蛋白質磷酸化(phosphorylation)與乙醯化(acetylation)都是重要的轉譯後修飾(post-translational modification)，在生物體內參與調控著許多功能。雖然目前有許多分析方法被提出，但分析蛋白質磷酸化仍然具有挑戰性。在本篇論文中，我們提出一種分析策略從複雜的數據中篩選出磷酸化胜肽(phosphopeptide)的訊號。此分析方法結合去磷酸化反應(dephosphorylation)、準確的質量量測、及電腦計算方法來找出具有因為磷酸根被移除而產生“質量偏移(mass shift)”的訊號。這些會因為加入去磷酸酶(phosphatase)而偏移的訊號會被認為是可能的磷酸化胜肽訊號，在接下來的液相層析串連式質譜儀(LC-MS/MS)會針對其滯留時間(retention time)、質荷比(m/z)來做分析，有利於得到有效的磷酸化胜肽鑑定。我們驗證了此方法可以比過去傳統的液相層析串連式質譜技術得到更多的酪蛋白(casein)磷酸化胜肽訊號。另外也在鼻咽癌細胞(NPC cell)中鑑定到221個磷酸化位置。此分析技術說明了結合計算方法可以有效的從複雜的資料數據中選出磷酸化胜肽訊號。接著我們發展將高解析度質譜數據引進到我們的分析策略中的流程，比起過去用飛行時間質譜儀(Q-TOF)，此高解析度質譜數據又得到更多酪蛋白磷酸化胜肽訊號。並且藉由結合去磷酸化反應及計算方法，我們得到具有高重複性與高信心度鑑定結果的肺癌細胞的酪胺酸磷酸化蛋白質體(tyrosine phosphoproteome)。此外，產生質量偏移與計算方法的概念也用在分析蛋白質乙醯化的分析上。我們將有同位素標定與未標定的乙醯基等量的轉移到受質蛋白質上，再接著利用電噴灑質譜分析(ESI-MS)，經由電腦計算方法，可以選出具有特定質量差異與強度相當的訊號，針對這些訊號做串連式質譜分析，可以得到乙醯化胜肽的鑑定。我們成功的使用了組蛋白(histone)的兩段胜肽(H3, aa 1-20與H2B, aa 1-21)及組蛋白混合液(H2A與H2B)驗證了此方法可以找到體外的乙醯化位置，在未來也許可以用來大量的選擇會被乙醯化的受質蛋白。本篇論文提出的分析方法，結合質譜儀量測質量偏移與電腦計算方法，針對被修飾的訊號做分析，幫助得到有效的身份鑑定。

Protein phosphorylation and acetylation are key post-translational modifications that govern biological processes. Despite a number of analytical strategies have been exploited for the characterization of protein phosphorylation, the identification of protein phosphorylation sites is still challenging. We proposed here an alternative approach to mine phosphopeptide signals in a mixture of proteins. The approach combined dephosphorylation reaction, accurate mass measurements, and a computing algorithm to differentiate possible phosphopeptide signals in the LC-MS analyses by taking advantage of the mass shift generated by alkaline phosphatase treatment. The retention times and m/z values of these selected signals were used to facilitate subsequent LC-MS/MS experiments for phosphorylation site determination. Unlike commonly used neutral loss scan experiments for phosphopeptide detection, this strategy may not bias against tyrosine-phosphorylated peptides. We have demonstrated the applicability of this strategy to sequence more, in comparison with conventional data-dependent LC-MS/MS experiments, number of phosphopeptides in a mixture of α- and β-caseins. The analytical scheme was applied to characterize the nasopharyngeal carcinoma (NPC) cellular phosphoproteome and yielded 221 distinct phosphorylation sites. Our data demonstrated the merits of computation in mining phosphopeptide signals from a complex mass spectrometric dataset. According to the above result, mass accuracy seems to play an important role in efficiently selecting out phosphopeptide signals. In recent years, hybrid linear ion trap (LTQ)/Orbitrap mass spectrometer, with high mass accuracy, has emerged as a powerful instrument in proteomic analysis. We developed a process to incorporate LTQ/Orbitrap LC-MS data into our strategy for taking advantage of the accurate mass measurement. More phosphopeptides in casein mixture, compared to the work finished by using a quadrupole/time-of-flight mass spectrometer, were identified by using LTQ/Orbitrap data. The characterization of the lung cancer cell tyrosine phosphoproteome revealed that the use of alkaline phosphatase treatment combined with accurate mass measurement in this strategy increased data repeatability and confidence.
The concept of “signal mining” was applied to develop an analytical strategy to determine the in vitro acetylation sites of proteins by tracing isotope labeling on acetyl groups using mass spectrometry. Isotope-labeled and unlabeled acetyl groups transferred onto the substrates in vitro resulted in specific “mass difference”. The identification of acetylation site is facilitated by conducting MS/MS experiments on those signals. Acetylation reactions of substrates were performed in the presence of acetyltransferase and equal molar amounts of isotope-labeled acetyl coenzyme A ([13C2-2-D3] acetyl-CoA) and unlabeled acetyl-CoA. Signals with 5-Da (or their multiples) mass differences and equal responses were selected out by program computation. Those potential acetylated peptide signals were subjected to MS/MS analyses for determination of acetylation sites. We have used histone H3 peptide (aa 1-20), histone H2B peptide (aa 1-21), histone H2A and histone H2B proteins as the model compounds to demonstrate the applicability of this analytical scheme for the characterization of in vitro acetylation sites.

