Currently used elution methods for strong cation exchange (SCX) chromatography are based on two principles: salt and pH gradient. In this paper, we report the first observation of peptide elution by acid gradient. The degree of peptide separation using C18-SCX StageTip was greatly improved by our acid and salt-based elution method compared with a salt-based elution method. This development enabled us to identify over 22 000 phosphopeptides from 2 mg of protein without labor-intensive sample preparation. Our method is simple, robust, scalable, and low-cost and can be easily implemented without any special equipment or techniques.

Phosphorylation is a major post-translational modification that regulates protein function, with phosphotyrosine (pY) modifications being implicated in oncogenesis. However, global profiling of pY statuses without treatment with a tyrosine phosphatase inhibitor such as pervanadate is still challenging due to the low occupancy of pY sites. In this study, we greatly improved the identification of pY sites by liquid chromatography-tandem mass spectrometry (LC-MS/MS) by optimization of both the pY-immunoprecipitation (pY-IP) protocol and the LC-MS/MS parameters. Our highly sensitive method reproducibly identified more than 1000 pY sites from 8 mg of protein lysate without the need for tyrosine phosphatase inhibitor treatment. Furthermore, >30% of the identified pY sites were not assigned in the PhosphositePlus database. We further applied our method to the comparison of pY status between PC3 cells with and without treatment using the epidermal growth factor receptor (EGFR) inhibitor Erlotinib. Under Erlotinib treatment, we observed not only a decrease in well-known modes of EGFR downstream signaling but also modulations of kinases that are not relevant to the EGFR cascade, such as PTK6 and MAPK13. Our newly developed method for pY proteomics has the potential to reveal unknown pY signaling modes and to identify novel kinase anticancer targets.

The nonreceptor tyrosine kinase v-Src is an oncogene first identified in Rous sarcoma virus. The oncogenic effects of v-Src have been intensively studied; however, its effects on chromosomal integrity are not fully understood. Here, using HeLa S3/v-Src cells having inducible v-Src expression, we found that v-Src causes mitotic slippage in addition to cytokinesis failure, even when the spindle assembly checkpoint is not satisfied because of the presence of microtubule-targeting agents. v-Src's effect on mitotic slippage was also observed in cells after a knockdown of C-terminal Src kinase (Csk), a protein-tyrosine kinase that inhibits Src-family kinases and was partially inhibited by PP2, an Src-family kinase inhibitor. Proteomic analysis and kinase assay revealed that v-Src phosphorylates cyclin-dependent kinase 1 (Cdk1) at Tyr-15. This phosphorylation attenuated Cdk1 kinase activity, resulting in a decrease in the phosphorylation of Cdk1 substrates. Furthermore, v-Src-induced mitotic slippage reduced the sensitivity of the cells to microtubule-targeting agents, and cells that survived the microtubule-targeting agents exhibited polyploidy. These results suggest that v-Src causes mitotic slippage by attenuating Cdk1 kinase activity via direct phosphorylation of Cdk1 at Tyr-15. On the basis of these findings, we propose a model for v-Src-induced oncogenesis, in which v-Src-promoted mitotic slippage due to Cdk1 phosphorylation generates genetic diversity via abnormal cell division of polyploid cells and also increases the tolerance of cancer cells to microtubule-targeting agents.

Abnormality in cellular phosphorylation is closely related to oncogenesis. Thus, kinase inhibitors, especially tyrosine kinase inhibitors (TKIs), have been developed as anti-cancer drugs. Genomic analyses have been used in research on TKI sensitivity, but some types of TKI resistance have been unclassifiable by genomic data. Therefore, global proteomic analysis, especially phosphotyrosine (pY) proteomic analysis, could contribute to predict TKI sensitivity and overcome TKI-resistant cancer. In this study, we conducted deep phosphoproteomic analysis to select active kinase candidates in colorectal cancer intrinsically resistant to Cetuximab. The deep phosphoproteomic data were obtained by performing immobilized metal-ion affinity chromatography-based phosphoproteomic and highly sensitive pY proteomic analyses. Comparison between sensitive (LIM1215 and DLD1) and resistant cell lines (HCT116 and HT29) revealed active kinase candidates in the latter, most of which were identified by pY proteomic analysis. Remarkably, genomic mutations were not assigned in most of these kinases. Phosphorylation-based signaling network analysis of the active kinase candidates indicated that SRC-PRKCD cascade was constitutively activated in HCT116 cells. Treatment with an SRC inhibitor significantly inhibited proliferation of HCT116 cells. In summary, our results based on deep phosphoproteomic data led us to propose novel therapeutic targets against cetuximab resistance and showed the potential for anti-cancer therapy.