Selective Zirconium Dioxide-Based Enrichment of Phosphorylated

Feb 7, 2006 - Recently, titanium dioxide-based columns have been successfully employed for phosphopeptide enrichment by several research groups. Here,...
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Anal. Chem. 2006, 78, 1743-1749

Selective Zirconium Dioxide-Based Enrichment of Phosphorylated Peptides for Mass Spectrometric Analysis 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 of protein phosphorylation, enrichment of phosphorylated peptides from proteolytic mixtures is often necessary prior to their characterization by mass spectrometry. Several phosphopeptide isolation strategies have been presented in the literature, including immobilized metal ion affinity chromatography. However, that technique suffers from poor selectivity and reproducibility. Recently, titanium dioxide-based columns have been successfully employed for phosphopeptide enrichment by several research groups. Here, we present, to our knowledge, the first demonstration of the utility of zirconium dioxide microtips for phosphopeptide isolation prior to mass spectrometric analysis. These microtips display similar overall performance as TiO2 microtips. However, more selective isolation of singly phosphorylated peptides was observed with ZrO2 compared to TiO2 whereas TiO2 preferentially enriched multiply phosphorylated peptides. Thus, these two chromatographic materials possess complementary properties. For r- and β-casein, Glu-C digestion provided no evident advantage compared to trypsin digestion when combined with TiO2 or ZrO2 phosphopeptide enrichment.

Reversible phosphorylation of proteins catalyzed by kinases and phosphatases is recognized as a primary regulatory mechanism in eukaryotic cells.1,2 This mechanism controls a wide variety of cellular events, including signal transduction, gene expression, metabolism, and cell growth, division, and differentiation. To achieve detailed insights into phosphorylation-controlled cellular regulation, it is important to identify phosphorylated proteins and determine the precise sites of phosphorylation within those proteins as well as the phosphorylation residency at a certain metabolic stage. However, phosphorylation profiling still remains a challenge due to its low and dynamic stoichiometry. Mass spectrometry (MS) has been widely applied as a powerful tool to characterize protein modifications, including phosphorylation, due to its high sensitivity and capability of rapid sequencing by tandem mass spectrometric (MSn) techniques.3-8 However, the * To whom correspondence should be addressed. E-mail: [email protected]. 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. 10.1021/ac0522355 CCC: $33.50 Published on Web 02/07/2006

© 2006 American Chemical Society

commonly low occurrence of phosphorylation is still an issue in mass spectrometric analysis for localization of protein phosphorylation sites. Thus, prior isolation and enrichment of phosphopeptides from a proteolytic peptide mixture (resulting from an enzymatic digest of a protein) is often required. This procedure serves to eliminate interferences and to enhance the signal from phosphopeptides, which often exhibit signal suppression in MS. Commonly used enrichment strategies include immobilized metal ion affinity chromatography (IMAC)9-11 incorporating Fe3+, Ga3+, or other metal ions (including Zr4+; however, that approach is markedly different from the use of zirconium oxide, which is presented here), immunoprecipitation with phosphoproteinspecific antibodies,12,13 and the addition of an affinity tag to phosphorylated amino acids through chemical reactions.14 Of these methods, IMAC is the most widely used, both on-line and off-line.11,15-17 However, nonspecific binding of nonphosphorylated acidic peptides and the complexity of factors affecting phosphopeptide binding and release often result in low specificity and sensitivity for target phosphopeptides. Recently, highly specific phosphopeptide isolation has been demonstrated with titanium dioxide columns in both off-line18 and on-line19,20 liquid chroma(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.