Aggarwal BD, Calvi BR. 2004. Chromatin regulates origin activity in Drosophila follicle cells. Nature 430:372–376.

Alland L, Muhle R, Hou HJr, Potes J, Chin L, Schreiber-Agus N, DePinho RA. 1997. Role for N-CoR and histone deacetylase in Sin3-mediated transcriptional repression. Nature 387:49–55.

Amanchy R, Kalume DE, Iwahori A, Zhong J, Pandey A. 2005. Phosphoproteome analysis of HeLa cells using stable isotope labeling with amino acids in cell culture (SILAC). J Proteome Res 4:1661–1671.

Andersson L, Porath J. 1986. Isolation of phosphoproteins by immobilized metal (Fe3+) affinity chromatography. Anal Biochem 154:250–254.

Archer SY, Hodin RA. 1999. Histone acetylation and cancer. Curr Opin Genet Dev 9:171–174.

Beausoleil SA, Jedrychowski M, Schwartz D, Elias JE, Villen J, Li J, Cohn MA, Cantley LC, Gygi SP. 2004. Large-scale characterization of HeLa cell nuclear phosphoproteins. Proc Natl Acad Sci USA 101:12130–12135.

Bellew M, Coram M, Fitzgibbon M, Igra M, Randolph T, Wang P, May D, Eng J, Fang R, Lin C, Chen J, Goodlett D, Whiteaker J, Paulovich A, McIntosh M. 2006. A suite of algorithms for the comprehensive analysis of complex protein mixtures using high-resolution LC-MS. Bioinformatics 22:1902–1909.

Bennett KL, Stensballe A, Podtelejnikov AV, Moniatte M, Jensen ON. 2002. Phosphopeptide detection and sequencing by matrix-assisted laser desorption/ionization quadrupole time-of-flight tandem mass spectrometry. J Mass Spectrom 37:179–190.

Bergström Lind S, Molin M, Savitski MM, Emilsson L, Aström J, Hedberg L, Adams C, Nielsen ML, Engström A, Elfineh L, Andersson E, Zubarev RA, Pettersson U. 2008. Immunoaffinity enrichments followed by mass spectrometric detection for studying global protein tyrosine phosphorylation. J Proteome Res 7:2897–2910.

Bernardi R, Scaglioni PP, Bergmann S, Horn HF, Vousden KH, Pandolfi PP. 2004. PML regulates p53 stability by sequestering Mdm2 to the nucleolus. Nat Cell Biol 6:665–672.

Bestor TH. 1998. Gene silencing. Methylation meets acetylation. Nature 393:311–312.

Bolden JE, Peart MJ, Johnstone RW. 2006. Anticancer activities of histone deacetylase inhibitors. Nat Rev Drug Discov 5:769–784.

Borchers C, Parker CE, Deterding LJ, Tomer KB. 1999. Preliminary comparison of precursor scans and liquid chromatography-tandem mass spectrometry on a hybrid quadrupole time-of-flight mass spectrometer. J Chromatogr A 854:119–130.

Boutillier AL, Trinh E, Loeffler JP. 2003. Selective E2F-dependent gene transcription is controlled by histone deacetylase activity during neuronal apoptosis. J Neurochem 84:814–828.

Brehm A, Miska EA, McCance DJ, Reid JL, Bannister AJ, Kouzarides T. 1998. Retinoblastoma protein recruits histone deacetylase to repress transcription. Nature 391:597–601.

Burns KL, Gelbaum LT, Sullards MC, Bostwick DEA. 2005. Iso-coenzyme A. J Biol Chem 280:16550–16558.

Carr SA, Huddleston MJ, Annan RS. 1996. Selective detection and sequencing of phosphopeptides at the femtomole level by mass spectrometry. Anal Biochem 239:180–192.

Chen A, Kaganovsky E, Rahimipour S, Ben-Aroya N, Okon E, Koch Y. 2002. Two forms of gonadotropin-releasing hormone (GnRH) are expressed in human breast tissue and overexpressed in breast cancer: a putative mechanism for the antiproliferative effect of GnRH by down-regulation of acidic ribosomal phosphoproteins P1 and P2. Cancer Res 62:1036–1044.

Chen CT, Chen YC. 2005. Fe3O4/TiO2 core/shell nanoparticles as affinity probes for the analysis of phosphopeptides using TiO2 surface-assisted laser desorption/ionization mass spectrometry. Anal Chem 77:5912–5919.

Chen Y, Kwon SW, Kim SC, Zhao Y. 2005. Integrated approach for manual evaluation of peptides identified by searching protein sequence databases with tandem mass spectra. J Proteome Res 4:998–1005.

