Detection of in Vitro Kinase Generated Protein Phosphorylation Sites

Anal. Chem. 2007, 79, 7603-7610

Detection of in Vitro Kinase Generated Protein Phosphorylation Sites Using γ[18O4]-ATP and Mass Spectrometry Ming Zhou,† Zhaojing Meng,† Andrew G. Jobson,‡ Yves Pommier,‡ and Timothy D. Veenstra*,†

SAIC-Frederick, Inc., National Cancer Institute at Frederick, Frederick, Maryland 21702-1201, and Laboratory of Molecular Pharmacology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892-4255

A novel stable-isotope labeling approach for identification of phosphopeptides that utilizes adenosine triphosphate, in which four oxygen-16 atoms attached to the terminal phosphate group are substituted with oxygen-18 [γ(18O4)ATP], has been developed. The ability to use γ(18O4)-ATP to monitor phosphorylation modification within various proteins was conducted by performing in vitro kinase reactions in the presence of a 1:1 mixture of γ(18O4)-ATP and normal isotopic abundance ATP (ATP). After tryptic digestion, the peptides were analyzed using mass spectrometry (MS). Phosphorylated peptides are easily recognized within the MS spectrum owing to the presence of doublets separated by 6.01 Da; representing versions of the peptide modified by ATP and γ(18O4)-ATP. Standard peptides phosphorylated using γ(18O4)-ATP via in vitro kinase reactions showed no exchange loss of 18O with 16O. The identity of these doublets as phosphorylated peptides could be readily confirmed using tandem MS. The method described here provides the first direct stable-isotope labeling method to definitely detect phosphorylation sites within proteins.

Phosphorylation plays an essential role in transmitting signals from the outside to the inside of a cell and is known to regulate a diversity of cellular processes such as metabolism, growth, proliferation, motility, differentiation, and division.1 A variety of different proteins such as receptors, adaptor proteins, kinases, and phosphatases play a key role in regulating these processes. With the developments in DNA cloning and sequencing that have culminated with the near completion of the human genome, scientists are now able to predict the number of different kinases that may be present within human cells. Most estimates predict that the human genome encodes for just over 500 different kinases, representing ∼2% of all genes.2 To unravel the connection between the structure of these proteins and their function, investigators have compared the homology of these sequences * To whom correspondence should be addressed. E-mail: veenstra@ ncifcrf.gov. † SAIC-Frederick, Inc. ‡ Center for Cancer Research. (1) Cohen, P. Trends Biochem. Sci. 2000, 25, 596-601. (2) Manning, G.; Whyte, D. B.; Martinez, R.; Hunter, T.; Sudarsanam, S. Science 2002, 298, 1912-1934. 10.1021/ac071584r CCC: $37.00 Published on Web 09/18/2007

© 2007 American Chemical Society

to those of known protein kinases.3-5 While a majority of those sequences share homology to kinases with known functions, there remains a group of proteins for which no function can be readily assigned.2,6 In addition, kinases with known functions can phosphorylate many different sites within a number of different proteins.7 The dissection of all of the sites within the human proteome that are phosphorylated by this collection of kinases is of profound importance to a variety of fields including cancer research, cell and developmental biology, and drug discovery. This importance is underscored by the fact that 30% of all drug discovery efforts target protein kinases, making this class of proteins the second most important group of drug targets, after G-protein-coupled receptors.8 Identifying phosphorylated residues within proteins has historically been accomplished using hypothesis-driven in vitro studies in which a kinase of interest is mixed with a potential substrate in the presence of 32P-labeled adenosine triphosphate (γ32P-ATP). After incubation, the reaction mixture is digested into peptides that are chromatographically separated and analyzed using scintillation counting. Radioactive peptides are then sequenced to reveal the identity of the phosphopeptide. While methods utilizing radioactive isotopes have been the gold standard for a number of years, it would be beneficial to eliminate the need for radioactive materials, primarily due to safety and regulatory issues. Mass spectrometry (MS) has recently played a prominent role in identifying phosphorylated residues through the use of peptide mapping or partial sequence information obtained using tandem MS (MS2) or MS3. The identification of phosphopeptides using peptide mapping relies on the correlation of empirical masses with those predicted for phosphorylated versions of peptides within a protein database. The success of identifying phosphopeptides using this approach is dependent on a number of factors including the ability to detect the peptide of interest and the mass accuracy at which the molecular weights of the peptide are measured. In MS2 and MS3, the success of identifying phosphopeptides is again (3) (4) (5) (6)

