Accurate Mass-Driven Analysis for the Characterization of Protein

After incubation at 37 °C for 1 h, the sample was applied to a Hypercarb Top Tip ... First, the theoretical mass lists of tryptic peptides and phosph...
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Anal. Chem. 2006, 78, 2171-2181

Accurate Mass-Driven Analysis for the Characterization of Protein Phosphorylation. Study of the Human Chk2 Protein Kinase Julie B. King,†,‡ Julia Gross,‡ Christine M. Lovly,† Henry Rohrs,§ Helen Piwnica-Worms,*,†,‡,| and R. Reid Townsend*,†,‡

Department of Cell Biology and Physiology, Department of Internal Medicine, Department of Chemistry, and Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, Missouri 63110

We describe the data-dependent analysis of protein phosphorylation using rapid-acquisition nano-LC-linear quadrupole ion trap Fourier transform ion cyclotron resonance mass spectrometry (nano-LC-FTMS). The accurate m/z values of singly, doubly, and triply charged species calculated from the theoretical protonated masses of peptides phosphorylated at all Ser, Thr, or Tyr residues of the human checkpoint 2 (Chk2) protein kinase were used for selected ion extraction and chromatographic analysis. Using a kinase-inactive Chk2 mutant as a control, accurate mass measurements from FTMS and collision-induced dissociation spectra, 11 novel Chk2 autophosphorylation sites were assigned. Additionally, the presence of additional Chk2 phosphorylation sites in two unique peptides was deduced from accurate mass measurements. Selected ion chromatograms of all Chk2 phosphopeptides gave single peaks except in three cases in which two closely eluting species were observed. These pairs of phosphopeptides were determined to be positional isomers from MS/MS analysis. In this study, it was also found that ions due to the neutral loss of phosphoric acid from the parent peptide ion were not prominent in 18 of 36 MS/MS spectra of O-linked Chk2 phosphopeptides. Thus, accurate mass-driven analysis and rapid parallel MS/MS acquisition is a useful method for the discovery of new phosphorylation sites that is independent of the signature losses from phosphorylated amino acid residues. Reversible protein phosphorylation contributes to a plethora of biological activities that ultimately determine whether a cell proliferates, differentiates, or dies.1 Mass spectrometry has been recognized for decades as an important technology for the analysis of protein phosphorylation,2 and with recent improvements in methods and instrumentation, the number of studies using this * Corresponding authors. E-mail: [email protected]. Tel: 314 362 7709. Fax: 314-362-8265. E-mail [email protected]. Tel: 314 362 6812. Fax: 314-362-3709. † Department of Cell Biology and Physiology. ‡ Department of Internal Medicine. § Department of Chemistry. | Howard Hughes Medical Institute. (1) Hunter, T. Cell 1995, 80, 225-236. (2) Gibson, B. W.; Cohen, P. Methods Enzymol. 1990, 193, 480-501. 10.1021/ac051520l CCC: $33.50 Published on Web 02/28/2006

© 2006 American Chemical Society

technology has risen dramatically.3 Ultimately, determining the biological significance of reversible phosphorylation of an individual protein requires identification and stoichiometric quantification of all phosphorylation sites. Comprehensive analysis of protein phosphorylation presents significant analytical challenges.4,5 Peptides, representing the entire protein sequence, must be produced.6 Phosphopeptides, often present in trace amounts, must be recovered from biological samples, separated by highperformance liquid chromatography, and analyzed in femtomole quantities by mass spectrometry. Quality peptide fragmentation spectra must be acquired to deduce the amino acid residues that are modified with phosphate groups. Further, the phosphate bonds of serine and threonine residues are chemically more labile than peptide bonds, and collision-induced dissociation (CID) spectra of O-linked phosphopeptides are often dominated by neutral loss ions [M + nH - H3PO4 ]n+.7 The facile neutral loss of chemical moieties from phosphate groups has been extensively used for selectively detecting and analyzing phosphopeptides using precursor ion scanning and multistage tandem mass spectrometry (MS2 and MS3).8-16 (3) Loyet, K. M.; Stults, J. T.; Arnott, D. Mol. Cell. Proteomics 2005, 4, 235245. (4) McLachlin, D. T.; Chait, B. T. Curr. Opin. Chem. Biol. 2001, 5, 591-602. (5) Mann, M.; Ong, S. E.; Gronborg, M.; Steen, H.; Jensen, O. N.; Pandey, A. Trends Biotechnol. 2002, 20, 261-268. (6) Schlosser, A. V.;Vanseloro, J. T.; Kramer, A. Anal. Chem. 2005, 77, 52435250. (7) Tholey, A.; Reed, J.; Lehmann, W. D. J. Mass Spectrom. 1999, 34, 117123. (8) Carr, S. A.; Huddleston, M. J.; Annan, R. S. Anal. Biochem. 1996, 239, 180192. (9) Wilm, M.; Neubauer, G.; Mann, M. Anal. Chem. 1996, 68, 527-533. (10) Steen, H.; Ku ¨ ster, B.; Fernandez, M.; Pandey, A.; Mann, M. Anal. Chem. 2001, 73, 1440-1448. (11) Ma, Y.; Lu, Y.; Zeng, H.; Ron, D.; Mo, W.; Neubert, T. A. Rapid Commun. Mass Spectrom. 2001, 15, 1693-1700. (12) Schlosser, A.; Pipkorn, R.; Bossemeyer, D.; Lehmann, W. D. Anal. Chem. 2001, 73, 170-176. (13) Bennett, K. L.; Stensballe, A.; Podtelejnikov, A. V.; Moniatte, M.; Jensen, O. N. J. Mass Spectrom. 2002, 37, 179-190. (14) Schroeder, M. J.; Shabanowitz, J.; Schwartz, J. C.; Hunt, D. F.; Coon, J. J. Anal. Chem. 2004, 76, 3590-3598. (15) Gruhler, A.; Olsen, J. V.; Mohammed, S.; Mortensen, P.; Faergeman, N. J.; Mann, M.; Jensen, O. N. Mol. Cell. Proteomics 2005, 4, 310-327. (16) Chang, E. J.; Archambault, V.; McLachlin, D. T.; Krutchinsky, A. N.; Chait, B. T. Anal. Chem. 2004, 76, 4472-4483.