Chu YW, Yang PC, Yang SC, Shyu YC, Hendrix MJ, Wu R, Wu CW. 1997. Selection of invasive and metastatic subpopulations from a human lung adenocarcinoma cell line. Am J Respir Cell Mol Biol 17:353–360.

Clayton AL, Rose S, Barratt MJ, Mahadevan LC. 2000. Phosphoacetylation of histone H3 on c-fos- and c-jun-associated nucleosomes upon gene activation. EMBO J 19:3714–3726.

Clough RC, Barnum SR, Jaworski JG. 1989. Synthesis of radiolabeled acetyl-coenzyme A from sodium acetate. Anal Biochem 176:82–84.

Cohen P. 2000. The regulation of protein function by multisite phosphorylation--a 25 year update. Trends Biochem Sci 25:596–601.

Collins MO, Yu L, Campuzano I, Grant SG, Choudhary JS. 2008. Phosphoproteomic analysis of the mouse brain cytosol reveals a predominance of protein phosphorylation in regions of intrinsic sequence disorder. Mol Cell Proteomics 7:1331–1348.

Connor PA, McQuillan AJ. 1999. Phosphate Adsorption onto TiO2 from Aqueous Solutions: An in Situ Internal Reflection Infrared Spectroscopic Study. Langmuir 15:2916–2921.

Dass C, Mahalakshmi P, Grandberry DJ. 1994. Manipulation of ion-pairing reagents for reversed-phase high-performance liquid chromatographic separation of phosphorylated opioid peptides from their non-phosphorylated analogues. Chromatography A 678:249–257.

DeGiorgis JA, Jaffe H, Moreira JE, Carlotti CG Jr, Leite JP, Pant HC, Dosemeci A. 2005. Phosphoproteomic analysis of synaptosomes from human cerebral cortex. J Proteome Res 4:306–315.

DeGnore JP, Qin J. 1998. Fragmentation of phosphopeptides in an ion trap mass spectrometer. J Am Soc Mass Spec 9:1175–1188.

Di Gennaro E, Bruzzese F, Caraglia M, Abruzzese A, Budillon A. 2004. Acetylation of proteins as novel target for antitumor therapy: review article. Amino Acids 26:435–441.

Ding J, Burkhart W, Kassel DB. 1994. Identification of phosphorylated peptides from complex mixtures using negative-ion orifice-potential stepping and capillary liquid chromatography/electrospray ionization mass spectrometry. Rapid Commun Mass Spectrom 8:94–98.

Dormeyer W, Dorr A, Ott M, Schnölzer M. 2003. Acetylation of the HIV-1 Tat protein: an in vitro study. Anal Bioanal Chem 376:994–1005.

Dormeyer W, Ott M, Schnölzer M. 2005. Probing lysine acetylation in proteins: strategies, limitations, and pitfalls of in vitro acetyltransferase assays. Mol Cell Proteomics 4:1226–1239.

Everett AD, Bushweller J. 2003. Hepatoma derived growth factor is a nuclear targeted mitogen. Current Drug Targets 4:367–371.

Ficarro SB, McCleland ML, Stukenberg PT, Burke DJ, Ross MM, Shabanowitz J, Hunt DF, White FM. 2002. Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nat Biotechnol 20:301–305.

Fraga MF, Ballestar E, Villar-Garea A, Boix-Chornet M, Espada J, Schotta G, Bonaldi T, Haydon C, Ropero S, Petrie K, Iyer NG, Pérez- Rosado A, Calvo E, Lopez JA, Cano A, Calasanz MJ, Colomer D, Piris MA, Ahn N, Imhof A, Caldas C, Jenuwein T, Esteller M. 2005. Loss of acetylation at Lys16 and trimethylation at Lys20 of histone H4 is a common hallmark of human cancer. Nature Genet 37:391–400.

Giorgianni F, Beranova-Giorgianni S, Desiderio DM. 2004. Identification and characterization of phosphorylated proteins in the human pituitary. Proteomics 4:587–598.

Goshe MB, Conrads TP, Panisko EA, Angell NH, Veenstra TD, Smith RD. 2001. Phosphoprotein isotope-coded affinity tag approach for isolating and quantitating phosphopeptides in proteome-wide analyses. Anal Chem 73:2578–2586.

Goshe MB, Veenstra TD, Panisko EA, Conrads TP, Angell NH, Smith RD. 2002. Phosphoprotein isotope-coded affinity tags: application to the enrichment and identification of low-abundance phosphoproteins. Anal Chem 74:607–616.

Grønborg M, Kristiansen TZ, Stensballe A, Andersen JS, Ohara O, Mann M, Jensen ON, Pandey A. 2002. A mass spectrometry-based proteomic approach for identification of serine/threonine-phosphorylated proteins by enrichment with phospho-specific antibodies: identification of a novel protein, Frigg, as a protein kinase A substrate. Mol Cell Proteomics 1:517–527.