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tography/MS and matrix-assisted laser desorption/ionizationMS.20 This approach is based on specific chemisorption of phosphate groups on the surface of titanium oxide and was shown to result in less nonspecific binding than IMAC. In addition, column preparation is more straightforward, eliminating the metal ion chelating and washing steps required for preparation of IMAC columns. An alternative strategy involving magnetic Fe3O4/TiO2 core/shell nanoparticles as affinity probes for phosphopeptides has also been recently demonstrated.21 Finally, utilization of a metal hydroxide, Al(OH)3, for phosphopeptide and phosphoprotein binding also proved effective and more selective than commercial phosphoprotein enrichment kits.22 Here, we present enrichment of phosphorylated peptides on the surface of zirconium dioxide loaded in a microtip. ZrO2 has drawn attention as chromatographic column 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 isolation has not previously been reported in the literature. We investigated the performance of these microtips for trypsin and Glu-C proteolytic digests of R- and β-casein and compared ZrO2 phosphopeptide binding specificity and recovery to TiO2-based enrichment. Mass spectrometric phosphopeptide analysis was performed with electrospray ionization (ESI) in both negative and positive ion modes with a Fourier transform ion cyclotron resonance (FT-ICR) instrument. EXPERIMENTAL SECTION Reagents and Sample Preparation. R-Casein and β-casein from bovine milk (Sigma, St. Louis, MO) were prepared in 25 mM 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 (25 or 50 µg) were either provided as a gift or purchased from Glygen (Columbia, MD) and used without further modification. For optimization of the phosphopeptide enrichment conditions, binding solutions of different pH were prepared by adding various concentrations (see below) of formic acid (Acros Organics, Fair Lawn, NJ) or ammonium bicarbonate to HPLC grade water (Fisher). Washing solutions consisted of pure water (pH 6.5), 250 mM 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 (pH 10.8 and 11, respectively) were used for eluting bound phosphopeptides. Peptide mixtures from the enzymatic digests were fractionated into 1-100-pmol aliquots, dried down in a vacuum concentrator (Eppendorf, Hamberg, Germany), reconstituted in binding solution, and loaded onto microtips that had been equilibrated with the same binding solution. Unbound peptides (21) Chen, C. T.; Chen, Y. C. Anal. Chem. 2005, 77, 5912-5919. (22) Wolschin, F.; Wienkoop, S.; Weckwerth, W. Proteomics 2005, 5, 43894397. (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., A 2004, 1028, 1-30. (25) Hoth, D. C.; Rivera, J. G.; Colon, L. A. J. Chromatogr., A 2005, 1079, 392396.

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were removed with washing solution, and bound peptides were eluted with the high-pH elution solutions. Eluted peptide samples were dried down and reconstituted in 2-propanol/acetonitrile/ water (1:1:2) with 0.25% piperidine for negative ion mode and in acetonitrile/water (1:1) with 0.1% formic acid for positive ion mode ESI-FT-ICR MS analysis. Mass Spectrometry and Data Analysis. Mass measurements were performed with a 7-T hybrid quadrupole (Q)-FT-ICR mass spectrometer (Bruker Daltonics, Billerica, MA) equipped with an Apollo electrospray ion source. The ESI flow rate was 50 µL/h, and nebulization was assisted by N2 gas. Peptide ions were externally accumulated for 0.5 s in a hexapole prior to being transferred to the ICR cell and captured by gated trapping. The external accumulation and transfer events were looped twice prior to excitation and detection. Spectra were acquired with XMASS (version 6.1, Bruker Daltonics) from m/z 250 to 2500 with 512k data points and summed over four or eight scans. Data analysis was performed with the MIDAS analysis software.26 Data were Hanning apodized and zero filled once prior to fast Fourier transformation and magnitude calculation. Lists of expected proteolytic peptide masses were generated with the Peptide Mass tool on the Expasy web site (www.us.expasy.org) using accession numbers P02662 and P02663 for the two forms (S1 and S2) of R-casein and P02666 for β-casein. Another tool on the Expasy web site, Compute pI, was used to compute pI values of certain peptides. ZrO2 and TiO2 phosphopeptide binding selectivity was determined by dividing the sum of the abundances of all phosphopeptide isotopomers with the sum of all observed isotopomers in the same spectrum before and after phosphopeptide enrichment. Only peaks with a signal-to-noise (S/N) ratio above three were included in the analysis. The noise level of a spectrum was determined from the root-mean-square signal in a region devoid of peptide peaks. The Peptide Mass tool automatically considers phosphorylation when that modification is present in the database. However, we identified additional phosphopeptides by allowing different numbers of phosphate groups. Those assignments were confirmed by collision-activated dissociation in the external hexapole with argon as collision gas. Neutral loss of 98 Da (corresponding to phosphoric acid) was used as a marker ion. RESULTS AND DISCUSSION Optimization of Zirconium Dioxide Phosphopeptide Enrichment Conditions. Zirconium oxide is known to have amphoteric properties; that is, it can react either as a Lewis acid or base depending on the pH of the reaction solution. This property is a result of unsatisfied valencies of both oxygen and zirconium atoms in the surface layer.23 In acidic solution, ZrO2 behaves as a Lewis acid with positively charged zirconium atoms, thereby displaying anion-exchange properties.27 For example, high binding affinity for polyoxy anions, including phosphate, borate, carboxylate, and sulfate, has been demonstrated.28 The binding constant of phosphate ions is markedly higher than for other Lewis bases,29,30 suggesting that high binding selectivity of phosphory(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, The Netherlands, 1964. (28) Kraus, K. A.; Philips, H. O.; Carlson, T. A.; Johnson, J. S. Proceedings of the Second International Conference on Peaceful Uses of Atomic Energy, Geneva, Switzerland, 1958; 3.