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In recent years, the developments of accurate mass-based proteomics17 and gas-phase fragmentation methods18 have provided unprecedented specificity for high-throughput, databaseassisted protein identification and characterization of posttranslational modifications. It is well recognized that increasing the accuracy of mass measurements reduces the number of possible peptide sequences, particularly within the constraints of sequence databases and endoprotease specificity.19-21 In addition, exact mass measurements using FTICR can identify the chemical nature of compounds and peptide modifications.22 For example, peptide sulfonation and phosphorylation can be distinguished by accurate mass measurements of the modified peptides.23 Recently, the capability to perform accurate mass measurements (∼2-5 ppm) and CID experiments with the same instrument and with greater sensitivity and speed has been achieved with a linear quadrupole ion trap Fourier transform ion cyclotron mass spectrometer (FTMS). The utility of this instrument configuration has recently been demonstrated for the analysis of histone modifications (acetylation and methylation)24 and protein phosphorylation.15,25 Here we apply accurate mass measurements and high-throughput MS/MS spectral acquisition to identify novel human checkpoint 2 (Chk2) autophosphorylation sites. Chk2 is a protein kinase that promotes cell cycle arrest or death in cells containing DNA double-strand breaks (DSBs).26 Thus, Chk2 functions to protect genome integrity, and Chk2 mutations are found in both familial and sporadic cancers.26,27 Chk2 is regulated, in part, by reversible phosphorylation. Exposure of cells to ionizing radiation (IR) or radiomimetic drugs induces Chk2 phosphorylation by the ATM protein kinase.28-32 This initiates a Chk2 autoactivation cycle involving Chk2 dimerization followed by Chk2 auto/trans phosphorylation and activation of Chk2 kinase activity.33-36 The Chk2 autoactivation cycle can be recapitulated (17) Bogdanov, B.; Smith, R. D. Mass Spectrom. Rev. 2005, 24, 168-200. (18) Aebersold, R.; Mann, M. Nature 2003, 422, 198-207. (19) Conrads, T. P.; Anderson, G. A.; Veenstra, T. D.; Pasa-Tolic, L.; Smith, R. D. Anal. Chem. 2000, 72, 3349-3354. (20) Smith, R. D.; Anderson, G. A.; Lipton, M. S.; Pasa-Tolic, L.; Shen, Y.; Conrads, T. P.; Veenstra, T. D.; Udseth, H. R. Proteomics 2002, 2, 513-523. (21) Clauser, K. R.; Baker, P.; Burlingame, A. L. Anal. Chem. 1999, 71, 28712882. (22) Zhang, L.-K.; Rempel, D.; Pramanik, B. N.; Gross, M. L. Mass Spectrom. Rev. 2005, 24, 286-309. (23) Bossio, R. E.; Marshall, A. G. Anal. Chem. 2002, 74, 1674-1679. (24) Syka, J. E.; Marto, J. A.; Bai, D. L.; Horning, S.; Senko, M. W.; Schwartz, J. C.; Ueberheide, B.; Garcia, B.; Busby, S.; Muratore, T.; Shabanowitz, J.; Hunt, D. F. J. Proteome Res. 2004, 3, 621-626. (25) Chalmers, M. J.; Hakansson, K.; Johnson, R.; Smith, R.; Shen, J.; Emmett, M. R.; Marshall, A. G. Proteomics 2004, 4, 970-981. (26) Ahn, J.; Urist, M.; Prives, C. DNA Repair (Amsterdam) 2004, 3, 10391047. (27) Bell, D. W., Variey, J. M., Szydlo, T. E., Kang, D. H., Wahrer-Doke, C. R., Shannon, K. E., Lubratovich, M., Versellls, S. J. Isselbacher, K. J., Fraumeni, J. F., Birch, J. M., Li, F. P., Garber, J. E., Haber, D. A. Science 1999, 286, 2528-2531. (28) Blasina, A.; Van de Weyer, I.; Laus, M. C.; Luyten, W. H. M. L.; Parker, A. E.; McGowan, C. H. Curr. Biol. 1998, 9, 1-10. (29) Brown, A.; Lee, C.-H.; Schwarz, J. K.; Mitiku, N.; Griffith, D.; Piwnica-Worms, H.; Chung, J. H. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 3745-3750. (30) Chaturvedi, P.; Eng, W. K.; Zhu, Y.; Mattern, M. R.; Mishra, R.; Hurle, M. R.; Zhang, X.; Annan, R. S.; Lu, Q.; Faucette, L. F.; Scott, G. F.; Li, X.; Carr, S. A.; Johnson, R. K.; Winkler, J. D.; Zhou, B.-B. S. Oncogene 1999, 18, 4047-4054. (31) Matsuoka, S.; Huang, M.; Elledge, S. J. Science 1998, 282, 1893-1897. (32) Ahn, J.-Y.; Schwarz, J. K.; Piwnica-Worms, H.; Canman, C. E. Cancer Res. 2000, 60, 5934-5936. (33) Xu, X.; Tsvetkov, L. M.; Stern, D. F. Mol. Cell. Biol. 2002, 22, 4419-4432.

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in bacteria in the absence of either DNA damage or ATM. As bacterial Chk2 protein levels rise, Chk2 oligomerization is facilitated, enabling Chk2 molecules to undergo auto/trans phosphorylation at residues normally phosphorylated in mammalian cells containing DNA DSBs. Thus, phosphorylated, active Chk2 can be isolated from bacteria for comprehensive phosphorylation analysis. In addition, kinase-inactive mutants can be used as an important control to discriminate between autophosphorylation and phosphorylation by other protein kinases present in the expression system. Metabolic labeling, two-dimensional phosphopeptide mapping, and site-directed mutagenesis have demonstrated that Chk2 is phosphorylated on Ser-19, Ser-33/35, Thr68, Thr-383/387, Thr-432, and Ser-516.28-31,34,37-39 We have applied electrospray ionization nano-LC-linear quadrupole ion trap-Fourier transform ion cyclotron resonance mass spectrometry (nano-LC-FTMS) to identify novel Chk2 autophosphorylation sites. Accurate mass measurements were used for comprehensive selective ion extraction of the spectra (MS and MS2) of masses for phophorylated tryptic and chymotryptic peptides, containing one, two, or three phosphate groups. Eleven novel phosphorylation sites were assigned from selected ion chromatograms using accurate mass measurements from FTICRMS and MS/MS spectra from CID in the linear ion trap. One site is present within the FHA domain (Ser-120), three are in the linker region between the FHA domain and kinase domain (Tyr-220, Ser223, Thr-225), six are within the kinase domain (Ser-228, Ser-260, Thr-272, Ser-372, Ser-379, Ser-435), and one is in the C-terminal region (Thr-532). Parallel analyses of a kinase-inactive Chk2 mutant (D368N) showed the absence of masses consistent with mono-, di-, or triphosphorylation at any of the Ser, Thr, or Tyr amino acid residues, supporting the conclusion that these novel sites result from Chk2 autophosphorylation. In addition, Ser-260 was identified not only as a site of Chk2 autophosphorylation in bacteria but also as a novel IR-inducible phosphorylation site in mammalian cells. This result confirms that nano-LC-FTMS can be used to identify biologically relevant Chk2 phosphorylation sites and validates the Chk2 bacterial expression system. EXPERIMENTAL SECTION Cell Culture. HeLa cells were routinely maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 100 units/mL penicillin and streptomycin, and 1 mM L-glutamine (complete DMEM). In some cases, cells were exposed to 10 Gy from a 60Co source. Antibodies and Western Blotting. Endogenous Chk2 was detected with mouse monoclonal antibody (Neomarkers, Fremont, CA). Flag3-Chk2 fusion proteins were precipitated with anti-Flag M2 antibody-agarose affinity gel (Sigma Chemical Co., St. Louis, MO) and detected by Western blotting with anti-Flag M2 monoclonal antibody (Sigma Chemical Co.). Bound primary antibodies (34) Schwarz, J. K.; Lovly, C. M.; Piwnica-Worms, H. Mol. Cancer Res. 2003, 1, 598-609. (35) Durocher, D.; Henckel, J.; Fersht, A. R.; Jackson, S. P. Mol. Cell 1999, 4, 387-394. (36) Durocher, D.; Taylor, I. A.; Sarbassova, D.; Haire, L. F.; Westcott, S. L.; Jackson, S. P.; Smerdon, S. J.; Yaffe, S. P. Mol. Cell 2000, 6, 1169-1182. (37) Matsuoka, S.; Rotman, G.; Ogawa, A.; Shiloh, Y.; Tamai, K.; Elledge, S. J. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 10389-10394. (38) Wu, X.; Chen, J. J. Biol. Chem. 2003, 278, 36163-36168. (39) Lee, C. H.; Chung, J. H. J. Biol. Chem. 2001, 276, 30537-30541.