Hanks, S. K.; Hunter, T. FASEB J. 1995, 9, 576-596. Rockey, W. M.; Elcock, A. H. Curr. Protein Pept. Sci. 2006, 7, 437-457. Drennan, D.; Ryazanov, A. G. Prog. Biophys. Mol. Biol. 2004, 85, 1-32. Bettencourt-Dias, M.; Giet, M.; Sinka, R.; Mazumdar, A.; Lock, W. G.; Balloux, F.; Zafiropoulos, P. J.; Yamaguchi, S.; Winter, S.; Carthew, R. W.; Cooper, M.; Jones, D.; Frenz, L.; Glover, D. M. Nature 2004, 432, 980987. (7) Martelli, A. M.; Sang, N.; Borgatti, P.; Capitani, S.; Neri, L. M. J. Cell. Biochem. 1999, 74, 499-521. (8) Cohen, P. Nat. Rev. Drug Discovery 2002, 1, 309-315.

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dependent on the ability to detect the peptide of interest and the quality of the fragmentation data obtained by collisional-induced dissociation (CID). Unfortunately, the fragmentation spectra of phosphoserine- and phosphothreonine-containing peptides, in particular, are often dominated by the loss of the phosphate group(s). When this event occurs, the tandem MS spectra lack the information necessary to identify the phosphopeptide sequence. In addition, sequence database search engines and statistical models for data validation are not optimized for the specific fragmentation properties of phosphorylated peptides. This results in large, indeterminable rates of false positive and false negative values for the identification of phosphopeptides.9 To increase confidence in the identification of phosphopeptides, we have developed a stable-isotope labeling method that incorporates the isotopic tag directly within the phosphate group. Stable-isotope labeling has become a common procedure within proteomics and is primarily used to measure the relative abundance of proteins in different samples.10-12 Stable-isotope labeling via the digestion of proteins in a 1:1 mixture of 16O- and 18O-labeled water has also been used to aid identification as y ions can be readily distinguished from b ions due to the incorporation of a heavy isotope of O at the C-terminus of the peptide.13 In this article, we report a stable-isotope labeling approach to aid in the identification of phosphorylated peptides. The method utilizes adenosine triphosphate in which four oxygen-16 atoms of the terminal phosphate group are substituted with oxygen-18 (γ(18O4)ATP). Standard peptides phosphorylated with γ(18O4)-ATP via in vitro kinase reactions showed no exchange loss of 18O with 16O. The ability to use γ(18O4)-ATP to monitor phosphorylation sites within various proteins was conducted by performing in vitro kinase reactions in the presence of a 1:1 mixture of γ(18O4)-ATP and normal isotopic abundance ATP. After digestion, the peptides are analyzed using MS. Phosphorylated peptides are readily recognized within the mass spectrum by their appearance as peaks separated by 6.01 Da due to the presence of both normal and 18O-labled phosphate groups. EXPERIMENTAL SECTION In Vitro Kinase Reaction Using γ(18O4)-ATP in the Presence of H218O and Unlabeled H2O. The reaction buffer was prepared by mixing 10 µL of kinase reaction buffer (87 mM TrisHCl, pH 7.2, 125 mM MgCl2, 25 mM MnCl2, 2 mM EGTA, 0.25 mM sodium orthovanadate, 2 mM dithiothreitol), and 10 µL of 0.6 mM γ(18O4)-ATP (adenosine 5′-triphosphate, disodium salt, γ-18O4, 97%, 88% pure, Cambridge Isotope Laboratories, Andover, MA) in ATP buffer 1 (100 mM Tris-HCl, pH 7.2, 75 mM MnCl2, 5 mM EGTA, and 1 mM sodium orthovanadate). The mixed solution was then lyophilized. The lyophilized sample was resuspended in 19.7 µL of H218O or regular distilled water, 0.2 µL of Src substrate (10 µg/µL in H2O, Upstate, Lake Placid, NY), and (9) Bodenmiller, B.; Mueller, L. N.; Pedrioli, P. G.; Pflieger, D.; Junger, M. A.; Eng, J. K.; Aebersold, R.; Tao, W. A. Mol. Biosyst. 2007, 3, 275-286. (10) Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R. Nat. Biotechnol. 1999, 17, 994-999. (11) Ong, S. E.; Blagoev, B.; Kratchmarova, I.; Kristensen, D. B.; Steen, H.; Pandey, A.; Mann, M. Mol. Cell Proteomics 2002, 1, 376-386. (12) Yao, X.; Freas, A.; Ramirez, J.; Demirev, P. A.; Fenselau, C. Anal. Chem. 2001, 73, 2836-2842. (13) Shevchenko, A.; Chernushevich, I.; Ens, W.; Standing, K. G.; Thomson, B.; Wilm, M.; Mann, M. Rapid Commun. Mass Spectrom. 1997, 11, 10151024.

