Analysis of tyrosine phosphorylation sites in signaling molecules by a phosphotyrosine-specific immonium ion scanning method.

Hanno Steen, Akhilesh Pandey, Jens S. Andersen, Matthias Mann

Research output: Contribution to journalArticle

40 Citations (Scopus)

Abstract

Signal transduction pathways involve cascades of events, such as formation of second messengers and protein complexes that alter the activities of proteins. This can ultimately lead to changes in gene expression in response to the stimuli. Reversible phosphorylation of proteins is an important mechanism for activating or inhibiting enzymes and for the assembly of multiprotein complexes. Here, we describe a mass spectrometry-based phosphotyrosine-specific immonium ion scanning (PSI scanning) method for selective detection of tyrosine-phosphorylated peptides. Once the tyrosine-phosphorylated peptides are identified, they can be directly sequenced in the same experiment to localize the phosphorylation site. We provide protocols for isolation and preparation of samples for analysis, and detailed instructions for operation of a quadrupole time-of-flight (TOF) mass spectrometer for this method. Because of its simplicity and specificity, PSI scanning is likely to become an important tool in proteomic studies of pathways involving tyrosine phosphorylation.

Original languageEnglish (US)
JournalScience's STKE : signal transduction knowledge environment
Volume2002
Issue number154
StatePublished - Jan 1 2002
Externally publishedYes

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Phosphotyrosine
Phosphorylation
Tyrosine
Ions
Scanning
Molecules
Multiprotein Complexes
Signal transduction
Peptides
Proteins
Second Messenger Systems
Mass spectrometers
Gene expression
Proteomics
Mass spectrometry
Signal Transduction
Mass Spectrometry
Gene Expression
Enzymes
Experiments

ASJC Scopus subject areas

  • Medicine(all)

Cite this

Analysis of tyrosine phosphorylation sites in signaling molecules by a phosphotyrosine-specific immonium ion scanning method. / Steen, Hanno; Pandey, Akhilesh; Andersen, Jens S.; Mann, Matthias.

In: Science's STKE : signal transduction knowledge environment, Vol. 2002, No. 154, 01.01.2002.

Research output: Contribution to journalArticle

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