were detected with horseradish peroxidase (HRP)-conjugated goat anti-mouse (Jackson Laboratory, Bar Harbor, ME) or HRP-goat anti-rabbit (Zymed, Carlsbad, CA) secondary antibodies, and proteins were visualized using ECL or ECL+ reagents (Amersham Biosciences, Piscataway, NJ). Antibodies specific for Chk2 phosphorylated on Ser-260 were generated by immunization of rabbits with the phosphopeptide CKFAIG-pS-ARE coupled to keyhole limpet hemocyanin. Antibodies specific for Chk2 phosphorylated on Thr-68 and Ser-516 have been described previously.34 Antibodies specific for Chk2 phosphorylated on Ser-19, Ser-33/35, and Thr-432 were purchased from Cell Signaling Technology (Beverly, MA). Specificity of the Phospho-Ser-260 Chk2 Antibody. HeLa cells were transfected with a Flag3-Chk2 expression plasmid using the Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA). At 44 h posttransfection, cells were washed once with ice cold PBS and lysed in mammalian cell lysis buffer (MCLB, 50 mM Tris-HCl pH 8, 150 mM NaCl, 0.5% NP-40, 5 mM EDTA, 2 mM DTT, 1 µM microcystin, 1 mM PMSF, 5 µg/mL leupeptin, 10 µg/ mL aprotinin). Ectopically expressed Chk2 was immunoprecipitated with anti-Flag M2 agarose. Immunoprecipitates were subjected to SDS-PAGE, transferred to nitrocellulose, and blotted with an antibody specific for the phospho-Ser-260 form of Chk2. Anti-Flag western blots were subsequently stripped and reprobed with an antibody specific for the Flag epitope. γ-Irradiation-Induced Phosphorylation of Ser-260. Asynchronously growing HeLa cells were either mock irradiated or irradiated with 10 Gy IR and harvested at 30 and 60 min post IR. Cell lysates were prepared in MCLB, and endogenous Chk2 was immunoprecipitated with a monoclonal antibody and protein A Sepharose beads (Pierce, Rockford, IL). Lysates were resolved by SDS-PAGE and transferred to nitrocellulose. The resulting membrane was blotted first with an antibody specific for phosphoSer-260 Chk2 and then with a monoclonal antibody specific for Chk2. Plasmids. pET15b-Chk2 WT, pET15b-D368N, and Flag3Chk2 have been described previously.34 Flag3-Chk2 (S260A) was generated by site-directed mutagenesis of Flag3-Chk2 WT using the Quick-Change site-directed mutagenesis kit (Stratagene, La Jolla, CA) and the following primers: 5′AGGAAGTTTGCTATTGGTGCAGCAAGAGAGGCAGACCCAGCT-3′ (forward) and 5′AGCTGGGTCTGCCTCTCTTGCTGCACCAATAGCAAACTTCCT3′ (reverse). Sequences of mutants were verified by DNA sequencing. Endoprotease Digestions. Wild-type and kinase-inactive (D368N) versions of human Chk2 were expressed and purified from bacteria as described previously.34 Purified kinase (20 µg) in 50 mM Tris-HCl, pH 7.4 (containing 10 mM MgCl2 and 1 mM DTT), was incubated for 30 min at 30 °C with 1 mM ATP. After concentration in a 5000 molecular weight cutoff filter device (Millipore, Bedford, MA), the protein (10 µg) was added to 100 µL of 0.2% Rapigest SF reagent (Waters, Milford, MA) in 10 mM Tris-HCl, pH 7.8. After addition of 5 mM DTT, the sample was placed at 60 °C for 30 min followed by addition of iodoacetamide to a final concentration of 15 mM. The sample was rocked in the dark at room temperature for 45 min. Either trypsin or chymotrypsin was added to a final concentration of 100 ng/µL, and the pH was adjusted to 8.0 with 0.75 M NH4OH. The sample was kept

at 37 °C for 2 h and then acidified with HCl to 30 mM. After incubation at 37 °C for 1 h, the sample was applied to a Hypercarb Top Tip (PolyLC Inc., Columbia, MD). The cartridge was washed with 0.1% formic acid (100 µL), and the peptides were eluted with 60% acetonitrile in 0.1% formic acid. The peptides were dried and dissolved in aqueous acetonitrile/formic acid (1%/1%). Nano-LC-FTMS Analysis. Mass spectrometry was performed using a linear quadrupole ion trap Fourier transform ion cyclotron resonance mass spectrometer (LTQ-FTMS, Thermoelectron, San Jose, CA). The nanoliquid chromatograph (Eksigent nano-LC, Eksigent, Livermore, CA) was interfaced to the LTQFTMS with a Pico-View nanocapillary source from New Objective (Woburn, MA). Sample injection was performed with an autosampler (Endurance, Spark, Plainsboro, NJ). The column was a C-18 PicoFrit (75 µm × 10 cm) (New Objective). The mobile phases were HPLC grade water (Fisher Scientific, Pittsburgh, PA) containing 1% formic acid (Sigma-Aldrich, St. Louis, MO) (Solvent A) and acetonitrile (Honeywell, Burdick & Jackson, Muskegon, MI) containing 1% formic acid (Solvent B). The sample (5 µL) was loaded at 600 nL/min at 1% B for 10 min. The flow was then decreased to 200 nL/min with isocratic elutions for 20 min followed by linear increase in Solvent B (2%/min) for 40 min. The LTQ-FTICR (7 T) mass spectrometer was operated in the data dependent mode. The survey scans (m/z ) 450-1500) were acquired using the FTICR-MS with resolution of ∼100,000 at m/z ) 421.75 after ion accumulation in the trap to a value of ∼1,000,000. The 10 most abundant ions were isolated and analyzed after reaching a target value of ∼40,000. The MS/MS isolation width was 2.5 Da, and the normalized collision energy was 35%. Electrospray ionization was accomplished with a spray voltage of 2.8-3.1 kV without sheath gas. The ion transfer tube temperature was 200 °C. Nano-LC-QTOF Mass Spectrometry. Capillary reversedphase HPLC-MS/MS was performed on an electrospray-quadrupole-time-of-flight (Q-TOF) mass spectrometer (Q-STAR XL, Applied Biosystems) interfaced to a low flow liquid chromatograph (Eksigent nano-LC, Eksigent, Livermore, CA). The nanocapillary source was an experimental version of the PicoView system from New Objective. Sample injection was performed with an autosampler (Endurance, Spark, Plainsboro, NJ). The gradient consisted of HPLC grade water (Fisher Scientific) in 1% formic acid (SigmaAldrich), solvent A, and acetonitrile (Honeywell, Burdick & Jackson) in 1% formic acid, solvent B. The column was a C-18 PicoFrit column (pore size, 5 µm; New Objective, 75 µm × 10 cm). Samples (5 µL) were injected at 2% B with a flow rate of 600 nL/min. The gradient was then held isocratically at 2% B for 20 min followed by an increase of solvent B by 0.67%/min for 90 min. The spray voltage was set to 2.7 kV with a curtain gas setting of 20 and a nebulizer gas setting of 15. The declustering potential was 90, and the focusing potential was 300. Collision energies were calculated by the Analyst QS software (MDS Sciex, Concord, ON, Canada) according to the following function with dependence on the m/z value of the parent ion: collision energy, 0.058 × m/z -2. MS/MS spectra were acquired for 8 s. The software was programmed to analyze the most intense ion from the inclusion list (independent data analysis mode). Data Analysis. Theoretical lists of the masses for singly, doubly, and triply protonated ions of phosphorylated and unmodiAnalytical Chemistry, Vol. 78, No. 7, April 1, 2006