Figure 1. Negative mode ESI FT-ICR mass spectra (8 scans) from 100 pmol of a tryptic digest of R-casein obtained prior to ZrO2 phosphopeptide enrichment (a) and following enrichment at various binding pH values (b-e). Phosphopeptides are labeled with numbers that are identified in Table 1. Nonphosphorylated peptides observed following enrichment are labeled with their corresponding amino acid residue numbers and R-casein isoform. *, noise.

lated peptides over nonphosphorylated acidic peptides should be achievable with ZrO2 upon careful selection of the pH. Figure 1a shows a negative mode ESI FT-ICR mass spectrum of a 100 pmol nonenriched tryptic digest of R-casein. Identified phosphopeptides are labeled with numbers from 1 to 12 and included in Table 1. Only the most abundant phosphopeptides are labeled in the spectrum. Spectra obtained following ZrO2 enrichment of phosphopeptides from the same digest at different binding solution pH values are shown in Figure 1b-e (100 pmol/spectrum). All these enrichment experiments involved water washing and elution in 0.5% piperidine. These data show that it is critical to use lowpH solutions to achieve high phosphopeptide binding selectivity. At pH 2 (Figure 1b), only one nonphosphorylated peptide (doubly deprotonated EPMIGVNQELAYFYPELFR, corresponding to amino acid residues 148-166 in the S1 form of R-casein) is detected with S/N > 3. This peptide contains three acidic glutamate residues and has a pI of 4.25. The data obtained at pH 3 (Figure 1c) do not differ drastically from those obtained at pH 2. However, already at this slightly higher pH, an increased abundance of nonphos(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.

phorylated peptides is seen with the peptide mentioned above being detected both in its doubly and triply deprotonated forms. In addition, a singly deprotonated peptide with one glutamate residue (VNELSK, corresponding to amino acid residues 52-57 in 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 compared to the spectrum obtained without the use of ZrO2 tips (Figure 1a). However, the selectivity is further compromised. At higher pH (Figure 1e) for which the Lewis acid property of ZrO2 diminishes, phosphopeptides are no longer the dominant species and a very limited amount of peptides binds to the tip. In chromatographic applications of zirconia, both the pH and the ionic strength of the mobile phase can influence retention properties due to a mixed-mode ligand- and ion-exchange analyte binding mechanism.23 We did not control the ionic strength in our pH-dependence experiments; however, the carboxylate ions originating from the formic acid added to lower the pH would be expected to decrease phosphopeptide binding due to competition for ZrO2 acidic sites. Thus, because we observed increased rather than decreased binding at lower pH, we believe the pH is far more important than ionic strength under our experimental conditions. To achieve maximum recovery of bound phosphopeptides, we also optimized the washing and elution conditions and found that pure water was the best washing solution. We also found that a pH as high as 10.8-11.5 was needed for optimum elution performance. The highest phosphopeptide recovery was achieved with 0.5% piperidine (pH 11.5) and that elution condition was used in all experiments presented below. ZrO2 and TiO2 Phosphopeptide Enrichment from Tryptic Digests of Phosphoproteins. To establish how ZrO2 phosphopeptide enrichment compares to TiO2 enrichment, we applied the protocol established above to both ZrO2 and TiO2 microtips (50 µg). Panels a-c in Figure 2 show this comparison for a tryptic digest of R-casein. In our hands, either technique is more selective than IMAC, similar to the results obtained previously by Larsen et al.20 However, we found that the relative abundance of singly versus multiply phosphorylated peptides varies between ZrO2 and TiO2 with singly phosphorylated peptides being enriched to a higher extent with ZrO2: For the R-casein tryptic digest, the most abundant signal following ZrO2 enrichment (Figure 2b) originates from the 2- charge state of the singly phosphorylated peptide YKVPQLEIVPNSAEER, corresponding to amino acid residues 119-134 in the S1 form of R-casein (peptide 7 in Table 1) followed by the 2- charge state of the doubly phosphorylated peptide DIGSESTEDQAMEDIK, corresponding to amino acid residues 58-73 in the S1 form of R-casein (peptide 6 in Table 1). Abundant signal (31% relative abundance) is also observed from the 3charge state of the singly phosphorylated peptide 7 as well as minor signal from its quadruply deprotonated form. In addition, a shorter version of peptide 7, corresponding to no missed trypsin cleavage sites (amino acid residues 121-134 in the S1 form of R-casein, peptide 4 in Table 1), is detected following ZrO2 enrichment. By contrast, the doubly phosphorylated peptide 6 is the most abundant species following TiO2 enrichment (Figure 2c), and the abundance for both the 2- and 3- charge states of the singly phosphorylated peptide 7 is lower in that spectrum (53 and 12%, respectively) compared to the spectrum obtained following Analytical Chemistry, Vol. 78, No. 6, March 15, 2006

