Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 106, Issue 4, Pages 995-1000Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0811964106
Keywords
phosphoproteomics; phosphorylation; MS/MS; PTM analysis; ion-ion chemistry
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Funding
- NHGRI NIH HHS [5T32HG002760, T32 HG002760] Funding Source: Medline
- NIGMS NIH HHS [P01 GM081629, R01 GM080148-02, R01GM080148, R01 GM080148, P01GM081629] Funding Source: Medline
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Protein phosphorylation is central to the understanding of cellular signaling, and cellular signaling is suggested to play a major role in the regulation of human embryonic stem (ES) cell pluripotency. Here, we describe the use of conventional tandem mass spectrometry-based sequencing technology-collision-activated dissociation ( CAD)-and the more recently developed method electron transfer dissociation (ETD) to characterize the human ES cell phosphoproteome. In total, these experiments resulted in the identification of 11,995 unique phosphopeptides, corresponding to 10,844 nonredundant phosphorylation sites, at a 1% false discovery rate (FDR). Among these phosphorylation sites are 5 localized to 2 pluripotency critical transcription factors-OCT4 and SOX2. From these experiments, we conclude that ETD identifies a larger number of unique phosphopeptides than CAD ( 8,087 to 3,868), more frequently localizes the phosphorylation site to a specific residue (49.8% compared with 29.6%), and sequences whole classes of phosphopeptides previously unobserved.
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