Steen Hanno, Fernandez Minerva, Ghaffari Saghi, Pandey Akhilesh, Mann Matthias
Center for Experimental Bioinformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense M, Denmark.
Mol Cell Proteomics. 2003 Mar;2(3):138-45. doi: 10.1074/mcp.M300001-MCP200. Epub 2003 Feb 25.
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.
Bcr/Abl是一种融合癌蛋白，在慢性粒细胞白血病和急性淋巴细胞白血病中至关重要。通过用抗磷酸酪氨酸抗体免疫沉淀和SDS-PAGE分离出Bcr/Abl的p185形式的酪氨酸磷酸化部分。凝胶分离蛋白的胰蛋白酶消化产物在POROS R2/OLIGO R3微柱上进行预分级，并在四极杆飞行时间串联质谱仪上以正离子模式利用磷酸酪氨酸的亚胺离子（也称为磷酸酪氨酸特异性亚胺离子扫描）进行磷酸酪氨酸图谱分析。总共在12个不同的前体离子中明确定位了9个不同的磷酸化酪氨酸残基。这些磷酸化位点对应于三个先前描述的磷酸酪氨酸残基和六个新的酪氨酸磷酸化位点，并且它们中的大多数未被磷酸化基序预测程序ProSite、NetPhos或ScanSite预测到。这项研究表明，当样品量有限时，磷酸酪氨酸特异性亚胺离子扫描在灵敏的磷酸酪氨酸图谱分析方面具有强大作用。