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

a

no.

peptide identity

enzyme

phosphorylation

monoisotopic mass

method

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

R-casein-S2:153-164 R-casein-S2:141-152 R-casein-S2:152-164(153-165) R-casein-S1:121-134 R-casein-S1:58-73 R-casein-S1:58-73 R-casein-S1:119-134 R-casein-S1:118-134 R-casein-S1:74-94 R-casein-S2:16-36 R-casein-S2:61-85 R-caseinS2:57-95 β-casein:48-63 β-casein:45-63 β-casein:17-40 β-casein:16-40 β-casein:16-40 R-casein-S1:126-133 R-casein-S1:55-65 R-casein-S1:126-140 R-casein-S1:55-70 R-casein-S1:77-92 R-casein-S2:67-83 R-casein-S2:16-33 β-casein:47-52 β-casein:47-57 β-casein:47-59 β-casein:21-36 β-casein:21-46 β-casein:21-46

trypsin trypsin trypsin trypsin trypsin trypsin trypsin trypsin trypsin trypsin trypsin trypsin trypsin trypsin trypsin trypsin trypsin Glu-C Glu-C Glu-C Glu-C Glu-C Glu-C Glu-C Glu-C Glu-C Glu-C Glu-C Glu-C Glu-C

1phos:158 2phos:144,146 1phos:158 1phos:130 1phos:61,63,68a 2phos:61,63,68a 1phos:130 1phos:130 1phos:79 4phos:23,24,25,31 4phos:71,71,73,76 4phos:71,72,73,76 1phos:50 1phos:50 4phos:30,32,33,34 3phos:30,32,33,34a 4phos:30,32,33,34 1phos:130 2phos:61,63 1phos:130 3phos:61,63,68 5phos:79,81,82,83,90 4phos:71,72,73,76 4phos:23,24,25,31 1phos:50 1phos:50 1phos:50 4phos:30,32,33,34 3phos:30,32,33,34a 4phos:30,32,33,34

1465.6047 1538.5902 1593.6997 1659.7868 1846.7179 1926.6842 1950.9451 2079.0401 2719.9055 2745.9923 3007.0221 4717.9274 2060.8211 2431.0430 2965.1571 3041.5000 3121.2582 937.3793 1324.4836 1818.8335 1978.6556 2075.6103 2093.6080 2353.7863 846.3160 1460.5820 1704.6516 2007.6804 3110.4225 3190.3888

w, Zr, Ti w, Zr, Ti w, Zr, Ti w, Zr, Ti w, Zr, Ti w, Zr, Ti w, Zr, Ti w, Zr, Ti w, Zr, Ti Zr, Ti Zr, Ti w, Zr, Ti w, Zr, Ti Zr w, Zr, Ti w, Zr, Ti w, Zr, Ti w, Zr, Ti w w, Zr, Ti w w, Zr, Ti w w w, Zr, Ti Zr, Ti w, Zr, Ti w, Zr, Ti w, Zr, Ti w, Zr, Ti

All possible (known) phosphorylation sites are listed.