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fied tryptic and chymotryptic peptides for human recombinant Chk2 kinase (gi 1328894) and copurifying Escherichia coli were prepared by in silico digestion using Protein Prospector (http:// prospector.ucsf.edu).21 Missed endoprotease cleavages (0, 1, and 2) and the following variable modifications were considered: carbamidomethylation of cysteine, oxidation of methionine, and phosphorylation of serine, threonine, and tyrosine residues. We determined the number of m/z values that would overlap between the peptides and phosphopeptides of Chk2 kinase with the four copurifying E. coli proteins, groEL (gi 41617), stringent starvation protein (gi 42999), FKBP-type peptidyl prolyl cis trans isomerase (gi 15833454), and putative formyl transferase (gi 16130190). First, the theoretical mass lists of tryptic peptides and phosphopeptides (0, 1, and 2 missed cleavages), from Chk2 and the four copurifying E. coli proteins were calculated with methionine oxidation and carbamidomethylation of Cys residues as variable modifications. Then the accurate m/z values (to seven significant figures) for the +1, +2, and +3 charges states were entered into a spreadsheet and compared using the experimentally determined mass accuracy (see below). The tables containing these values are in the Supporting Information II (Tables 1-5). The mass accuracy (as the absolute mean) and standard deviations were determined for each chromatographic analysis using the observed and theoretical m/z values of unmodified Chk2 kinase peptides: run 1, 0.77 ( 0.93 ppm (n ) 47); run 2, 1.05 ( 1.57 ppm (n ) 65); run 3, 1.03 ( 1.68 ppm (n ) 28), and run 4, 2.36 ( 2.49 ppm (n ) 28). For selected ion extraction, a mass of (0.01 Da was used (X-calibur, Thermoelectron). To determine the optimal mass tolerance setting for database searches with MASCOT, we compared the results from searches at 3, 10, and 50 ppm and 1.1 and 1.5 Da. The number of peptide and phosphopeptide sequences were the same for tolerances of g10 ppm. A single quadruply charged peptide was excluded in the search at 3 ppm, apparently due to “round off” error in determining the peptide mass in MASCOT. The MS data were collected in the profile mode, and the MS/ MS data were centroided during acquisition. The “raw” files were processed using LCQ-DTA software (Thermoelectron), and the resulting text files were exported to MASCOT 1.9.05 software (Matrix Science, Oxford, U.K.). The LCQ-DTA settings were as follows: grouping tolerance, 0.0001 Da; “intermediate scans”, 1; and “minimum number of scans per group”, 1. The tandem MS data were searched using the following settings and databases (considering 0, 1, or 2 missed endoprotease cleavages): (1) enzyme, trypsin or chymotrypsin, MS tolerance 3 ppm, MS/MS tolerance 0.8 Da, NCBI nonredundant database (July 26, 2005), and carbamidomethylation of cysteines and oxidation on methionines as “variable modifications”; (2) no enzyme, MS tolerance 3 ppm, MS/MS tolerance 0.8 Da, Chk2 database (gi code 13278894), and carbamidomethylation of cysteines, oxidation on methionines and phosphorylation of all serine, threonine, and tyrosine residues as variable modifications. RESULTS Expression and Purification of Phosphorylated Human Chk2. Kinase-active and -inactive versions of Chk2 protein kinase were expressed and purified from bacteria using Ni2+ affinity chromatography as described previously.34 As can be seen in Figure 1, the kinase-active (lane 1) but not kinase-inactive (lane 2174 Analytical Chemistry, Vol. 78, No. 7, April 1, 2006

Figure 1. Autophosphorylation of wild-type but not kinase-inactive Chk2 in bacteria. His6-Chk2 WT and His6-Chk2 D368N were purified from bacteria. Matched amounts of protein were resolved by SDSPAGE and blotted with the indicated antibodies.

2) Chk2 was phosphorylated on residues known to be phosphorylated in mammalian cells containing DNA DSBs. Thus, the bacterial expression system was used to produce phosphorylated Chk2 for analysis by nano-LC-FTMS/MS. Chk2 Sequence Coverage. The sequence of the human Chk2 protein kinase is shown in Figure 2. The sequences assigned from accurate mass measurements using nano-LC-FTMS/MS analysis are capitalized. Table 1 compares the theoretical and observed masses of the Chk2 peptides produced after reduction, alkylation, digestion with trypsin or chymotrypsin, and analysis using nanoLC-FTMS/MS. The absolute mean of the mass accuracy over four chromatographic runs ranged from 0.77 to 2.36 ppm with standard deviations that ranged from 0.93 to 2.49 ppm as detailed in the Experimental Section. Using selected ion extraction, we first analyzed the FTMS spectra using the accurate peptide masses (7 significant figures) of Chk2 peptides in either the +1, +2, or +3 charge states. Every FTMS spectrum containing signals corresponding to these accurate m/z values was then inspected to confirm the theoretical charge state and isotopic distribution. Peptide sequences that were not observed in +1, +2, or +3 charge

Figure 2. Amino acid sequence and phosphorylation sites of human Chk2. The capitalized residues were deduced from accurate mass measurements using nano-LC-FTMS and confirmed by tandem mass spectrometry. The yellow circled residues indicate the novel sites identified in this study, while the green boxed residues indicate previously identified sites of phosphorylation confirmed by nano-LC-FTMS/MS. The open boxed residues indicate known phosphorylation sites, which were not observed in this study.

states were analyzed by selected ion extraction considering higher charge states (+4 through +10). Although peptides of higher charge states were observed, additional peptide sequences were not obtained with the following exceptions. Sequence coverage was found from the N-terminal tryptic peptide, 4ESDVEAQQSHGSSSACSQPHGSVTQSQGSSSQSQGISSSTSTMPNSSQSSHSSSGTLSSLETVSTQELYSIPEDQEPEDQEPEEPTPAPWAR,95 and was detected as the [M + 10H]10+ ion. Partial sequences, 7VEAQ10 and 82DQEPEEP88, were deduced from the MS/MS spectrum of this multiply charged ion. Additional sequence coverage was obtained from the peptide 288IKNFFDAEDYYIVLEMEGGELFDKVVGNKR318 that was observed in the +4 charge state with sequence confirmation from the MS/MS spectrum. We also used database searching against a Chk2 “database” that was not constrained by endoprotease specificity (“No Enzyme” search). Two additional peptides, (L)ELMEGGELFDK(V) and (K)LYFYQML(L), were found that were a result of nonspecific endoprotease cleavages in tryptic digests. In addition, peptides that were the result of greater than two missed tryptic cleavages, 237AFERKTCKKVAIKIISKRKF256, 124CFDEPLLKRTDKY132, 288IKNFFDAEDYYIVLEMEGGELFDKVVGNKR318, and 319LKEATCKLYFYQMLLAVQYLHENGIIHR346 were observed. For all the observed parent masses in the FTMS spectra, fragmentation spectra were interpreted to deduce all the peptide

