Assessment of protein phosphorylation in cells activated by anti-β₂GPI antibodies. Endothelial cells were metabolically labeled with ³²P-orthophosphate in serum and phosphate-free media prior to addition of β₂GPI and either control IgG or affinity-purified anti-β₂GPI antibodies. Total cell lysates were subjected to 2D SDS-PAGE. (A) Coomassie staining (i,iii) and autoradiography (ii,iv) of total cell lysates from cells incubated with β₂GPI and control IgG (i-ii), or β₂GPI and anti-β₂GPI antibodies (iii-iv). Arrows point to the protein spot demonstrating consistently increased phosphorylation following incubation of cells with anti-β₂GPI antibodies. (B) Mass spectrometric profile of the differentially phosphorylated protein following tryptic digestion. The ions denoted with the ♦ represent H₂O loss peaks. (C) The 5 myosin RLC peptides identified in this analysis, including the Sequest Xcorr score for each peptide. (D) Sequence of myosin RLC with the peptides positively identified in the LC-MS/MS analysis shown in red.