ZrO2 enrichment (74 and 31%, respectively). In addition, the 4charge state of peptide 7 is not detected following TiO2 enrichment, nor is the singly phosphorylated peptide 4. These enrichments were performed from 100-pmol aliquots of the same tryptic digest. Thus, variations in the digestion conditions cannot explain the absence of peptide 4. We believe these results constitute an important finding because a plentitude of biological regulatory mechanisms involve a single phosphorylation event and our results imply that ZrO2 phosphopeptide enrichment would be preferred over other isolation strategies in the characterization of such cases. We do not believe the observed behavior is a result of irreversible binding of multiply phosphorylated peptides to ZrO2 because we could still detect such phosphopeptides in the left-over solution of tryptic peptides. Also, we do not believe the differentiation between singly and multiply phosphorylated peptides is a result of our specific enrichment protocol because changing the conditions did not result in varied ratios of, for example, peptides 6 and 7 for R-casein (Figure 1). The finding that ZrO2 more selectively isolates singly phosphorylated peptides as compared to TiO2 was confirmed with a tryptic digest of β-casein; see Figure 2d-f. Again, the most abundant peak following ZrO2 enrichment originates from a singly phosphorylated peptide (the 2- charge state of FQSEEQQQTEDELQDK, corresponding to amino acid residues 48-63 in β-casein (peptide 13 in Table 1) whereas its abundance following TiO2 enrichment is comparable to the quadruply phosphorylated peptide RELEELNVPGEIVESLSSSEESITR (peptide 17, triply deprotonated). The latter peptide displays similar S/N ratios 1746

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following both techniques, again indicating that its lower relative yield following ZrO2 enrichment is not due to poorer recovery from the column. The observed difference in phosphopeptide binding selectivity between zirconia and titania may be related to their different surface chemistry: First, zirconia is a stronger Lewis acid than titania,31 which could contribute to an enhanced binding of singly phosphorylated peptides. Second, the coordination numbers of Zr and Ti are different in the crystalline forms typically present in chromatographic materials, six for Ti and seven for Zr,31 which could result in different binding properties for singly and multiply phosphorylated peptides. However, because the surface properties of zirconia and titania are not well understood,24 these suggestions are rather speculative at this point. ZrO2 and TiO2 Phosphopeptide Enrichment from Glu-C Digests of Phosphoproteins. Regnier and co-workers have recently shown that the use of Glu-C rather than trypsin for phosphoprotein digestion prior to IMAC phosphopeptide enrichment significantly reduces nonspecific binding of acidic nonphosphorylated peptides.32 This behavior is a result of the specificity of Glu-C, which can cleave a protein (depending on the digestion conditions) at the C-terminal side of acidic amino acid residues (Asp and Glu). Thus, in theory, Glu-C digestion results in only one 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.

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

phosphopeptides), hence reducing nonspecific binding. We explored the use of Glu-C digestion prior to ZrO2 and TiO2 enrichment; see Figure 3. For R-casein (Figure 3a-c), Glu-C digestion resulted in lower overall (i.e., with or without enrichment) phosphopeptide signal as compared to trypsin digestion (see Table 2). With TiO2, the enrichment factor (i.e., the increase in relative phosphopeptide signal) following Glu-C digestion was about the same (∼2.5-fold) as for the R-casein trypsin digest whereas a higher enrichment factor (∼4-fold relative increase) was observed with ZrO2 for the Glu-C digest (compared to ∼2.5fold relative increase for the trypsin digest). From repeated experiments, the error of the tabulated selectivity values was estimated to be ∼8%. Again, one additional singly phosphorylated peptide (peptide 20 in Table 1) was detected following ZrO2 enrichment as compared to TiO2 enrichment. However, several multiply phosphorylated peptides (peptides 22-24 in Table 1) detected prior to TiO2 and ZrO2 treatment were not observed following the use of metal oxide microtips, rendering trypsin digestion the preferred strategy for R-casein. For β-casein, the enrichment factors following both TiO2 and ZrO2 treatment were similar for the Glu-C and trypsin digests (see Table 2). Additional evidence for the selective enrichment of singly phosphorylated peptides with ZrO2 is evident from Figure 3e and f: Following both ZrO2 and TiO2 enrichment, the most abundant species correspond to the 2- charge state of singly phosphorylated KFQSEEQQQTEDE (peptide 27 in Table 1). However, the relative abundance is 64% following ZrO2 treatment and 44% following TiO2 treatment. Again, TiO2 shows higher selectivity than ZrO2 for a