Gruhlew A, Olsen JV, Mohammed S, Mortensen P, Færgeman NJ, Mann M, Jensen ON. 2005. Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway. Mol Cell Proteomics 4:310–327.

Hassig CA, Fleischer TC, Billin AN, Schreiber SL, Ayer DE. 1997. Histone deacetylase activity is required for full transcriptional repression by mSin3A. Cell 89:341–347.

Hess-Stumpp H. 2005. Histone deacetylase inhibitors and cancer: from cell biology to the clinic. Eur J Cell Biol 84:109–121.

Hirschberg D, Jagerbrink T, Samskog J, Gustafsson M, Stahlberg M, Alvelius G, Husman B, Carlquist M, Jornvall H, Bergman T. 2004. Detection of phosphorylated peptides in proteomic analyses using microfluidic compact disk technology. Anal Chem 76:5864–5871.

Holmes MR, Giddings MC. 2004. Prediction of posttranslational modifications using intact-protein mass spectrometric data. Anal Chem 76:276–282.

Hubbard MJ, Cohen P. 1993. On target with a new mechanism for the regulation of protein phosphorylation. Trends Biochem Sci 18:172–1777.

Huddleston MJ, Annan RS, Bean MT, Carr SA. 1993. Selective detection of 5 phosphopeptides in complex mixtures by electrospray liquid chromatography Mass spectrom. J Am Soc Mass Spectrom 4:710–717.

Hunter AP, Games DE. 1994. Chromatographic and mass spectrometric methods for the identification of phosphorylation sites in phosphoproteins. Rapid Commun Mass Spectrom 8:559–570.

Hunter T. 2000. Signaling-2000 and Beyond. Cell 100:113–127.

Imanishi SY, Kochin V, Ferraris SE, de Thonel A, Pallari HM, Corthals GL, Eriksson JE. 2007. Reference-facilitated phosphoproteomics: fast and reliable phosphopeptide validation by microLC-ESI-Q-TOF MS/MS. Mol Cell Proteomics 6:1380–1391.

Is'harc H, Watling D, Kerr IM. 2001. Phosphotyrosine profiling to identify novel components of interferon and interleukin 6-family cytokine signaling. Proteomics 1:767–772.

Ishihama Y, Wei FY, Aoshima K, Sato T, Kuromitsu J, Oda Y. 2007. Enhancement of the efficiency of phosphoproteomic identification by removing phosphates after phosphopeptide enrichment. J Proteome Res 6:1139–1144.

Iyer NG, Chin SF, Ozdag H, Daigo Y, Hu DE, Cariati M, Brindle K, Aparicio S, Caldas C. 2004. p300 regulates p53-dependent apoptosis after DNA damage in colorectal cancer cells by modulation of PUMA/p21 levels. Proc Natl Acad Sci USA 101:7386–7391.

Jaffe H, Veeranna, Shetty KT, Pant HC. 1998. Characterization of the phosphorylation sites of human high molecular weight neurofilament protein by electrospray ionization tandem mass spectrometry and database searching. Biochemistry 37:3931–3940.

Jaffe H, Veeranna, Pant HC. 1998. Characterization of serine and threonine phosphorylation sites in beta-elimination/ethanethiol addition-modified proteins by electrospray tandem mass spectrometry and database searching. Biochemistry 37:16211–16224.

Kawakami T, Tateishi K, Yamano Y, Ishikawa T, Kuroki K, Nishimura T. 2005. Protein identification from product ion spectra of peptides validated by correlation between measured and predicted elution times in liquid chromatography/mass spectrometry. Proteomics 5:856–864.

Kim JY, Kim KW, Kwon HJ, Lee DW, Yoo JS. 2002. Probing lysine acetylation with a modification-specific marker ion using high-performance liquid chromatography/electrospray-mass spectrometry with collision-induced dissociation. Anal Chem 74:5443–5449.

Kim JE, Tannenbaum SR, White FM. 2005. Global phosphoproteome of HT-29 human colon adenocarcinoma cells. J Proteome Res 4:1339–1346.

Knoepfler PS, Eisenman RN. 1999. Sin meets NuRD and other tails of repression. Cell 99:447–450.

Kouzarides T. 1999. Histone acetylases and deacetylases in cell proliferation. Curr Opin Genet Dev 9:40–48.

Kuo MH, Allis CD. 1998. Roles of histone acetyltransferases and deacetylases in gene regulation. Bioessays 20:615–626.

Larsen MR, Sørensen GL, Fey SJ, Larsen PM, Roepstorff P. 2001. Phospho-proteomics: evaluation of the use of enzymatic de-phosphorylation and differential mass spectrometric peptide mass mapping for site specific phosphorylation assignment in proteins separated by gel electrophoresis. Proteomics 1:223–238.

Larsen MR, Thingholm TE, Jensen ON, Roepstorff P, Jørgensen TJD. 2005. Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol Cell Proteomics 4:873–886.

Lee CH, McComb ME, Bromirski M, Jilkine A, Ens W, Standing KG, Perrault H. 2001. On-membrane digestion of beta-casein for determination of phosphorylation sites by matrix-assisted laser desorption/ionization quadrupole/time-of-flight mass spectrometry. Rapid Commun Mass Spectrom 15:191–202.

