Large-scale characterization of HeLa cell nuclear phosphoproteins.

Determining the site of a regulatory phosphorylation event is often essential for elucidating specific kinase-substrate relationships, providing a handle for understanding essential signaling pathways and ultimately allowing insights into numerous disease pathologies. Despite intense research efforts to elucidate mechanisms of protein phosphorylation regulation, efficient, large-scale identification and characterization of phosphorylation sites remains an unsolved problem. In this report we describe an application of existing technology for the isolation and identification of phosphorylation sites. By using a strategy based on strong cation exchange chromatography, phosphopeptides were enriched from the nuclear fraction of HeLa cell lysate. From 967 proteins, 2,002 phosphorylation sites were determined by tandem MS. This unprecedented large collection of sites permitted a detailed accounting of known and unknown kinase motifs and substrates.

[1]  R. Aebersold,et al.  Mass spectrometry in proteomics. , 2001, Chemical reviews.

[2]  Matthias Mann,et al.  A Mass Spectrometry-based Proteomic Approach for Identification of Serine/Threonine-phosphorylated Proteins by Enrichment with Phospho-specific Antibodies , 2002, Molecular & Cellular Proteomics.

[3]  A. Pandey,et al.  Detection of tyrosine phosphorylated peptides by precursor ion scanning quadrupole TOF mass spectrometry in positive ion mode. , 2001, Analytical chemistry.

[4]  W. Lehmann,et al.  Analysis of protein phosphorylation by a combination of elastase digestion and neutral loss tandem mass spectrometry. , 2001, Analytical chemistry.

[5]  Hanno Steen,et al.  Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome. , 2002, Trends in biotechnology.

[6]  E. Reddy,et al.  Signaling by dual specificity kinases , 1998, Oncogene.

[7]  M. Mann,et al.  Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. , 2003, Analytical chemistry.

[8]  P. Andrews,et al.  Cation-exchange chromatography of peptides on poly(2-sulfoethyl aspartamide)-silica. , 1988, Journal of chromatography.

[9]  B. Chait,et al.  Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome , 2001, Nature Biotechnology.

[10]  Lewis C Cantley,et al.  Hitting the Target: Emerging Technologies in the Search for Kinase Substrates , 2002, Science's STKE.

[11]  Michael B. Yaffe,et al.  Scansite 2.0: proteome-wide prediction of cell signaling interactions using short sequence motifs , 2003, Nucleic Acids Res..

[12]  R. Aebersold,et al.  A systematic approach to the analysis of protein phosphorylation , 2001, Nature Biotechnology.

[13]  M. Senko,et al.  A two-dimensional quadrupole ion trap mass spectrometer , 2002, Journal of the American Society for Mass Spectrometry.

[14]  Steven P Gygi,et al.  A proteomics approach to understanding protein ubiquitination , 2003, Nature Biotechnology.

[15]  B. Chait,et al.  Analysis of phosphorylated proteins and peptides by mass spectrometry. , 2001, Current opinion in chemical biology.

[16]  Hanno Steen,et al.  Development of human protein reference database as an initial platform for approaching systems biology in humans. , 2003, Genome research.

[17]  S. Carr,et al.  A multidimensional electrospray MS-based approach to phosphopeptide mapping. , 2001, Analytical chemistry.

[18]  B. Chait,et al.  Improved beta-elimination-based affinity purification strategy for enrichment of phosphopeptides. , 2003, Analytical chemistry.

[19]  A. Stensballe,et al.  Large-scale Analysis of in Vivo Phosphorylated Membrane Proteins by Immobilized Metal Ion Affinity Chromatography and Mass Spectrometry* , 2003, Molecular & Cellular Proteomics.

[20]  S. Carr,et al.  Mapping phosphorylation sites in proteins by mass spectrometry. , 2002, Methods in enzymology.

[21]  J. Yates,et al.  An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database , 1994, Journal of the American Society for Mass Spectrometry.

[22]  J. Yates,et al.  Large-scale analysis of the yeast proteome by multidimensional protein identification technology , 2001, Nature Biotechnology.

[23]  A. Shevchenko,et al.  Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. , 1996, Analytical chemistry.

[24]  L. Pinna,et al.  One‐thousand‐and‐one substrates of protein kinase CK2? , 2003, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[25]  J. Shabanowitz,et al.  Phosphoproteome Analysis of Capacitated Human Sperm , 2003, The Journal of Biological Chemistry.

[26]  M. Mann,et al.  Stable Isotope Labeling by Amino Acids in Cell Culture, SILAC, as a Simple and Accurate Approach to Expression Proteomics* , 2002, Molecular & Cellular Proteomics.

[27]  M. Goethals,et al.  Identification of Tyr438 as the major in vitro c‐Src phosphorylation site in human gelsolin: A mass spectrometric approach , 2008, Protein science : a publication of the Protein Society.

[28]  D. Chan,et al.  Utilization of Oriented Peptide Libraries to Identify Substrate Motifs Selected by ATM* , 2000, The Journal of Biological Chemistry.

[29]  R. Roeder,et al.  Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. , 1983, Nucleic acids research.

[30]  J. Shabanowitz,et al.  Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae , 2002, Nature Biotechnology.

[31]  S. Gygi,et al.  Proteomics: the move to mixtures. , 2001, Journal of mass spectrometry : JMS.

[32]  Joshua E. Elias,et al.  Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for large-scale protein analysis: the yeast proteome. , 2003, Journal of proteome research.

[33]  F. Cross,et al.  Accurate quantitation of protein expression and site-specific phosphorylation. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[34]  T. Soderling,et al.  A structural basis for substrate specificities of protein Ser/Thr kinases: primary sequence preference of casein kinases I and II, NIMA, phosphorylase kinase, calmodulin-dependent kinase II, CDK5, and Erk1 , 1996, Molecular and cellular biology.