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0.1 µL of active Src kinase (0.1 µg/µL, Upstate). The reaction was run at 30 °C for 100 min and was stopped by adding 2 µL of 2% TFA. In Vitro Kinase Reaction with and without β-Glycerophosphate. The kinase reaction was run at the same fashion as that described above except that the reaction mixture is prepared with either ATP buffer 1 or ATP buffer 2 (20 mM MOPS, pH 7.2, 75 mM MnCl2, 5 mM EGTA, 25 mM β-glycerophosphate, and 1 mM sodium orthovanadate). In Vitro Kinase Reaction Using a γ(18O4)-ATP and ATP Mixture. The kinase reaction was run in the same fashion as that decribed above except that the reaction mitxture was prepared with Src kinase buffer, 10 µL of 0.3 mM γ(18O4)-ATP, and 0.3 mM unlabeled ATP (Roche Applied Science, Indianapolis, IN) in ATP buffer 1. Myelin Basic Protein and Histone H1.2 Phosphorylation by MAP and Checkpoint Kinase 2 (Chk2) Kinases. The reaction buffer was prepared by mixing 10 µL of kinase reaction buffer, 10 µL of 0.3 mM γ(18O4)-ATP, and 0.3 mM ATP in a buffer containing 100 mM Tris-HCl, pH 7.2, 125 mM MgCl2, 5 mM EGTA, and 1 mM sodium orthovanadate. The mixed solution was then added with 1.0 µL of myelin basic proteins (MBP; 5.0 µg/ µL, Upstate) and 0.2 µL of active MAP kinase (0.1 µg/µL, Upstate). The reaction was run at 30 °C for 100 min and was stopped by adding 2 µL of 2% TFA. The reaction sample was then lyophilized, resuspended in LDS sample buffer, run on SDS-PAGE, and stained by Simple Blue. (Invitrogen, Carlsbad, CA) The MBP band at ∼20 kDa was cut off. The MBP protein was in-gel digested, and the peptides were extracted. Human recombinant Chk2 was expressed and purified as previously described.24 In vitro kinase assays were performed at 37 °C for 30 min using 400 ng of recombinant human Chk2 with either 1 µg of recombinant human histone H1.2 (Calbiochem, San Diego, CA) or 1 µg of bovine MBP (Sigma, St. Louis, MO) as substrates in a 10-µL reaction mixture containing 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM DTT, and 10 µM 1:1 ATP/γ(18O4)-ATP mixture. Reactions were terminated by placing the samples at 95 °C for 5 min followed by tryptic digestion at 37 °C overnight. The samples were zip-tipped prior to being analyzed using nanoflow reversed-phase liquid chromatography (nanoRPLC) tandem mass spectrometry (MS2). Neutral loss of both 16O- and 18O-labeled phosphoric acid was included in the instrument method for data-dependent neutral loss-driven MS3 peptide identification. (14) Litwin, C. M.; Cheng, H. C.; Wang, J. H. J. Biol. Chem. 1991, 266, 25572566. (15) Cheng, H. C.; Nishio, H.; Hatase, O.; Ralph, S.; Wang, J. H. J. Biol. Chem. 1992, 267, 9248-9256. (16) Hirschberg, D.; Radmark, O.; Jornvall, H.; Bergman, T. J. Protein Chem. 2003, 22, 177-181. (17) Ahn, J.; Urist, M.; Prives, C. DNA Repair (Amsterdam) 2004, 3, 10391047. (18) Chang, E. J.; Archambault, V.; McLachlin, D. T.; Krutchinsky, A. N.; Chait, B. T. Anal. Chem. 2004, 76, 4472-4483. (19) Garcia, B. A.; Shabanowitz, J.; Hunt, D. F. Methods 2005, 35, 256-264. (20) Sun, X.; Chiu, J. F.; He, Q. Y. Expert Rev. Proteomics 2005, 2, 649-657. (21) Pinkse, M. W.; Uitto, P. M.; Hilhorst, M. J.; Ooms, B.; Heck, A. J. Anal. Chem. 2004, 76, 3935-3943. (22) Goshe, M. B.; Conrads, T. P.; Panisko, E. A.; Angell, N. H.; Veenstra, T. D.; Smith, R. D. Anal. Chem. 2001, 73, 2578-2586. (23) Oda, Y.; Nagasu, T.; Chait, B. T. Nat. Biotechnol. 2001, 19, 379-382. (24) Yu, Q.; LaRose, J.; Zhang, H.; Takemura, H.; Kohn, K. W.; Pommier, Y. Cancer Res. 2002, 62, 5743-5748.