Lees-Miller SP, Anderson CW. 1989. Two human 90-kDa heat shock proteins are phosphorylated in vivo at conserved serines that are phosphorylated in vitro by casein kinase II. J Biol Chem 264:2431–2437.

Lees-Miller SP, Anderson CW. 1989. The human double-stranded DNA-activated protein kinase phosphorylates the 90-kDa heat-shock protein, hsp90 alpha at two NH2-terminal threonine residues. J Biol Chem 264:17275–17280.

Liao PC, Leykam J, Andrews PC, Gage DA, Allison J. 1994. An approach to locate phosphorylation sites in a phosphoprotein: mass mapping by combining specific enzymatic degradation with matrix-assisted laser desorption/ionization mass spectrometry. Anal Biochem 219:9–20.

Lucas J, Henschen AJ. 1986. Identification and assay of phosphoserine and tyrosine-O-sulphate in fibrinopeptides by reversed-phase high-performance liquid chromatography. Chromatography 369:357–364.

Ma Y, Lu Y, Zeng H, Ron D, Mo W, Neubert TA. 2001. Characterization of phosphopeptides from protein digests using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nanoelectrospray quadrupole time-of-flight mass spectrometry. Rapid Commun Mass Spectrom 15:1693–1700.

Madison DL, Yaciuk P, Kwok RP, Lundblad JR. 2002. Acetylation of the adenovirus-transforming protein E1A determines nuclear localization by disrupting association with importin-alpha. J Biol Chem 277:38755–38763.

Magnaghi-Jaulin L, Groisman R, Naguibneva I, Robin P, Lorain S, Le Villain JP, Troalen F, Trouche D, Harel-Bellan A. 1998. Retinoblastoma protein represses transcription by recruiting a histone deacetylase. Nature 391:601–605.

Mai A, Massa S, Rotili D, Cerbara I, Valente S, Pezzi R, Simeoni S, Ragno R. 2005. Histone deacetylation in epigenetics: an attractive target for anticancer therapy. Med Res Rev 25:261–309.

Mann M, Meng CK, Fenn JB. 1989. Interpreting mass spectra of multiply charged ions. Anal Chem 61:1702–1708.

Mann M, Jensen ON. 2003. Proteomic analysis of post-translational modifications. Nat Biotechnol 21:255–261.

Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S. 2002. The protein kinase complement of the human genome. Science 298:1912–1934.

Marcantonio M, Trost M, Courcelles M, Desjardins M, Thibault P. 2008. Combined enzymatic and data mining approaches for comprehensive phosphoproteome analyses: application to cell signaling events of interferon-gamma-stimulated macrophages. Mol Cell Proteomics 7:645–660.

Marks P, Rifkind RA, Richon VM, Breslow R, Miller T, Kelly WK. 2001. Histone deacetylases and cancer: causes and therapies. Nat Rev Cancer 1:194–202.

McDonald WH, Yates JR. 2003. Shotgun proteomics: integrating technologies to answer biological questions. Curr Opin Mol Ther 5:302–309.

Moser K, White FM. 2006. Phosphoproteomic analysis of rat liver by high capacity IMAC and LC-MS/MS. J Proteome Res 5:98–104.

Mu JJ, Tsay YG, Juan LJ, Fu TF, Huang WH, Chen DS, Chen PJ. 2004. The small delta antigen of hepatitis delta virus is an acetylated protein and acetylation of lysine 72 may influence its cellular localization and viral RNA synthesis. Virology 319:60–70.

Muda M, Worby CA, Simonson-Leff N, Clemens JC, Dixon JE. 2002. Use of double-stranded RNA-mediated interference to determine the substrates of protein tyrosine kinases and phosphatases. Biochem J 366:73–77.

Nesvizhskii AI, Keller A, Kolker E, Aebersold R. 2003. A statistical model for identifying proteins by tandem mass spectrometry. Anal Chem 75:4646–4658.

Nousiainen M, Sillje HHW, Sauer G, Nigg EA, Koerner R. 2006, Phosphoproteome analysis of the human mitotic spindle. Proc Natl Acad Sci USA 103:5391–5396.
Oda Y, Nagasu T, Chait BT. 2001. Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome. Nat Biotechnol 19:379–382.

Orr JA, Hamilton PW. 2007. Histone acetylation and chromatin pattern in cancer. A review. Anal Quant Cytol Histol 29:17–31.

Ouaissi M, Ouaissi A. 2006. Histone Deacetylase Enzymes as Potential Drug Targets in Cancer and Parasitic Diseases. J Biomed Biotechnol 2006:1–10.

Pasqualini JR, Mercat P, Giambiagi N. 1989. Histone acetylation decreased by estradiol in the MCF-7 human mammary cancer cell line. Breast Cancer Res Treat 14:101–105.

