Analysis of tyrosine phosphorylation sites in signaling molecules by a phosphotyrosine-specific immonium ion scanning method.
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.
Steen
Hanno
H
Harvard Medical School, Department of Cell Biology, 240 Longwood Avenue, Boston, MA 02115, USA. hanno_steen@hms.harvard.edu
Pandey
Akhilesh
A
Andersen
Jens S
JS
Mann
Matthias
M
D000478:Alkylation; D000906:Antibodies / Q000378:metabolism; D001665:Binding Sites / Q000502:physiology; D002460:Cell Line; D002846:Chromatography, Affinity; D016895:Culture Media, Serum-Free; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells / Q000737:chemistry; D006801:Humans; D007097:Imines / Q000032:analysis / Q000378:metabolism*; D010084:Oxidation-Reduction; D010447:Peptide Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D016622:Silver Staining; D012967:Sodium Dodecyl Sulfate / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D014443:Tyrosine / Q000378:metabolism* / Q000502:physiology
D000906:Antibodies; D016895:Culture Media, Serum-Free; D007097:Imines; D011506:Proteins; D019000:Phosphotyrosine; D012967:Sodium Dodecyl Sulfate; D014443:Tyrosine; D010447:Peptide Hydrolases