Mass Spectrometry Analysis. For maxtix-assisted laser desorption/ionization (MALDI)-MS analysis, the reaction mixture was desalted by C-18 ZipTip (Millipore, Bedford, MA). The peptides were eluted to 10 µL of 50% acetonitrile, 0.1% TFA. An aliqout of 0.5 µL of the eluted peptides was spotted on a MALDI plate using 0.5 µL of R-cyano-4-hydroxycinnamic acid (Agilent Technologies, Santa Clara, CA) as matrix. Mass spectra were acquired using a 4700 MALDI TOF/TOF mass spectrometer (Applied Biosystems Inc., Foster City, CA). For samples analyzed by electrospray ionization (ESI)-MS2, nanoRPLC-MS/MS was performed using an Agilent 1100 nanoflow LC system (Agilent Technologies, Palo Alto, CA) coupled online with an linear ion trap MS (LTQ, ThermoElectron, San Jose, CA). NanoRPLC columns were slurry-packed in-house with 5-µm, 300-Å-pore size C-18 phase (Jupiter, Phenomenex, CA) in a 75 µm i.d. × 10 cm fused-silica capillary (Polymicro Technologies, Phoenix, AZ) with a flame-pulled tip. After sample injection, the column was washed for 20 min with 98% mobile phase A (0.1% formic acid in water) at 0.5 µL/min and peptides were eluted using a linear gradient of 2% mobile phase B (0.1% formic acid in acetonitrile) to 42% mobile phase B in 40 min at 0.25 µL/min, and then to 98% B in an additional 10 min. The IT-MS was operated in a data-dependent mode in which each full MS scan was followed by seven tandem MS scans wherein the seven most abundant molecular ions were dynamically selected for CID using a normalized collision energy of 35%. The neutral loss of both 16O- and 18O-labeled phosphoric acid were included in the instrument method for data-dependent neutral loss-driven MS3 scan. A dynamic exclusion time of 1 min was applied to minimize repeated selection of peptides previously selected for CID. The capillary temperature and electrospray voltage were set to 160 °C and 1.5 kV, respectively. RESULTS In Vitro Phosphorylation Using γ(18O4)-ATP. To test the ability to phosphorylate proteins using γ(18O4)-ATP, a series of reactions were performed using a known kinase/substrate in vitro reaction. A synthetic peptide (KVEKIGEGTYGVVYK), representing residues 6-20 of Cdc2, was incubated with the tyrosine kinase Src in the presence of γ(18O4)-ATP. While there is no evidence that Src phosphorylates Cdc2 at Y15, this peptide has been widely used as an artificial substrate for Src in vitro since the reaction has a relatively low Km and very high Vmax.14,15 An obvious concern when using stable isotopes is the possibility of exchange loss. Although it did not seem likely that the 18O atoms of γ(18O4)-ATP would naturally exchange for 16O when dissolved in H2O, it was not clear whether kinase activity had any affect on exchange of the phosphate oxygen atoms. The kinase reaction was performed using γ(18O4)-ATP in both native H2O (i.e., H216O) and H218O. The spectra of the phosphorylated peptide are identical regardless of whether the kinase reactions are run in H216O or H218O (Figure 1A and B, respectively). Two small peaks at m/z 1753.0 and 1754.0 in Figure 1A and B result from the purity level (i.e., 97%) of heavy isotope incorporation of γ(18O4)-ATP used in this study. The in vitro kinase reactions shown in Figure 1 were repeated in the presence of β-glycerolphosphate. β-glycerolphosphate is commonly added to kinase reactions to inhibit serine/threonine phosphatase activity. Although only γ(18O4)-ATP was used, analysis of the reaction products using MALDI-TOF-MS revealed two peptide signals separated by 6.02 Da, regardless of whether the