Pandey A, Fernandez MM, Steen H, Blagoev B, Nielsen MM, Roche S, Mann M, Lodish HF. 2000. Identification of a novel immunoreceptor tyrosine-based activation motif-containing molecule, STAM2, by mass spectrometry and its involvement in growth factor and cytokine receptor signaling pathways. J Biol Chem 275:38633–38639.

Pandey A, Podtelejnikov AV, Blagoev B, Bustelo XR, Mann M, Lodish HF. 2000. Analysis of receptor signaling pathways by mass spectrometry: identification of vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors. Proc Natl Acad Sci USA 97:179–184.

Pawson T, Scott JD. 2005. Protein phosphorylation in signaling--50 years and counting. Trends Biochem Sci 30:286–290.

Pedrioli PG, Eng JK, Hubley R, Vogelzang M, Deutsch EW, Raught B, Pratt B, Nilsson E, Angeletti RH, Apweiler R, Cheung K, Costello CE, Hermjakob H, Huang S, Julian RK, Kapp E, McComb ME, Oliver SG, Omenn G, Paton NW, Simpson R, Smith R, Taylor CF, Zhu W, Aebersold R. 2004. A common open representation of mass spectrometry data and its application to proteomics research. Nat Biotechnol 22:1459–1466.

Peters EC, Brock A, Ficarro SB. 2004. Exploring the phosphoproteome with mass spectrometry. Mini Rev Med Chem 4:313–324.

Peterson CL, Laniel MA. 2004. Histones and histone modifications. Curr Biol 14:R546–R551.

Pinkse MW, Uitto PM, Hilhorst MJ, Ooms B, Heck AJ. 2004. Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumns. Anal Chem 76:3935–3943.

Posewitz MC, Tempst P. 1999. Immobilized gallium(III) affinity chromatography of phosphopeptides. Anal Chem 71:2883–2892.

Rajarhia VB, Priestley ND, Strohl WR. 1995. Efficient synthesis of radiolabeled propionyl-coenzyme A and acetyl-coenzyme A. Anal Biochem 224:159–162.

Raska CS, Parker CE, Dominski Z, Marzluff WF, Glish GL, Pope RM, Borchers CH. 2002. Direct MALDI-MS/MS of phosphopeptides affinity-bound to immobilized metal ion affinity chromatography beads. Anal Chem 74:3429–3433.

Rinalducci S, Larsen MR, Mohammed S, Zolla L. 2006. Novel protein phosphorylation site identification in spinach stroma membranes by titanium dioxide microcolumns and tandem mass spectrometry. J Proteome Res 5:973–982.

Robinson DR, Wu YM, Lin SF. 2000. The protein tyrosine kinase family of the human genome. Oncogene 19:5548–5557.

Rodriguez P, Pelletier J, Price GB, Zannis-Hadjopoulos M. 2000. NAP-2: histone chaperone function and phosphorylation state through the cell cycle. J Mol Biol 298:225–238.

Roth SY, Denu JM, Allis CD. 2001. Histone acetyltransferases. Annu Rev Biochem 70:81–120.

Rush J, Moritz A, Lee KA, Guo A, Goss VL, Spek EJ, Zhang H, Zha XM, Polakiewicz RD, Comb M. 2005. Immunoaffinity profiling of tyrosine phosphorylation in cancer cells. J Nat Biotechnol 3:94–101.

Santini V, Gozzini A, Ferrari G. 2007. Histone deacetylase inhibitors: molecular and biological activity as a premise to clinical application. Curr Drug Metab 8:383–393.

Schlosser A, Pipkorn R, Bossemeyer D, Lehmann WD. 2001. Analysis of protein phosphorylation by a combination of elastase digestion and neutral loss tandem mass spectrometry. Anal Chem 73:170–176.

Schlosser A, Vanselow JT, Kramer A. 2005. Mapping of phosphorylation sites by a multi-protease approach with specific phosphopeptide enrichment and NanoLC-MS/MS analysis. Anal Chem 77:5243–5250.

Scott BF, McCleland ML, Stukenberg PT, Burke DJ, Ross MM, Shabanowitz J, Hunt DF, White FM. 2002. Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nat Biotechnol 20:301–305.

Senko MW, Beu SC, McLafferty FW. 1995. Automated assignment of charge states from resolved isotopic peaks for multiply charged ions. J Am Soc Mass Spectrom 6:52–56.

Senko MW, Beu SC, McLafferty FW. 1995. Determination of monoisotopic masses and ion populations for large biomolecules from resolved isotopic distributions. J Am Soc Mass Spectrom 6:229–233.

Seo J, Lee KJ. 2004. Post-translational modifications and their biological functions: proteomic analysis and systematic approaches. J Biochem Mol Biol 37:35–44.

Steen H, Küster B, Mann M. 2001. Quadrupole time-of-flight versus triple-quadrupole mass spectrometry for the determination of phosphopeptides by precursor ion scanning. J Mass Spectrom 36:782–790.

Steen H, Küster B, Fernandez M, Pandey A, Mann M. 2001. Detection of tyrosine phosphorylated peptides by precursor ion scanning quadrupole TOF mass spectrometry in positive ion mode. Anal Chem 73:1440–1448.

