Cell cycle of tumor cells are often dysregulated, and its regulation differs in each tumor cell line. Thus, a single cell line analysis cannot determine whether the changes induced by cell cycle progression are general events occurred in various tumor cell lines or cell-type-specific events observed in a particular cell type. To identify general M-phase specific phosphoproteins, we compared phosphorylation status and changes upon nocodazole treatment in 3 distinct tumor cell lines, HeLa, HCT-116, and NCI-H460, all of which are arrested at M-phase by nocodazole treatment. Phosphopeptides from 3 distinct tumor cell lines with or without nocodazole treatment were enriched by MassPrepTM (Waters) and analyzed by LC-MS-based protein identification technology and data analysis (Fig.1). In total, 1,570 unique phosphopeptides assigning to 726 phosphoproteins were identified with false discovery rate at 0.8 % in average. Although there are some common phosphoproteins in all 3 tumor cell lines, the identified phosphopeptides varied among cell lines (Fig.2). Distinct phosphopeptides include representative M-phase proteins, such as kinesin family members, microtubule-associated proteins, and nuclear mitotic apparatus protein 1, as well as some signaling molecules, such as Src, raf, Erk, and PDK1. These results suggest that distinct signaling cascades are activated or inactivated in 3 tumor cell lines. In spite of the variability between cell lines and LC-MS analyses, phosphorylation of nucleophosmin S254 and phosphoribosylaminoimidazole carboxylase S27 were identified in all the independent LC-MS analyses of 3 tumor cell lines only when treated with nocodazole. Thus, our results suggest that phosphorylation of these 2 proteins serve as M-phase specific biomarkers, which may be used to monitor the efficacy of M-phase inhibitors treated as anti-cancer drugs.