sequences (Table 1). Spectra from only two peptides, 419ICLSGYPPFSEHR431 and 137RTYSKKHFR145, were not observed. In summary, four data-dependent nano-LC-FTMS analyses, consuming ∼1 µg per analysis, yielded 95% coverage of the Chk2 amino acid residues. Phosphopeptide Analysis. In this study, each nano-LCFTMS analysis produced ∼6000-12 000 MS/MS spectra in the data-dependent mode. We investigated the utility of finding MS/ MS spectra of Chk2 phosphopeptides in this large data set using the accurate mass measurements from the FTICR spectra. We determined the overlap (within 3 ppm) of m/z values (+1, +2, and +3 charge states) for tryptic peptides (0, 1, and 2 missed cleavages and variable modifications for oxidized Met and carbamidomethylation of Cys residues) for Chk2 (peptides and phosphopeptides) and the four copurifying E. coli proteins, groEL (gi 41617), stringent starvation protein (gi 42999), FKBP-type peptidyl prolyl cis trans isomerase (gi 15833454), and putative formyl transferase (gi 16130190). We found 15 overlapping values. We then performed selected ion extraction analysis and inspected the FTMS spectra that contained signals corresponding to the overlapping values. Only one of the values extracted gave an FTMS spectrum of sufficient quality to determine the charge state and judge the isotopic distribution. The MS/MS spectrum for this m/z value was interpreted and found to be attributable to a Chk2 Analytical Chemistry, Vol. 78, No. 7, April 1, 2006

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Table 1. Peptides Identified in Endoprotease Digestions of His6-WT Chk2 Using Nano-LC-FTMS startend

sequencea ESDVEAQQSHGSSACSQPHGSVTQSQ GSSSQSQGISSSSTSTMPNSSQSSHSSS GTLSSLETVSTQELYSIPEDQEPEDQE PEEPTPAPWAR SIPEDQEPEDQEPEEPTPAPWc LWALQDGFANLECVNDNYWFGR DKSCEYDFDEPLLK DKSCEYDFDEPLLKR SCEYDFDEPLLK SCEYCFDEPLLKR CFDEPLLKRTDKY4 IFREVGPK NSYIAYIEDHSGNGTFVNTELVGK GKRRPLNNNSEIALSLSR RRPLNNNSEIALSLSR RPLNNNSEIALSLSR NKVFVFFDLTVDDQVYPKc VFVFFDLTVDDQSVYPK ALRDEYIMSK ALRDEYIMSK DEYIMSKc TLGSGACEGEVK LAFER AFERKTCKKVAIKIISKRKFd,e KKVAIKIISKRd,e RKFAIGSAR FAIGSAR EADPALNVETEIEILK KLNHPCIIK LNHPCIIK IKNFFDAEDYYIVLEMEGGELFDKVVGNKRe ELMEGGELFDKe LKEATCKLYFYQMLLAVQYLHENGIIHR KEATCKLYd LYFYQMLe DLKPENVLLSSQEEDCLIK ITDFGHSK ILGETSLMR TLCGTPTYLAPEVLVSVGTAGYNR NRAVDCWd SLGVILFd TQVSLKc DQITSGKc ALDLVKc LLVVDPKc FTTEEALR HPWLQDEDMK HPWLQDEDMKR FTTEEALRHPWLQDEDMK KFQDLLSEENESTALPQVLAQPSTSR FQDLLSEENESTALPQVLAQPSTSRK EGEAEGAETTKc RPAVCAAVL EGEAEGAETTKRPAVCAAVL

missed cleavages

4-95

0

73-93 96-117 118-131 118-132 120-131 120-132 124-132 146-153 154-177 178-195 180-195 181-195 196-214 198-214 215-224 215-224 218-224 225-235 236-240 237-256 244-254 254-262 256-262 263-278 279-287 280-287 288-318 302-312 319-346 320-327 326-332 347-365 366-373 374-382 383-406 405-411 412-418 432-437 438-444 459-464 466-472 475-482 483-492 483-493 475-492 494-519 495-520 524-534 535-543 524-543

0 0 1 2 0 1 1 1 0 2 1 0 1 0 1 1 0 0 0

modification CAM

CAM 2 CAM 2 CAM 2 CAM 2 CAM 1 CAM

Met-ox CAM CAM

2 0 0 1 0

CAM CAM

2 0

CAM

0 0 0 0 0 1 0 0 0 0 0 0 1 1 1 1 0 0 1

CAM CAM

CAM CAM

theoretical mass

calculated massb

9649.1980

9649.1830

2419.0339 2687.2226 1802.7854 1958.8866 1559.6635 1715.7647 1683.8290 944.5443 2627.2503 2024.1238 1839.0074 1682.9063 2260.1415 2018.0036 1224.6172 1240.6121 884.3949 1077.5124 634.3438 2451.4750 1283.8891 1004.5879 720.3918 1782.9250 1121.6379 993.5429 3650.8456 1266.5801 3451.8023 1011.5058 976.4726 2229.1198 903.4450 1018.5480 2538.2791 919.3970 747.4530 674.3962 747.3762 657.4061 782.4901 965.4817 1297.5760 1453.6772 2245.0473 2887.4563 2887.4563 1120.4883 955.5272 2058.0051

2419.0410 2687.2318 1802.7866 1958.8900 1560.6638 1715.7647 1683.8300 944.5440 2627.2518 2024.1237 1839.0084 1682.9076 2260.1373 2018.0040 1224.6176 1240.6128 884.3957 1077.5122 634.3440 2451.4769 1283.8904 1004.5880 720.3920 1782.9296 1121.6382 993.5428 3650.8368 1266.5820 3451.8054 1011.5082 976.4739 2229.1124 903.4455 1018.5482 2538.2814 919.4012 747.4536 674.3964 747.3766 657.4077 782.492 965.4814 1297.5768 1453.6772 2245.0494 2887.4619 2887.4577 1120.4882 955.5274 2058.0072

a All sequences were confirmed from manual interpretation of the MS/MS spectra. b Calculated masses were determined from the observed m/z values of singly, doubly, or triply charged species. c Indicates peptides that were detected in Chk2 kinase that was not subjected to reduction and alkylation prior to digestion with trypsin. d These peptides were found from a chymotryptic digest of reduced/carbamidomethylated Chk2 kinase. e These peptides were resulted from a search of the data against a Chk2 database with no enzyme specified.

kinase peptide. Using selective ion chromatograms, we next analyzed all possible theoretical m/z values of the singly, doubly, and triply charged species from peptides with one, two, or three phosphate groups. Using selected ion extraction, we first analyzed the FTMS spectra using the accurate peptide masses (7 significant figures) of Chk2 phosphopeptides in either +1, +2, or +3 charge states. Every FTMS spectra containing signals corresponding to these accurate m/z (s/n ∼ 4) values were then inspected to confirm 2176

Analytical Chemistry, Vol. 78, No. 7, April 1, 2006

the theoretical charge state and isotopic distribution. Table 2 shows the phosphopeptides that were observed using both accurate mass determination and MS/MS spectra to deduce both the peptide sequence and site(s) of phosphorylation. Phosphopeptides that were not detected as singly, doubly, or triply charged ions were considered by analyzing the spectral data for each charge state up to +10. One additional phosphorylated peptide was found corresponding to the N-terminal peptide described above in its monophosphorylated form. Parallel data analyses were