Steen H, Jebanathirajah JA, Rush J, Morrice N, Kirschner MW. 2006. Phosphorylation analysis by mass spectrometry: myths, facts, and the consequences for qualitative and quantitative measurements. Mol Cell Proteomics 5:172–181.

Steiner MS, Pound CR. 2003. Phase IIA clinical trial to test the efficacy and safety of Toremifene in men with high-grade prostatic intraepithelial neoplasia. Clin Prostate Cancer 2:24–31.

Stensballe A, Anderson S, Jensen ON. 2001. Characterization of phosphoproteins from electrophoretic gels by nanoscale Fe(III) affinity chromatography with off-line mass spectrometry analysis. Proteomics 1:207–222.

Sterner DE, Berger SL. 2000. Acetylation of histones and transcription-related factors. Microbiol Mol Biol Rev 64:435–459.

Stevens SM, Chung AY, Chow MC, McClung SH, Strachan CN, Harmon AC, Denslow ND, Prokai L. 2005. Enhancement of phosphoprotein analysis using a fluorescent affinity tag and mass spectrometry.Rapid Commun Mass Spectrom 19:2157–2162.

Smith CM, Gafken PR, Zhang Z, Gottschling DE, Smith JB, Smith DL. 2003. Mass spectrometric quantification of acetylation at specific lysines within the amino-terminal tail of histone H4. Anal Biochem 316:23–33.

Smith CM. 2005. Quantification of acetylation at proximal lysine residues using isotopic labeling and tandem mass spectrometry. Methods 36:395–403.

Sobel RE, Cook RG, Allis CD. 1994. Non-random acetylation of histone H4 by a cytoplasmic histone acetyltransferase as determined by novel methodology. J Biol Chem 269:18576–18582.

Su X, Zhang L, Lucas DM, Davis ME, Knapp AR, Green-Church KB, Marcucci G, Parthun MR, Byrd JC, Freitas MA. 2007. Histone H4 acetylation dynamics determined by stable isotope labeling with amino acids in cell culture and mass spectrometry. Anal Biochem 363:22–34.

Takenaga K. 1996. Suppression of metastatic potential of high-metastatic Lewis lung carcinoma cells by vanadate, an inhibitor of tyrosine phosphatase, through inhibiting cell-substrate adhesion. Invasion Metastasis 16:97–106.

Torres MP, Thapar R, Marzluff WF, Borchers CH. 2005. Phosphatase–directed phosphorylation-site determination: a synthesis of methods for the detection and identification of phosphopeptides. J Proteome Res 4:1628–1635.

Tourriere H, Gallouzi IE, Chebli K, Capony JP, Mouaikel J, van der Geer P, Tazi J. 2001. RasGAP-associated endoribonuclease G3Bp: selective RNA degradation and phosphorylation-dependent localization. Mol Cell Biol 21:7747–7760.

Tourriere H, Chebli K, Zekri L, Courselaud B, Blanchard JM, Bertrand E, Tazi J. 2003. The RasGAP-associated endoribonuclease G3BP assembles stress granules. J Cell Biol 160:823–831.

Townsend R, Lipniunas P, Tulk B, Verkman A. 1996. Identification of protein kinase A phosphorylation sites on NBD1 and R domains of CFTR using electrospray mass spectrometry with selective phosphate ion monitoring. Protein Sci 5:1865–1873.

Tsay YG, Wang YH, Chiu CM, Shen BJ, Lee SC. 2000. A strategy for identification and quantitation of phosphopeptides by liquid chromatography/tandem mass spectrometry. Anal Biochem 287:55–64.

Vaziri H, Dessain SK, Ng Eaton E, Imai SI, Frye RA, Pandita TK, Guarente L, Weinberg RA. 2001. hSIR2(SIRT1) functions as an NAD-dependent p53 deacetylase. Cell 107:149–159.

Vidali G, Gershey EL, Allfrey VG. 1968. Chemical studies of histone acetylation. The distribution of epsilon-N-acetyllysine in calf thymus histones. J Biol Chem 243:6361–6366.

Villén J, Beausoleil SA, Gerber SA, Gygi SP. 2007. Large-scale phosphorylation analysis of mouse liver. Proc Natl Acad Sci USA 104:1488–1493.

Wang YH, Tsay YG, Tan BC, Lo WY, Lee SC. 2003. Identification and characterization of a novel p300-mediated p53 acetylation site, lysine 305. J Biol Chem 278:25568–25576.

Weatherly DB, Atwood JA 3rd, Minning TA, Cavola C, Tarleton RL, Orlando RA. 2005. Heuristic method for assigning a false-discovery rate for protein identifications from Mascot database search results. Mol Cell Proteomics 4:762–772.

White DA, Belyaev ND, Turner BM. 1999. Preparation of site specific antibodies to acetylated histones. Methods 19:417–424.

