Phosphoproteome analysis of Drosophila melanogaster embryos.

Protein phosphorylation is a key regulatory event in most cellular processes and development. Mass spectrometry-based proteomics provides a framework for the large-scale identification and characterization of phosphorylation sites. Here, we used a well-established phosphopeptide enrichment and identification strategy including the combination of strong cation exchange chromatography, immobilized metal affinity chromatography, and high-accuracy mass spectrometry instrumentation to study phosphorylation in developing Drosophila embryos. In total, 13,720 different phosphorylation sites were discovered from 2702 proteins with an estimated false-discovery rate (FDR) of 0.63% at the peptide level. Because of the large size of the data set, both novel and known phosphorylation motifs were extracted using the Motif-X algorithm, including those representative of potential ordered phosphorylation events.

[1]  I. Hariharan,et al.  The Drosophila Mst Ortholog, hippo, Restricts Growth and Cell Proliferation and Promotes Apoptosis , 2003, Cell.

[2]  B. Wang,et al.  The Drosophila Ste20 family kinase dMST functions as a tumor suppressor by restricting cell proliferation and promoting apoptosis. , 2003, Genes & development.

[3]  P J Bryant,et al.  The Drosophila tumor suppressor gene warts encodes a homolog of human myotonic dystrophy kinase and is required for the control of cell shape and proliferation. , 1995, Genes & development.

[4]  Shian Wu,et al.  hippo Encodes a Ste-20 Family Protein Kinase that Restricts Cell Proliferation and Promotes Apoptosis in Conjunction with salvador and warts , 2003, Cell.

[5]  K. Harvey,et al.  The Salvador–Warts–Hippo pathway — an emerging tumour-suppressor network , 2007, Nature Reviews Cancer.

[6]  Steven P Gygi,et al.  Large-scale characterization of HeLa cell nuclear phosphoproteins. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[7]  D. Montell.  The social lives of migrating cells in Drosophila. , 2006, Current opinion in genetics & development.

[8]  S. Gygi,et al.  An iterative statistical approach to the identification of protein phosphorylation motifs from large-scale data sets , 2005, Nature Biotechnology.

[9]  Steven P Gygi,et al.  Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry , 2007, Nature Methods.

[10]  Jianbin Huang,et al.  The Hippo Signaling Pathway Coordinately Regulates Cell Proliferation and Apoptosis by Inactivating Yorkie, the Drosophila Homolog of YAP , 2005, Cell.

[11]  S. Cohen,et al.  The Hippo Pathway Regulates the bantam microRNA to Control Cell Proliferation and Apoptosis in Drosophila , 2006, Cell.

[12]  R. Cagan,et al.  Drosophila models for cancer research. , 2006, Current opinion in genetics & development.

[13]  P. Lasko.  The Drosophila melanogaster Genome: Translation Factors and RNA Binding Proteins , 2000 .

[14]  Steven P Gygi,et al.  A probability-based approach for high-throughput protein phosphorylation analysis and site localization , 2006, Nature Biotechnology.

[15]  Steven P. Gygi,et al.  Large-scale phosphorylation analysis of mouse liver , 2007, Proceedings of the National Academy of Sciences.

[16]  Jay Z. Parrish,et al.  The tumour suppressor Hippo acts with the NDR kinases in dendritic tiling and maintenance , 2006, Nature.

[17]  K. White,et al.  Regulation and execution of apoptosis during Drosophila development , 2000, Developmental dynamics : an official publication of the American Association of Anatomists.

[18]  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.

[19]  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.

[20]  G. Halder,et al.  The bantam MicroRNA Is a Target of the Hippo Tumor-Suppressor Pathway , 2006, Current Biology.

[21]  Scott A Gerber,et al.  Large-scale phosphorylation analysis of alpha-factor-arrested Saccharomyces cerevisiae. , 2007, Journal of proteome research.

[22]  T. Xu,et al.  Identifying tumor suppressors in genetic mosaics: the Drosophila lats gene encodes a putative protein kinase. , 1995, Development.

[23]  Stephen M. Mount,et al.  The genome sequence of Drosophila melanogaster. , 2000, Science.

[24]  Ryan S. Udan,et al.  Hippo promotes proliferation arrest and apoptosis in the Salvador/Warts pathway , 2003, Nature Cell Biology.

[25]  P. Roepstorff,et al.  Highly Selective Enrichment of Phosphorylated Peptides from Peptide Mixtures Using Titanium Dioxide Microcolumns* , 2005, Molecular & Cellular Proteomics.

[26]  N. Tapon,et al.  Dmp53 Activates the Hippo Pathway to Promote Cell Death in Response to DNA Damage , 2006, Current Biology.

[27]  D. Pan,et al.  Hippo signaling in organ size control. , 2007, Genes & development.

[28]  Ruedi Aebersold,et al.  PhosphoPep—a phosphoproteome resource for systems biology research in Drosophila Kc167 cells , 2007, Molecular systems biology.

[29]  Steven P Gygi,et al.  Comparative evaluation of mass spectrometry platforms used in large-scale proteomics investigations , 2005, Nature Methods.

[30]  E. Myers,et al.  Finishing a whole-genome shotgun: Release 3 of the Drosophila melanogaster euchromatic genome sequence , 2002, Genome Biology.