Figure 1. MALDI-TOF spectra of the phosphorylated synthetic peptide (KVEKIGEGTYGVVYK; amino acid residues 6-20 of Cdc2) by Src protein tyrosine kinase. (A) In vitro kinase reaction using γ(18O4)-ATP in H2O. (B) In vitro kinase reaction using γ(18O4)-ATP in H218O.

Figure 2. MALDI-TOF spectra of the phosphorylated synthetic peptide (KVEKIGEGTYGVVYK; amino acid residues 6-20 of Cdc2) by Src protein tyrosine kinase using γ(18O4)-ATP in the presence of β-glycerolphosphate. (A) Reaction in H2O. (B) Reaction in H218O.

reaction was conducted in H216O or H218O (Figure 2). The masses of the two signals correspond to the peptide substrate modified with 18O3PO- (m/z 1755.02) and 16O3PO- (m/z 1748.99). Comparison of this study to that shown in Figure 1 reveals β-glycerAnalytical Chemistry, Vol. 79, No. 20, October 15, 2007

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Figure 4. Peptide mapping of in vitro kinase reaction products of MAP kinase and MBP in the presence of a 1:1 mixture of ATP and γ(18O4)-ATP. A) Mono- and di-phosphorylated versions of the MBP tryptic peptide, NIVTPRTPPPSQGK were observed in the mass spectrum (insets).

Figure 3. MALDI-TOF spectra of the phosphorylated synthetic peptide (KVEKIGEGTYGVVYK; amino acid residues 6-20 of Cdc2) by Src protein tyrosine kinase. (A) In vitro kinase reaction in H2O using a 1:1 mixture of ATP and γ(18O4)-ATP. (B) In vitro kinase reaction in H218O using a 1:1 mixture of ATP and γ(18O4)-ATP.

