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Vorapaxar,a novel antagonist of the protease-activated receptor 1 (PAR-1 ),can inhibit the clotting process. Deuterium-labeled vorapaxar was required for the analysis of clinical sample as an internal stand-ard. Starting for unlabeled vorapaxar,four-step reactions including hydrolysis,condensation,transesterification and hydrogen-deuterium exchange were carried out to synthesize [D8]vorapaxar effectively for the first time. All intermediates and final products were confirmed by NMR and high resolution mass spectrometry (HRMS).Impor-tantly,the prepared [D8]vorapaxar could meet the requirements of sample analysis as the internal standard.
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AIM:To identify and analyze tyrosine-phosphorylated proteins regulated by protein tyrosine phos-phatase-like A domain containing protein 1 ( PTPLAD1) in colon cancer cells by phosphoproteomics.METHODS: The expression of PTPLAD1 in colon cancer cell line HCT-116 was knocked down by small interfering RNAs, and the differenti-al expression of tyrosine-phosphorylated proteins in response to the konckdown of PTPLAD1 in HCT-116 cells was identified by stable isotope labeling with amino acid in cell culture ( SILAC) , coupled with the tyrosine phosphorylation antibody im-munoprecipitation and LC-MS/MS analysis.The Ingenuity Pathway Analysis ( IPA) software was employed for bioinformat-ics analysis on the differentially-expressed proteins.RESULTS:A total of 20 differentially-expressed tyrosine-phosphoryla-ted proteins were identified by MS, including 8 markedly up-regulated and 10 evidently down-regulated proteins.IPA soft-ware suggested that these proteins were mainly associated with the disease of cancer, tissue development and function, and cell death and survival.CONCLUSION:We successfully identified PTPLAD1-regulated differentially-expressed tyrosine-phosphorylated proteins in colon cancer cell line HCT-116.Our analysis suggests that PTPLAD1-regulated proteins in colon cancer are closely correlated with colon cancer.
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Stable isotope labeling by amino acids in cell culture (SILAC) is an in vivo metabolic labeling technique based on the abundance ratio of mass spectrometry. Combined with different methods of proteins affinity enrichment, SILAC can obtain sensitive, accurate and quantitative information of proteins. This article reviewed the history, rationale, characteristics and application of SILAC, with special focus on its role in studying protein interactions with small molecules, quantitative proteomics and post-translational modification of proteins.
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In order to optimize the ~(18)O labeling method, two key aspects, peptide dispersion and trypsin deac tivation were discussed o The addition of Rapigest SF in H_2~('8)O and microwave heating enhanced labeling efficiency of α-casein digested peptides(~(18)O/~(16)O) ratio >99%).Chemical modification with tris(2-carboxyeth yl) phosphine (TCEP) and iodoacetamide (IAA) resulted in trypsin deactivated completely.No significant back-exchange from ~(18)O to ~(16)O was observed after labeling in 6 days.The experiment result with peptide mixture from showed that the improved method could be effectively used to label protein and peptide.