Table 2. Phosphopeptides Identified in Endoprotease Digests of His6 WT-Chk2 Using Nano-LC-FTMS startend

no. of PO4

theor mass

calcd massb

TQVSLK TQVSLK FAIGSAR TQVSLK

432-437 432-437 256-262 432-437

1 1 1 2

754.3621 754.3621 800.3581 834.3289

754.3633 754.3639 800.3593 834.3303

KFAIGSAR DEYIMSK DEYIMSK ITDFGHSK ILGETSLMR TLGSGACGEVK TLGSGACGEVK EGEAEGAETTK ALRDEYIMSK ALRDEYIMSK DKSCEYCFDEPLLK RPLNN NSEIALSLSR EADPALNVETEIELK RRPLNN NSEIALSLSR DKSCEYDFDEPLLKR EGEAEGAETTKRPAVCAAVL ALRDEYIMSKTLGSGACGEVK ALRDEYIMSKTLGSGACGEVK NSYIAYIEDHSGNGTFVNTELVGK FQDLLSEENESTALPQVLAQPSTSR FQDLLSEENESTALPQVLAQPSTSRK KFQDLLSEENESTALPQVLAQPSTSR KFQDLLSEENESTALPQVLAQPSTSR KFQDLLSEENESTALPQVLAQPSTSRK RKFQDLLSEENESTALPQVLAQPSTSR EVGPKNSYIAYIEDHSGNGTFVNTELVGK EVGPKNSYIAYIEDHSGNGTFVNTELVGK ILGETSLMRTLCGTPTYLAPEVLVSVGTAGYNR ILGETSLMRTLCGTPTYLAPEVLVSVGTAGYNR ILGETSLMRTLCGTPTYLAPEVLVSVGTAGYNR ESDVEAQQSHGSSACSQPHGSVTQSQGSSSQSQGISSSSTSTMPNSSQSSHSSSGTLSSLETVSTQELYSIPEDQEPEDQEPEEPTPAPWAR

255-262 218-224 218-224 366-373 374-382 225-235 225-235 524-534 215-224 215-224 118-131 181-195 263-278 180-195 118-132 524-553 215-235 215-235 154-177 495-519 495-520 494-519 494-519 494-520 493-519 149-177

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 2 1 1 1

928.4531 964.3612 964.3612 983.4113 1098.514 1157.479 1157.479 1200.455 1304.583 1304.583 1882.752 1762.873 1863.889 1918.974 2038.853 2137.971 2364.085 2444.052 2707.217 2839.328 2967.423 2967.423 3047.389 3095.517 3123.524 3217.497

149-177

2

374-406

sequencea

site localized

found by MASCOT

[MHH3PO4]1+

site confirmed

yes yes yes no

yes yes yes yes

yes yes yes yes

928.4552 964.3614 964.3614 983.4112 1098.514 1157.48 1157.48 1200.453 1304.584 1304.584 1882.752 1762.873 1863.903 1918.975 2038.855 2137.974 2364.084 2444.0559 2707.222 2839.328 2967.431 2967.422 3047.394 3095.524 3123.527 3217.497

T-423 S-435 S-260 T-432, S-435 S-260 Y-220 S-223 S-372 S-379 T-225 S-228 T-532 Y-220 S-223 S-120 S-192 T-272 no S-120 T-532 T-225 no Y-156 S-516 S-516 T-517 No S-516 S-516 S-155

yes yes no yes yes yes yes yes yes yes yes yes yes no yes yes yes no yes yes yes yes no yes yes yes

yes no no yes no yes yes no no yes yes yes no yes yes no yes yes yes no no no no yes no no

yes yes yes yes yes yes yes yes yes yes yes no yes no yes yes yes no no yes yes no no yes yes no

3297.463

3297.463

no

no

no

no

1

3618.783

3618.788

Y-390

yes

no

no

374-406

2

3698.749

3698.755

yes

no

no

374-406

2

3698.749

3698.755

yes

no

no

4-95

1

9729.164

9729.1227

T-378, T-401 T-387, T-389 no

no

no

no

a All sequences were confirmed from manual interpretation of the MS/MS spectra. b Calculated masses were determined from the observed m/z values of singly, doubly, or triply charged species.

performed on a tryptic digest of a kinase-inactive mutant of Chk2 protein. Figure 3A shows the selected ion chromatogram for an m/z value of 801.3660, the theoretical mass of the protonated molecular ion for the FAIGpSAR phosphopeptide (Ser-260). A single peak (peak width (PW) ) 6 s) was observed at a retention time (tr) of 14.15 min. The partial FTICR mass spectrum is detailed in the inset of panel A. The expected isotopic distribution for a peptide of this mass was observed with differences of 1.0037 and 1.0032 Da between the isotopic peaks. The low-energy CID spectrum obtained in the linear trap is shown in Figure 3B. A prominent neutral loss ion ([M - H3PO4 + H]+) at m/z ) 703.51 dominates the MS/MS spectrum. However, phosphate-bearing fragment ions were observed at m/z ) 654.47, 583.34, and 470.35 for y6, y5, and y4, respectively, confirming the only possible location of the phosphate group. The list of observed ions is given in Supporting Information, Table 1. The selected ion chromatogram for the protonated molecular ion of FAIGpSAR peptide extracted from the nano-FTMS analysis of the tryptic digest of the kinase-

inactive mutant of the Chk2 protein (D368N) is shown in Figure 3C. A weak signal was observed at tr ) 30.45 min with a PW of 3.6 s. Although there are minor signals present (Figure 3C inset), the mass differences between the signals do not correspond to an expected isotopic pattern for a peptide. Selected ion extraction of the singly charged ion of the nonphosphorylated peptide from kinase-inactive Chk2 digest showed a single peak (PW ) 3 s; tr ) 14.47 min) and an associated MS/MS spectrum consistent with the nonphosphorylated sequence, FAIGSAR (data not shown). Selected ion extraction of all theoretical accurate m/z values corresponding to the masses of the mono-, di-, and triphosphorylated peptides demonstrated that kinase-inactive Chk2 was not phosphorylated. The facile neutral loss of phosphoric acid [M + nH - H3 PO4 ]n+ from the protonated molecular ion of O-linked phosphopeptides is often the only apparent ion in CID spectra and underpins the method of neutral loss scanning to locate phosphopeptides in complex peptide mixtures during LC-MS/MS. Figure 4 shows Analytical Chemistry, Vol. 78, No. 7, April 1, 2006

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Figure 4. Nano-LC-FTMS of a tryptic phosphopeptide containing Thr-532 of Chk2. The MS/MS spectrum was acquired from the [M + 2H]2+ ion (inset) from the linear trap as described in the Experimental Section. The complete list of assigned ions is given in the Supporting Information, Table 2.