olphosphate as being responsible for this effect. When the Src phosphorylation reaction is run in the absence of β-glycerolphosphate, only the heavy isotope-labeled phosphopeptide peaks are observed (Figure 1). A possible explanation for the presence of the light isotope-labeled peptide is a swapping mechanism in which the phosphate group of β-glycerolphosphate is exchanged with the γ-phosphate of γ(18O4)-ATP or is transferred to ADP generated from the kinase reaction, resulting in a pool of ATP molecules containing a single 18O atom between the β- and γ-phosphate groups within ATP. During the kinase reaction, Src has the option of transferring the fully 18O-labeled phosphate moiety of γ(18O4)-ATP or the unlabeled phosphate moiety of the partially 18O-labeled ATP to the peptide substrate. This pool of mixed γ(18O4)-ATP and γ(18O1)-ATP results in a mixture containing heavy and light isotopically labeled phosphopeptides. To avoid complication, it is recommended that sodium orthovanadate, instead of β-glycerolphosphate, be used to inhibit phosphatase activity whenever necessary. In Vitro Phosphorylation Using an ATP/γ(18O4)-ATP Mixture. As mentioned in the introduction, the false positive and negative rates for the identification of phosphorylated peptides using tandem MS are high. To provide a mass spectral indicator that absolutely indicates the phosphorylation status of a peptide, the Src kinase reactions were conducted in a 1:1 mixture of γ(18O4)-ATP and ATP. Using this combination of ATP reagents results in a peptide doublet separated by 6.02 Da when analyzed using MALDI-MS (Figure 3). The presence of this doublet separated by 6.02 Da provides direct conclusive evidence of a phosphate group on the peptide. 7606 Analytical Chemistry, Vol. 79, No. 20, October 15, 2007

To test the utility of this method in identifying phosphorylated peptides within proteins, MBP was phosphorylated in vitro by MAP kinase in the presence of a 1:1 mixture of ATP and γ(18O4)ATP. The mass spectrum of the products of this in vitro kinase reaction acquired using MALDI-TOF/MS is shown in Figure 4. Examination of the spectrum revealed a doublet of signals at m/z ratios 1571.79 and 1577.81 (∆m/z ) 6.02). Both the mass measurement and tandem MS data (shown in Figure 5) confirmed the doublet as 16O3PO- and 18O3PO-labeled versions of the NIVpTPRTPPPSQGK peptide. The location of the phosphorylation site was determined by the 6.0-Da difference between the b7 fragment ions of the 16O3PO- and 18O3PO-labeled versions of the peptide (m/z 862.42 and 868.42, respectively), while no differences were observed in the positions of the y7 (m/z 710.3938) and y10 (m/z 1064.5739) fragment ions of the heavy- and light-labeled phosphopeptides (Figure 5). The MALDI-TOF-MS spectrum shown in Figure 4 also contains a triplet of signals with m/z ratios of 1651.76, 1657.77, and 1663.79. The mass measurement and tandem MS data (Figure 6) confirm the identity of this triplet as the doubly phosphorylated peptide, NIVpTPRpTPPPSQGK. The peaks at m/z values 1651.76, 1657.77, and 1663.79 correspond to the peptide with two lightisotopic (i.e., 16O3PO) phosphate groups, the peptide with one light- and one heavy-isotopic (i.e., 18O3PO) phosphate groups, and the peptide with two heavy isotopic phosphate groups, respectively. The tandem MS data were able to localize the phosphate modifications to Thr 94 and Thr97 of MBP by virtue of the different m/z values observed for the b7 fragment for the three peptides. These modifications have previously been identified via phosphorylation by ERK2 kinase in vitro,16 thus validating the technique. Identification of Phosphorylation Sites within Chk2 Substrates. Chk2 is a Ser/Thr kinase that plays an important role in cell cycle arrest and apoptosis upon DNA damage.17 The strategy of using a combined ATP/γ(18O4)-ATP mixture to assist phosphorylation mapping by MS was applied to identify phosphorylation sites by the Chk2 using MBP and histone H1.2 as substrates. Two in vitro kinase reactions were performed in the presence of

