Mouse liver phosphoproteome methodology optimization and kinase analysis / 军事医学

Objective To analyze the construction of mouse liver phosphoproteome and phosphorylated kinases to provide useful information for integrating mouse kinase phosphorylation regulatory networks.Methods A new method was established to identify phosphoproteome from the mouse liver.First of all, liver protein was digested with trypsin before the resulting peptides were subjected to a two-step phosphopeptide enrichment and separation procedure consisting of TiO2 chro-maphy enrichment combined with high pHHPLC separation.Samples were injected onto aNanolC-Ultra-2Dplus system cou-pled to an AB-Sciex 5600 Triple TOF mass spectrometer instrument.Then data analysis was performed to provide information of new identified phosphorylation sites of kinase.Results and Conclusion Using our efficient and high-throughput platform, we reported the identification of 5386 phosophorylation sites and 4553 phosphopeptides from 1533 proteins of the mouse liver.126 new phosphorylation sites were identified from 116 kinases, which provides valuable infor-mation for phosphorylation networks in the mouse liver.

Construction and application of cell fines screening Mycobacterium tuberculosis-specific tetramers of CD4+α/β T cell receptor / 中华微生物学和免疫学杂志

Objective To construct and apply a cell line screening Mycobacterium tuberculosis (Mtb)-specific tetramers of CD4+α/β T cell receptor(TCR). Methods The β chains of HLA class Ⅱ (DR) were amplified from tuberculosis patients by PCR. The pMT-HLA-DRB expression vectors that carries the HLA-DR 13 chain and pMT-HLA-DRA-P expression vectors which carries the genes of HLA-DR α chain loaded with Mtb antigen were transfected into S2 cells with the method of calcium phosphate transfection. The expressed Mtb peptide/HLA-DR complexes were primarily identified by the method of cell immunohistochemistry. The cell lines expressing Mtb peptide/HLA-DR complexes were used to screen tetramers of CD4+ TCR by flow cytometry. Results S2 cell lines expressing Mtb peptide/HLA-DR complexes on the cell surface were obtained, two kinds of Mtb specific tetramers of CD4+α/β TCR were screened. Conclusion S2 cell lines expressing Mtb peptide/HLA-DR complexes on the cell surface provide the solid basis of the further research on the TCR tetramers and are helpful for exploring new diagnostic study methods about tuberculosis and developing new vaccines.