Magnetic Enrichment And Detection Of Circulating Tumor Cells And Tumor-associated N-phosphorylated Proteomics

Early diagnosis,intervention and treatment of cancer is the key to improve the survival rate of cancer patients.As an important marker of tumor genesis and early metastasis,CTCs detection provides a powerful means for the early diagnosis of cancer.However,the scarcity of CTCs makes the detection of CTCs challenging.In the experiment,folate functionalized magnetic nanoclusters with high magnetization were synthesized and prepared,and then one-step-based enrichment and separation method for CTCs was constructed.The magnetic nanoclusters can specifically target to the surface of folate receptor-positive tumor cells,and can realize the enrichment and separation of CTCs under the low magnetic field gradient.The enrichment and separation efficiency of CTCs can reach more than 80%,and the tumor cells still have high activity.In order to facilitate the study and analysis of magnetic captured CTCs,a DTT responsive magnetic nanoprobe was constructed in the experiment to achieve efficient capture and non-damaging release of CTCs.The release efficiency of CTCs reached more than 80%and 98.6%of the released cells were alive,which was conducive to the specificity detection,counting and activity analysis of CTCs downstream.Protein phosphorylation is one of the most common post-translational modifications in organisms.However,studies of phosphorylation in mammals have focused on O-phosphorylation(pSer,pThr,and pTyr),while the existence of Nphosphorylation(pLys,pArg,and pHis)has been neglected.The large-scale phosphorylation proteomics in the experiment confirmed that N-phosphorylation is a common form of protein phosphorylation in mammalian cells,which is widely distributed in the nucleus,cytoplasm and membrane.Molecular functional enrichment analysis showed that N-phosphorylated modified proteins were enriched in chromatin binding and transcription coregulation,and were involved in the processing of mRNA,chromatin remodeling and cell division.Phosphorylproteomics of clinical tumor samples showed abnormal N-phosphorylation in tumor tissues,suggesting that N-phosphorylation is involved in the development of tumors.The function of histidine kinase in mammalian protein was further studied.It was found that knocking down NDPK-A promoted the migration and metastasis of HepG2 cells,but had little effect on cell growth.Knockdown NDPK-B significantly affected the proliferation and clonal formation of HepG2 cells,and inhibited the migration and metastasis of cells.Further,163 potential NDPK-A and 119 NDPK-B substrate proteins were identified based on quantitative proteomics and phosphorylproteomics,respectively.NDPK-A and NDPKB regulated and modified substrate sites overlapped,accounting for 11.8%～16.5%of the number of potential substrate sites.There may be kinase redundancy in NDPK-A and NDPK-B.