Figure 5. MALDI-TOF/TOF spectra of the monophosphorylated peptide, NIVpTPRTPPPSQGK. (A) Peptide with natural isotopic abundance phosphate group at m/z 1571.79 and (B) the same peptide with an 18O-labeled phosphate group at m/z 1577.81. The fragmentation patterns of the peptides unambiguously pinpoint the phosphorylation site as T94.

a 1:1 mixture of ATP and γ(18O4)-ATP. After digestion of the kinase reaction products, the samples were analyzed using reversedphase liquid chromatography coupled directly on-line with ESIMS2. Phosphorylated peptides were easily recognized in the mass spectra as doublets separated by 3 or 2 m/z units, representing doubly charged or triply charged versions of the phosphopeptide modified by normal ATP and γ(18O4)-ATP. An example of three phosphopeptides identified for MBP is shown in Figure 7. A complete list of phosphopeptides identified within MBP and histone H1.2 is shown in Table 1 along with the m/z values of the corresponding light- and heavy-isotope-labeled versions of each peptide. DISCUSSION Mass spectrometry is widely used in the identification of phosphorylation sites within proteins. This technology, however,

still has a number of limitations when it comes to identifying this type of post-translational modification. Successfully identifying any post-translational modification is often dictated by the level of sequence coverage obtained during an MS experiment. The selection of any peptide for MS2 in a data-dependent experiment is based on its signal intensity. This selection criterion exacerbates the selection of phosphopeptides since this class of peptides is generally present in lower abundance compared to their unmodified counterpart. In addition, higher abundance unmodified peptides can suppress the MS signal of phosphopeptides while the negative charge on the phosphate group can reduce the MS response observed in positive ion mode. To overcome the undersampling of phosphopeptides observed during an LC-MS experiment, Chait’s laboratory developed an approach termed hypothesis-driven multiple-stage (HMS) MS.18 This method emAnalytical Chemistry, Vol. 79, No. 20, October 15, 2007

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Figure 6. MALDI-TOF/TOF spectra of the di-phosphorylated peptide, NIVTPRTPPPSQGK. (A) The peptide with two natural isotopic abundance phosphate groups at m/z 1651.76, (B) the peptide with one natural isotopic abundance and one 18O labeled phosphate group at m/z 1657.77, and (C) the peptide with two 18O-labeled phosphate groups at m/z 1663.79. The fragmentation patterns of the peptides indicate phosphorylation at residues T94 and T97.

ploys a list of the calculated m/z values for every theoretical phosphopeptide that could be produced from a given protein. The mass spectrometer is then instructed to use this inclusion list and sample these m/z values for the presence of a phosphopeptide. Many proteins, however, depending on their size and Ser, Thr, and Tyr content, can result in an extremely large number of theoretical phosphopetides. While this method is extremely valuable, it would benefit from being able to construct an inclusion list based on the presence of known phosphopeptides. An experiment in which the reaction products generated from an in vitro kinase reaction performed using a 1:1 mixture ATP and γ(18O4)-ATP could be initially analyzed using MS to generate an inclusion list containing m/z values of known phosphopeptides 7608 Analytical Chemistry, Vol. 79, No. 20, October 15, 2007

based on their characteristic 6.01-Da separation. A subsequent experiment using an inclusion list containing m/z values of these peptides could be performed to exclusively target the MS2 (and MS3) analysis to signals that are known to be phosphorylated. There have been a number of different sample preparation and MS approaches presented for increasing the accuracy of identifying phosphopeptides.19 Most of the sample preparation methods rely on chromatographic approaches, such as immobilized metal affinity20 or titanium dioxide chromatography,21 to extract phosphopeptides from a complex mixture prior to MS analysis. All of these approaches suffer from various levels of nonspecificity and do not provide any direct evidence that a specific signal observed using MS is a legitimate phosphopeptide. To increase the

Figure 7. Liquid chromatography-electrospray ionization-tandem MS analysis of the reaction products of an in vitro kinase reaction between Chk2 and MBP in the presence of a 1:1 mixture of ATP and γ(18O4)-ATP. A number of peaks separated by 2 and 3 m/z units, depending on the charge state of the peptide, were observed. The sequences of the peptides were confirmed, using tandem MS, as phophopeptides originating from MBP.

