Analysis of protein tyrosine phosphorylation by nanoelectrospray ionization high-resolution tandem mass spectrometry and tyrosine-targeted product ion scanning.
A novel highly sensitive strategy is introduced for analysis of tyrosine phosphorylation in previously identified proteins channelling for this aim all analytical and sequence information available. Nanoelectrospray high-resolution MS/MS analysis is targeted to precalculated m/z values corresponding to phosphotyrosine-containing tryptic peptides. Identification of these peptides is supported by the occurrence of the phosphotyrosine immonium ion at m/z 216, neutral loss of 79.97/z (= loss of HPO3), and similarity of the fragmentation patterns of phosphotyrosine-containing peptides with their nonphosphorylated analogues. This tyrosine-targeted tandem mass spectrometry strategy is demonstrated for epidermal growth factor receptor showing that phosphotyrosine-containing tryptic peptides invisible in the survey spectrum can be safely identified.
Salek
Mogjiborahman
M
Central Spectroscopy Unit, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany.
Alonso
Angel
A
Pipkorn
R
R
Lehmann
Wolf D
WD
D000595:Amino Acid Sequence; D002460:Cell Line; D066246:ErbB Receptors / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry; D010750:Phosphoproteins / Q000032:analysis* / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000032:analysis / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D014357:Trypsin / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*
D010446:Peptide Fragments; D010750:Phosphoproteins; D019000:Phosphotyrosine; D014443:Tyrosine; D066246:ErbB Receptors; D014357:Trypsin