multiply phosphorylated peptide (quadruply phosphorylated LNVPGEIVESLSSSEE, peptide 28 in Table 1): The 2- charge state of this peptide is detected at a 27% relative abundance following TiO2 enrichment versus 15% for ZrO2. Also, its triply deprotonated form is only observed following TiO2 enrichment. Sensitivity of ZrO2 and TiO2 Microtip Phosphopeptide Enrichment. All spectra presented above were obtained following enrichment of 100 pmol of a phosphoprotein digest employing microtips containing 50 µg of ZrO2 or TiO2. Table 3 shows the selectivity of these microtips with decreasing amounts of an R-casein tryptic digest (50 and 25 pmol, respectively). Again, the error of these values was found to be ∼8%. It is clear that, for R-casein, the phosphopeptide selectivity is not compromised when the sample amount is decreased. However, because our mass spectrometer had not been optimized for high sensitivity (e.g., we did not use nanospray or an ion funnel for improved ion transmission), we obtained poor S/N values with lower than 25 pmol of sample. To allow utilization of the ZrO2 microtips for sample amounts compatible with proteomic applications, it will be necessary to scale down the size of these columns. An initial attempt to half the amount of metal oxide to 25 µg resulted in improved S/N ratios of phosphopeptides from a tryptic digest of 1 pmol of R-casein (Figure 4). These experiments were performed with a digest different from the one used for the experiments shown in Figures 1 and 2. Thus, the relative abundance of the detected phosphopeptides is somewhat different. However, again, singly phosphorylated peptides (peptides 4 and 7) are more abundant Analytical Chemistry, Vol. 78, No. 6, March 15, 2006

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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 β-casein obtained prior to phosphopeptide enrichment (a, d) and following ZrO2 (b, e) and TiO2 phosphopeptide enrichment (c, f). Phosphopeptides were bound in 3.3% formic acid (pH 2), washed with water, and eluted in 0.5% piperidine (pH 11.5). For R-casein, poorer phosphopeptide signal is observed as compared to trypsin digestion (Figure 2) whereas similar performance is seen for β-casein with selective enrichment of a singly phosphorylated peptide (27, see Table 1) following ZrO2 treatment. Nonphosphorylated peptides observed following enrichment are labeled with their corresponding amino acid residue numbers. *, noise. Table 2. Selectivitya (%) of ZrO2 and TiO2 Microtips for Phosphopeptide Enrichment of Proteolytic Digests of r- and β-Casein

without enrichment ZrO2 enrichment TiO2 enrichment a

trypsin digest of R-casein

trypsin digest of β-casein

Glu-C digest of R-casein

Glu-C digest of β -casein

27 67 62

22 61 65

8 31 22

22 62 77

Table 3. Selectivitya (%) of 50-µg ZrO2 and TiO2 Microtips for Phosphopeptide Enrichment of a Tryptic Digest of r-casein as a Function of Sample Amount

without enrichment ZrO2 enrichment TiO2 enrichment aDefined

100 pmol

50 pmol

25 pmol

27 67 62

29 85 77

29 83 74

as relative phosphopeptide signal; see text.