Figure 3. Selected ion chromatogram of Ser-260 phosphopeptide from wild type and kinase-inactive Chk2. Panel A, selected ion chromatogram for m/z ) 801.3660 from nano-LC-FTMS analysis of a tryptic digest of wild-type Chk2 kinase with FTICR partial spectrum as the inset. Panel B, low-energy CID spectrum of the FAIGpSAR phosphopeptide ([M + H]+ ) 801.37) from wild-type Chk2 kinase. The complete list of assigned ions is given in the Supporting Information, Table 1. Panel C, selected ion chromatogram for m/z ) 801.3660 from nano-LC-FTMS analysis of a tryptic digest of kinase dead Chk2 kinase with the FTICR spectrum as the inset.

the CID spectrum acquired from the doubly charged ion (m/z ) 601.2354) of the phosphopeptide that contains Thr-532 of Chk2 kinase. The neutral loss of phosphoric acid from the doubly 2178 Analytical Chemistry, Vol. 78, No. 7, April 1, 2006

charged parent ion, [M + 2H - H3PO4 ]2+, was observed as a minor signal at m/z ) 552.37. A complete y and b series of phosphate-bearing ions was observed (except for b6). Complementary, phosphate-containing b9 and y3 ions were observed from cleavage at the -E-pT- peptide bond, consistent with phosphorylation at Thr-532. The list of assigned ions is given in Supporting Information, Table 2. We found that the neutral losses from the phosphorylated residues ions were not prominent signals in 18 of 36 low-energy fragmentation spectra of phosphopeptides from Chk2 kinase (see Supporting Information Figures 2, 11, and 12). Selected ion chromatograms using accurate m/z values of all theoretical mono-, di, and triphosphorylated peptides gave single peaks in all but three cases, m/z ) 579.7474, 653.2995, and 755.3712. Figure 5A shows the selected ion chromatogram using the theoretical mass of a doubly charged phosphopeptide with the sequence 215ALRDEYIMSK224. Two peaks with tr ) 13.95 and 14.19 min and PW ) 4.2 and 6.6 s, respectively, were observed. Figure 5B shows the MS/MS spectrum of the peptide eluting at 14.19 min. The spectrum is dominated by a parent neutral loss ion (m/z ) 604.58). Phosphate-containing fragment ions consistent with cleavage at the M-pSer peptide bond were observed (b9 and for y2), as well as phosphorylated fragments, y3 and y4. The data indicate that the spectrum containing an intense neutral loss ion is phosphorylated at the Ser residue (Ser-223), ALRDEYIMpSK. The MS/MS spectrum of the earlier eluting peptide is shown in Figure 5C. A complete suite of y and b ions was observed, localizing the phosphate moiety to the Tyr residue (Tyr-220), ALRDEpYIMSK. Ions consistent with the neutral loss of phosphoric acid were not observed in the spectrum of the peptide eluting at 13.95 min. The list of assigned fragment ions is given in the Supporting Information, Tables 3 and 4. To confirm phosphorylation at the Tyr residue, an aliquot of the sample was analyzed for the phosphorylated Tyr immonium ion by nano-LCQTOF mass spectrometry. Figure 5D shows the partial MS/MS spectrum of the triply charged ion (m/z ) 435.68) of the monophosphorylated 215ALRDEYIMSK224 peptide. An ion was observed at m/z ) 216.039 that agreed with the mass of the phosphorylated Tyr immonium ion (m/z ) 216.043). Interpretation of the additional four MS/MS spectra associated with two closely

eluting peaks in individual selected ion chromatograms revealed positional isomers of the following phosphopeptide pairs: pTLGSGACGEVK, TLGpSGACGEVK; and pTQVSLK, TQVpSLK, revealing phosphorylation at Thr-225, Ser-228; and Thr-432, and Ser435 (Figure 2). The spectra and assigned ions are given in Supporting Information, Figures 4, 5, 8, and 9. Positional isomers of the three mono-phosphorylated peptide pairs eluted with similar retention times, within ∼30 s. The phosphopeptides and assigned phosphorylation sites from the nano-LC-FTMS analysis of endoprotease digests of Chk2 are summarized in Table 2. Selected ion chromatograms using accurate m/z values and interpretation of MS/MS spectra resulted in the localization of phosphate groups to Ser-120, Tyr-220, Ser223, Thr-225, Ser-228, Ser-260, Thr-272, Ser-372, Ser-379, Thr-432, Ser-435, Ser-516, and Thr-532. Twelve phosphopeptides were found in which the modification sites could not be unambiguously assigned. Two of these phosphopeptides, 149EVGPKNSYIAYIEDHSGNGTFVNTELVGK177 and 181RPLNNNSEIALSLSR195, assigned by accurate mass, cover residues of Chk2 where there are no reported phosphorylation sites. The MS/MS spectra for both phosphopeptides contained sequencing ions to confirm the identity of the peptide, and the MS/MS spectrum for the latter peptide contained a prominent neutral loss ion at m/z ) 833.67 (see Supporting Information, Figures 2 and 3). The identification of phosphate-containing sequences from accurate mass measurements is important for devising strategies to produce smaller peptides that can be analyzed by CID to pinpoint the phosphorylated residues. Chk2 Is Phosphorylated on Ser-260 in Mammalian Cells. Western blot analysis (Figure 1) and nano-LC-FTMS demonstrated that Chk2 is capable of phosphorylating itself on physiologically relevant sites when it is overproduced in bacteria. In addition to known phosphorylation sites, nano-LC-FTMS identified 11 novel sites. To determine if Chk2 was phosphorylated on any of these new sites in mammalian cells, a phosphospecific antibody was produced for Ser-260. As seen in Figure 6A, this antibody was specific to phosphorylated Chk2 because it recognized wild-type Chk2 (lane 1) but not a Chk2 mutant containing Ala in place of Ser at position 260 (lane 2). This antibody was used to monitor the phosphorylation of endogenous Chk2 on Ser260 in both the absence and the presence of DNA DSBs (Figure 6B). In the absence of DNA damage, endogenous Chk2 was not phosphorylated on Ser-260 (lane 1). However, exposure of cells to ionizing radiation induced the phosphorylation of Chk2 on Ser260. Thus, analysis of bacterial Chk2 by nano-LC-FTMS identified a new physiologically relevant Chk2 phosphorylation site.

Figure 5. Nano-LC-FTMS and nano-LC-QTOF-MS of isobaric peptides containing sites Tyr-220 and Ser-223 from Chk2. Panel A, selected ion chromatogram for m/z ) 653.2995 with the detail of the eluting peak shape as the inset. Panel B, fragmentation spectrum of the [M + 2H]2+ ) 653.2995 with tr ) 14.21 min. Panel C, fragmentation spectrum of the [M + 2H]2+ ) 653.2995 with tr ) 13.93 min. Panel D, immonium ion region of the tandem spectrum from 215ALRDEYIMSK224 acquired by nano-LC-QTOF mass spectrometry as described in the Experimental Section. The precusor ion was the triply charged species (m/z ) 435.86). The complete list of assigned ions is given in the Supporting Information, Tables 3 and 4.