Wilkins MR, Gasteiger E, Gooley AA, Herbert BR, Molloy MP, Binz PA, Ou K, Sanchez JC, Bairoch A, Williams KL, Hochstrasser DF. 1999. High-throughput mass spectrometric discovery of protein post-translational modifications. J Mol Biol 289:645–657.

Wilm M, Neubauer G, Mann M. 1996. Parent ion scans of unseparated peptide mixtures. Anal Chem 68:527–533.

Wolffe AP, Guschin D. 2000. Review: chromatin structural features and targets that regulate transcription. J Struct Biol 129:102–122.

Wolschin F, Wienkoop S, Weckwerth W. 2005. Enrichment of phosphorylated proteins and peptides from complex mixtures using metal oxide/hydroxide affinity chromatography (MOAC). Proteomics 5:4389–4397.

Wu HY, Tseng VS, Liao PC. 2007. Mining phosphopeptide signals in liquid chromatography-mass spectrometry data for protein phosphorylation analysis. J Proteome Res 6:1812–1821.

Wu J, Shakey Q, Liu W, Schuller A, Follettie MT. 2007. Global profiling of phosphopeptides by titania affinity enrichment. J Proteome Res 6:4684–4689.

Xu CF, Lu Y, Ma J, Mohammadi M, Neubert TA. 2005. Identification of phosphopeptides by MALDI Q-TOF MS in positive and negative ion modes after methyl esterification. Mol Cell Proteomics 4:809–818.

Yan JX, Packer NH, Gooley AA, Williams KL. 1998. Protein phosphorylation: technologies for the identification of phosphoamino acids. J Chromatogr A 808:23–41.

Yang XJ, Ogryzko VV, Nishikawa J, Howard BH, Nakatani Y. 1996. A p300/CBP-associated factor that competes with the adenoviral oncoprotein E1A. Nature 382:319–324.

Yang XJ. 2004. Lysine acetylation and the bromodomain: a new partnership for signaling. Bioessays 26:1076–1087.

Yang F, Stenoien LD, Strittmatter EF, Wang J, Ding L, Lipton MS, Monroe ME, Nicora CD, Gristenko MA, Tang K, Fang R, Adkins JN, Camp IIDG, Chen DJ, Smith RD. 2006. Phosphoproteome profiling of human skin fibroblast cells in response to low- and high-dose irradiation. J Proteome Res 5:1252–1260.

Zhang ZQ, Guan SH, Marshall AG. 1997. Enhancement of the Effective Resolution of Mass Spectra of High-Mass Biomolecules by Maximum Entropy-Based Deconvolution to Eliminate the Isotope Natural Abundance Distribution. J Am Soc Mass Spectrom 8:659–670.

Zhang X, Herring CJ, Romano PR, Szczepanowska J, Brzeska H, Hinnebusch AG, Qin J. 1998. Identification of phosphorylation sites in proteins separated by polyacrylamide gel electrophoresis. Anal Chem 70:2050–2059.

Zhang ZQ, Marshall AG. 1998. A universal algorithm for fast and automated charge state deconvolution of electrospray mass-to-charge ratio spectra. J Am Soc Mass Spectrom 9:225–233.

Zhang Q, Yao H, Vo N, Goodman RH. 2000. Acetylation of adenovirus E1A regulates binding of the transcriptional corepressor CtBP. Natl Acad Sci USA 97:14323–14328.

Zhang K, Tang H, Huang L, Blankenship JW, Jones PR, Xiang F, Yau PM, Burlingame AL. 2002. Identification of acetylation and methylation sites of histone H3 from chicken erythrocytes by high-accuracy matrix-assisted laser desorption ionization-time-of-flight, matrix-assisted laser desorption ionization-postsource decay, and nanoelectrospray ionization tandem mass spectrometry. Anal Biochem 306:259–269.

Zhang K, Williams KE, Huang L, Yau P, Siino JS, Bradbury EM, Jones PR, Minch MJ, Burlingame AL. 2002. Histone acetylation and deacetylation: identification of acetylation and methylation sites of HeLa histone H4 by mass spectrometry. Mol Cell Proteomics 1:500–508.

Zhang N, Li XJ, Ye M, Pan S, Schwikowski B, Aebersold R. 2005. ProbIDtree: an automated software program capable of identifying multiple peptides from a single collision-induced dissociation spectrum collected by a tandem mass spectrometer. Proteomics 5:4096–4106.

Zhou M, Halanski MA, Radonovich MF, Kashanchi F, Peng J, Price DH, Brady JN. 2000. Tat modifies the activity of CDK9 to phosphorylate serine 5 of the RNA polymerase II carboxyl-terminal domain during human immunodeficiency virus type 1 transcription. Mol Cell Biol 20:5077–5086.

Zhou W, Merrick BA, Khaledi MG, Tomer KB. 2000. Detection and sequencing of phosphopeptides affinity bound to immobilized metal ion beads by matrix-assisted laser desorption/ionization mass spectrometry. J Am Soc Mass Spectrom 11:273–282.

Zhou H, Watts JD, Aebersold R. 2001. A systematic approach to the analysis of protein phosphorylation. Nat Biotechnol 19:375–378.