Table 1. Mass-to-Charge (m/z) Ratios of Phosphopeptides Detected in the LC/ESI-MS Analysis of Histone H1.2 and MBP Phosphorylated by ChK2 Kinase Using a 1:1 Mixture of ATP and γ(18O4)-ATP. protein

phosphopeptide

light

m/z heavy

H1.2

K.KPAAATVpTKK K.SLVSKGpTLVQTK K.SLVpSKGTLVQTK R.KApSGPPVSELITK R.pSGVSLAALKK R.GpTLVQTK K.GHDAQGpTLSK K.GHDAQGTLpSK R.SKYLASASpTMDHAR

548.2 671.0 671.1 704.3 527.7 414.2 547.8 547.6 540.5 809.7 702.2 468.8 702.2 710.8 474.6 405.2 606.7

551.2 674.0 673.9 707.2 530.4 417.2 551.1 550.7 542.5 812.8 705.2 470.8 705.0 713.8 476.5 407.1 609.6

MBP

K.YLASASpTMDHAR K.YLASApSTMDHAR R.HRDpTGILDSLGR R.pTTHYGSLPQK

charge state 2 2 2 2 2 2 2 2 3 2 2 3 2 2 3 3 2

extraction efficiency, other groups have utilized chemical modification schemes in which the phosphate group is removed from the protein creating a reactive site that can be modified using a reagent containing a biotin tag.22,23 While the use of avidin chromatography increases the enrichment efficiency over other chromatographic methods, the chemistry involved is complicated and affects sites of O-linked glycosylation as well as phosphorylation. In addition, these chemistry-based methods are limited by side reactions and insufficient sensitivity and have not yet attained

widespread use. The method presented here provides direct physical evidence that a particular m/z value results from a phosphorylated peptide. This 18O-labeling method can be combined with any of the phosphopeptide enrichment techniques mentioned above. As described in the proceeding paragraph, however, the signature produced by the differentially labeled phosphopeptides can be specifically targeted using an HMS MS strategy, obviating the need for enrichment in most cases. As with peptide identification, the characterization of phosphorylation sites is conducted using MS2, often via CID in an ion trap. Ion trap-based CID of pSer- and pThr-containing peptides, in particular, often results in a dominant peak corresponding to a neutral loss of phosphoric acid (H3PO4, 98 Da). This phenomenon limits the amount of fragmentation observed throughout the peptide backbone, preventing the sequence of the peptide from being identified. In addition, the MS2 spectrum of any phosphopeptide is a mixture of fragment ions containing and lacking the phosphate group, thus complicating the spectral interpretation. The inclusion of a dynamic modification on Ser, Thr, and Tyr residues dramatically increases the size of the database that the raw MS2 data is searched against. Increasing the number of possibilities to which a MS spectrum can be matched, immediately increases the chances of obtaining a false positive identification. Each of these factors negatively impacts the ability to unambiguously identify phosphopeptides. The functional significance of putative phosphorylation sites identified using MS often requires site-directed mutational studies, which are time-consuming and expensive. The strength of using an ATP/γ(18O4)-ATP labeling method is that it provides a simple way to recognize signals arising from phosphopeptides within the mass spectrum and affords Analytical Chemistry, Vol. 79, No. 20, October 15, 2007

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absolute confidence in the modification state of the peptide, eliminating false positive identifications. The use of a nonradioactive phosphate tag for the identification of phosphorylation sites eliminates a number of problems associated with γ32P-ATP. Besides all of the associated health issues and precautions that must be taken, experiments using γ32P-ATP require extremely careful planning and timing so that the precise amount needed for a specific experiment is ordered and consumed within a relatively short (e.g.,