Identification of the quantitative phosphoproteome in mitotic HeLa cells with or without G6PD inhibitors.

HeLa cells were synchronized with a double thymidine block procedure. Briefly, the exponentially growing HeLa cells were maintained with 2 mM thymidine for 18 h, followed by a release of 9 h in fresh medium, and then cells were re-cultured in 2 mM thymidine for additional 15 h. After a release of 7.5 h in fresh medium, DMSO or G6PD inhibitor was added to the medium. 1 h later, the cells entered into M phase. Mitotic cells were harvested by mitotic shake-off. Then, the samples were subjected to LC-MS/MS analysis. Finally, a Kinase-Substrate Enrichment was performed, which could infer the changes of upstream kinase activity upon the treatment of G6PD inhibitors.