Systems-wide analysis of a phosphatase knock-down by quantitative proteomics and phosphoproteomics

Maximiliane Hilger, Tiziana Bonaldi, Florian Gnad, Matthias Mann

Research output: Contribution to journalArticle

82 Citations (Scopus)

Abstract

Signal transduction in metazoans regulates almost all aspects of biological function, and aberrant signaling is involved in many diseases. Perturbations in phosphorylation-based signaling networks are typically studied in a hypothesis-driven approach, using phospho-specific antibodies. Here we apply quantitative, high-resolution mass spectrometry to determine the systems response to the depletion of one signaling component. Drosophila cells were metabolically labeled using stable isotope labeling by amino acids in cell culture (SILAC) and the phosphatase Ptp61F, the ortholog of mammalian PTB1B, a drug target for diabetes, was knocked down by RNAi. In total we detected more than 10,000 phosphorylation sites in the phosphoproteome of Drosophila Schneider cells and trained a phosphorylation site predictor with this data. SILAC-based quantitation after phosphatase knock-down showed that apart from the phosphatase, the proteome was minimally affected whereas 288 of 6,478 high-confidence phosphorylation sites changed significantly. Responses at the phosphotyrosine level included the already described Ptp61 F substrates Stat92E and Abi. Our analysis highlights a connection of Ptp61F to cytoskeletal regulation through GTPase regulating proteins and focal adhesion components.

Original languageEnglish
Pages (from-to)1908-1920
Number of pages13
JournalMolecular and Cellular Proteomics
Volume8
Issue number8
DOIs
Publication statusPublished - 2009

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Phosphorylation
Phosphoric Monoester Hydrolases
Proteomics
Drosophila
Phospho-Specific Antibodies
Isotope Labeling
Signal transduction
Phosphotyrosine
Focal Adhesions
GTP Phosphohydrolases
Proteome
Medical problems
RNA Interference
Cell culture
Isotopes
Labeling
Mass spectrometry
Signal Transduction
Mass Spectrometry
Adhesion

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Analytical Chemistry

Cite this

Systems-wide analysis of a phosphatase knock-down by quantitative proteomics and phosphoproteomics. / Hilger, Maximiliane; Bonaldi, Tiziana; Gnad, Florian; Mann, Matthias.

In: Molecular and Cellular Proteomics, Vol. 8, No. 8, 2009, p. 1908-1920.

Research output: Contribution to journalArticle

Hilger, Maximiliane ; Bonaldi, Tiziana ; Gnad, Florian ; Mann, Matthias. / Systems-wide analysis of a phosphatase knock-down by quantitative proteomics and phosphoproteomics. In: Molecular and Cellular Proteomics. 2009 ; Vol. 8, No. 8. pp. 1908-1920.
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