively enriches multiplyphosphorylated peptides. Thus, these two column materials havecomplementary properties and the choice of phosphopeptideenrichment strategy can be tailored according to the specificapplication. For R- and â-casein investigated here, the use of Glu-Crather than trypsin for proteolytic digestion did not appear toprovide an advantage. Current commercially available 50-µgmicrotips are limited to sample amounts above 1 pmol althoughfurther miniaturization of these columns appears promising.

ACKNOWLEDGMENTThis work was supported by the Searle Scholars Program and

the University of Michigan. We also thank Ashok Shukla forvaluable discussions and for providing the 25-µg microtips.

Received for review December 18, 2005. AcceptedJanuary 23, 2006.

AC0522355

Figure 4. Negative mode ESI FT-ICR mass spectra (8 scans) from1 pmol of a trypsin digest of R- casein obtained following phospho-peptide enrichment with a 50-µg ZrO2 microtip (a) and a 25-µg ZrO2

microtip (b). Downscaling the column size results in increasedsensitivity. Identified nonphosphorylated peptides are labeled withtheir corresponding amino acid residue numbers and R-caseinisoform. *, noise.

Figure 5. Positive mode ESI FT-ICR mass spectra (4 scans) from100 pmol of a tryptic digest of R-casein obtained prior to phospho-peptide enrichment (a) and following ZrO2 phosphopeptide enrichment(b). Phosphopeptides are labeled with numbers identified in Table 1,except for peptide 31 (singly phosphorylated NMAINPSKENLCST-FCK), which was not observed in negative ion mode. The relativephosphopeptide signal increased a factor of 3 following enrichment.

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