Defined as relative phosphopeptide signal; see text.

than the doubly phosphorylated peptide 6. All three peptides are more abundant following enrichment with the smaller as compared to the larger column. However, it should be noted that the phosphopeptide selectivity was compromised at 1 pmol, even with the 25-µg tip: Several singly deprotonated nonphosphorylated peptides 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 at higher sample amounts (Table 3). ZrO2 Phosphopeptide Enrichment for Positive Ion Mode Mass Spectrometry. All data discussed above were obtained in negative ion mode. Although negative ionization generally provides higher sensitivity for phosphopeptides, many phosphoproteomics experiments are performed in positive ion mode because the dissociation behavior of peptide cations is much better 1748 Analytical Chemistry, Vol. 78, No. 6, March 15, 2006

understood than that of peptide anions.33,34 However, the major fragmentation pathway of serine- and threonine-phosphorylated peptides in both positive and negative modes is loss of phosphoric acid rather than backbone cleavage,3,4,6 and thus, their sequencing can be difficult. Phosphate loss is not observed to a large extent in 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, 105116. (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. (36) Shi, S. D.-H.; Hemling, M. E.; Carr, S. A.; Horn, D. M.; Lindh, I.; McLafferty, F. W. Anal. Chem. 2001, 73, 19-22. (37) Chalmers, M. J.; Hakansson, K.; Johnson, R.; Smith, R.; Shen, J.; Emmett, M. R.; Marshall, A. G. Proteomics 2004, 4, 970-981.

Figure 4. Negative mode ESI FT-ICR mass spectra (8 scans) from 1 pmol of a trypsin digest of R- casein obtained following phosphopeptide enrichment with a 50-µg ZrO2 microtip (a) and a 25-µg ZrO2 microtip (b). Downscaling the column size results in increased sensitivity. Identified nonphosphorylated peptides are labeled with their corresponding amino acid residue numbers and R-casein isoform. *, noise.

can therefore facilitate phosphopeptide sequencing and localize sites of phosphorylation. However, ECD also requires high signalto-noise ratios of precursor ions, and thus, enrichment of phosphopeptides may be crucial to its applicability in phosphoproteomics. Figure 5 shows positive ion mode ESI-FT-ICR mass spectra obtained prior to and following ZrO2 enrichment of a tryptic digest of R-casein. Only three singly phosphorylated peptides (peptides 1, 4, and 7 in Table 1) are observed without enrichment (Figure 5a) whereas one more charge state of peptide 7 and four additional phosphopeptides are detected following ZrO2 enrichment (Figure 5b). Three of these additional phosphopeptides (peptides 2, 3, and 6 in Table 1), of which two are doubly phosphorylated, were also observed in negative ion mode (Figure 2). However, the fourth additional phosphopeptide (labeled 31 in Figure 5b) was identified as the 2+ charge state of the singly phosphorylated peptide NMAINPSKENLCSTFCK, corresponding to amino acid residues 40-56 in the S2 form of R-casein. No peptides including that region of the protein were observed in negative ion mode. There are also several peaks (mainly singly charged) in Figure 5b that could not be identified as either phosphorylated or nonphosphorylated peptides from R-casein; however, R-casein phosphopeptides dominate the enriched spectrum. The relative phosphopeptide signal increased a factor of 3 following enrichment.

Figure 5. Positive mode ESI FT-ICR mass spectra (4 scans) from 100 pmol of a tryptic digest of R-casein obtained prior to phosphopeptide enrichment (a) and following ZrO2 phosphopeptide enrichment (b). Phosphopeptides are labeled with numbers identified in Table 1, except for peptide 31 (singly phosphorylated NMAINPSKENLCSTFCK), which was not observed in negative ion mode. The relative phosphopeptide signal increased a factor of 3 following enrichment.

CONCLUSIONS In our hands, both zirconium oxide and titanium oxide microtips provide highly selective phosphopeptide enrichment from proteolytic peptide mixtures. However, the zirconium oxide columns possess a unique selectivity for singly phosphorylated peptides whereas titanium oxide selectively enriches multiply phosphorylated peptides. Thus, these two column materials have complementary properties and the choice of phosphopeptide enrichment strategy can be tailored according to the specific application. For R- and β-casein investigated here, the use of Glu-C rather than trypsin for proteolytic digestion did not appear to provide an advantage. Current commercially available 50-µg microtips are limited to sample amounts above 1 pmol although further miniaturization of these columns appears promising. ACKNOWLEDGMENT This work was supported by the Searle Scholars Program and the University of Michigan. We also thank Ashok Shukla for valuable discussions and for providing the 25-µg microtips.

Received for review December 18, 2005. Accepted January 23, 2006. AC0522355

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