DISCUSSION Comprehensive phosphorylation analysis of phosphoproteins using mass spectrometry requires measurements of phosphopeptides representing all potentially modifiable residues. Complete amino acid sequence coverage by mass spectrometry is sequence dependent and has been difficult to achieve, even if the quantity of purified phosphoprotein is not limiting. The choice of endoproteases, the necessity for denaturation and reduction/alkylation of Cys residues, the different ionization potentials of peptides, the substoichiometric nature of phosphorylation, and the recovery of multiphosphorylated and hydrophobic peptides are recognized issues and difficulties.4,5 Multiprotease methods have been used Analytical Chemistry, Vol. 78, No. 7, April 1, 2006

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Figure 6. Ser-260, an IR-inducible Chk2 autophosphorylation site. (A) Lysates from HeLa cells expressing Flag3-Chk2 WT or Flag3S260A were incubated with an antibody specific for the Flag epitope. Immunoprecipitates were resolved by SDS-PAGE and blotted with an antibody specific for Chk2 phosphorylated on Ser-260. The blot was then stripped and reprobed with an antibody specific for the Flag epitope. (B) Total lysates from HeLa cells that had been mockirradiated (lane 1) or exposed to 10 Gy IR (lanes 2,3) were resolved by SDS-PAGE and blotted with an antibody specific for Chk2 phosphorylated on Ser-260. The blot was then stripped and probed with an antibody specific for Chk2.

to increase amino acid sequence coverage by producing peptides more amenable to sequencing by CID analysis,40 and this approach has been successfully extended to phosphoprotein analysis.6,12 The combination of resolution and sensitivity afforded by FTICR has been shown to significantly increase protein sequence coverage by enabling sequence assignments to be deduced from the accurate measurements of large multiply charged peptides.25,41 In the present study of the Chk2 protein kinase, we observed the N-terminal tryptic peptide as the [M + 10H]10+ species and acquired fragmentation spectra consistent with the sequence of this peptide. Quadrupole linear ion trap-FTICR mass spectrometry gives both accurate mass measurements (∼5 ppm) and rapid low-energy CID analysis in the ion trap (∼100 ms) of peptides separated by nano-LC.24,42 In agreement with a previous report, we found that for phosphopeptides (masses of ∼2500) were observed as multiply charged species using FTICR-MS; however, as previously reported, we were unable (40) Gatlin, C. L.; Eng, J. K.; Cross, S. T.; Detter, J. C.; Yates, J. R., 3rd. Anal. Chem. 2000, 72, 757-763. (41) Bruce, J. E.; Anderson, G. A.; Wen, J.; Harkewicz, R.; Smith, R. D. Anal. Chem. 1999, 71, 2595-2599. (42) Belov, M. E.; Anderson, G. A.; Wingerd, M. A.; Udseth, H. R.; Tang, K.; Prior, D. C.; Swanson, K. R.; Buschbach, M. A.; Strittmatter, E. F.; Moore, R. J.; Smith, R. D. J. Am. Soc. Mass Spectrom. 2004, 15, 212-232.

2180 Analytical Chemistry, Vol. 78, No. 7, April 1, 2006

to deduce the location of the phosphate groups.43 Alternative endoprotease or new fragmentation methods are being pursued to determine the location of additional Chk2 phosphorylation sites. Simultaneous FTICR mass measurements and low-energy CID spectral acquisition with greater speed significantly increases the probability of acquiring spectra from a greater number of peptides within the chromatographic time frame.15 In this study, we acquired ∼17 400 MS and 35 985 MS/MS spectra of peptides from a sample containing human Chk2 in 4 nano-LC-FTMS analyses. Of the 87 potential phosphorylation sites that were included in our sequence coverage of the protein (103 total sites), we found that 3 Thr, 7 Ser, and 1 Tyr residues were phosphorylated. To determine if the kinase-inactive Chk2 protein was phosphorylated, we performed selected ion extraction using the accurate peptide masses (7 significant figures) of Chk2 phosphopeptides (Table 2) (+1, +2, or +3 charge states). We manually inspected all FTMS spectra for signals corresponding to the accurate m/z values of identified Chk2 phosphopeptides and interpreted the FTMS spectra for charge state and isotopic distribution. We found no spectral evidence for Chk2 phosphopeptides in the tryptic digest of the kinase-inactive mutant. We concluded that the identified phosphorylation sites were a result of Chk2 autophosphorylation activity. This study utilized recombinant human Chk2 that was overproduced in bacteria, and although the authenticity of bacterially produced Chk2 was confirmed by identifying known phosphorylation sites, it is a concern that the newly identified phosphorylation sites may not be physiologically relevant. For example, overproduction of Chk2 could facilitate aberrant auto/trans phosphorylation events due to the high levels of Chk2 that accumulate and oligomerize in bacteria. Chk2 is a serine/threonine protein kinase; yet Tyr-220 was identified as a novel autophosphorylation site. Future studies will be directed at determining whether Chk2 is phosphorylated on each of the novel sites in mammalian cells in a DNA damage-inducible manner. As a first step toward this goal, we generated a phosphospecific antibody that recognizes Chk2 only when it is phosphorylated on Ser-260. Ser-260 was one of the novel sites identified by nano-LC-FTMS/MS in this study. Chk2 was inducibly phosphorylated on Ser-260 in mammalian cells containing DNA DSBs due to exposure to ionizing radiation. Similar approaches will be required to determine whether the other 10 sites are also inducibly phosphorylated in mammalian cells. In addition, another site identified in this study, Ser-379, is conserved in several Chk2 orthologs, including Rad53, the budding yeast ortholog. Rad53 also undergoes autophosphorylation, and Ser-350 (Ser-379 equivalent) has been identified as one of the autophosphorylations sites.44 Reversible phosphorylation regulates Chk2 kinase activity, intracellular localization, and association with other proteins.26 Determining the contribution made by each of the novel phosphorylation sites to Chk2 function is anticipated to provide important insights into how cells respond to genotoxic stress and how cancer cells subvert these regulatory pathways. (43) Shi, S. D.; Hemling, M. E.; Carr, S. A.; Horn, D. M.; Lindh, I.; McLafferty, F. W. Anal. Chem. 2001, 73, 19-22. (44) Sweeney, F. D.; Yang, F.; Chi, A.; Shabanowitz, J.; Hunt, D. F.; Durocher, D. Curr. Biol. 2005, 15, 1364-1375.

CONCLUSIONS Rapid acquisition nano-LC-FTMS is an efficient method for the comprehensive structural analysis of phosphoproteins, as demonstrated by the above study of a cell cycle human kinase (Chk2) with 103 potential Ser, Thr, or Tyr phosphorylation sites. Selected ion extraction and accurate mass measurements (∼3 ppm) in combination with data-dependent acquisition of ∼10 000 MS/MS spectra for each chromatographic analysis is an efficient strategy for discovering novel phosphorylation sites and deducing the presence of phosphorylated residues in large (>2500 Da) phosphopeptides. The signature neutral loss of phosphoric acid from the parent ion of O-linked phosphopeptides is not always a prominent signal in low-energy CID spectra using electrospray ionization. Comparative nano-LC-FTMS analysis of wild-type and inactive kinase is useful not only to locate spectra of phosphopeptides in large MS data sets but also to determine the biological specificity of site-specific phosphorylation. ACKNOWLEDGMENT The authors thank Janis Watkins for contributing Figure 1. The authors thank Paul H. Davis for expert assistance with the

database search algorithms and mass spectrometric software. This work was supported, in part, by the National Centers of Research Resources of the National Institutes of Health (P41RR00954), by the Siteman Cancer Center, by institutional resources provided to the Proteomics Center by Washington University, and by a grant from the National Institutes of Health to H.P.-W. C.M.L. is a member of the Medical Scientist Training Program at Washington University School of Medicine and was supported in part by a Pre- and Postgraduate Training in Molecular Hematology (NIH/NHLBI Grant T32 HL07088). H.P.-W. is an Investigator of the Howard Hughes Medical Institute. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review August 24, 2005. Accepted January 13, 2006. AC051520L

Analytical Chemistry, Vol. 78, No. 7, April 1, 2006

2181