Phosphotyrosine mapping in Bcr/Abl oncoprotein using phosphotyrosine-specific immonium ion scanning

Bcr/Abl is a fusion oncoprotein that is of paramount importance in chronic myelogenous leukemia and acute lymphocytic leukemia. The tyrosine-phosphorylated fraction of the p185 form of Bcr/Abl was isolated by immunoprecipitation with an anti-phosphotyrosine antibody and SDS-PAGE. The tryptic digest of the gel-separated protein was prefractionated on POROS R2/OLIGO R3 microcolumns and subjected to phosphotyrosine mapping by precursor ion scanning in positive ion mode utilizing the immonium ion of phosphotyrosine, also called phosphotyrosine-specific immonium ion scanning, on a quadrupole time-of-flight tandem mass spectrometer. In total, nine different phosphorylated tyrosine residues were unambiguously localized in 12 different precursor ions. These phosphorylation sites correspond to three previously described phosphotyrosine residues and six novel tyrosine phosphorylation sites, and most of them were not predicted by the phosphorylation motif prediction programs ProSite, NetPhos, or ScanSite. This study shows the power of phosphotyrosine-specific immonium ion scanning for sensitive phosphotyrosine mapping when limited amounts of samples are available.