TY - JOUR
T1 - Analysis of tyrosine phosphorylation sites in signaling molecules by a phosphotyrosine-specific immonium ion scanning method.
AU - Steen, Hanno
AU - Pandey, Akhilesh
AU - Andersen, Jens S.
AU - Mann, Matthias
PY - 2002/10/15
Y1 - 2002/10/15
N2 - 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.
AB - 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.
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U2 - 10.1126/scisignal.1542002pl16
DO - 10.1126/scisignal.1542002pl16
M3 - Article
C2 - 12381836
AN - SCOPUS:0037107685
SN - 1937-9145
VL - 2002
SP - PL16
JO - Science's STKE : signal transduction knowledge environment
JF - Science's STKE : signal transduction knowledge environment
IS - 154
ER -