2 DE Coupled HPLC-CHIP/MS Analysis Of Differently Expressed Proteins In R/S-Atenolol Treated VSMC

Objective:β-receptor blocker is the a series of chiral characteristics drugs. The majority ofβ-receptor blocker, (S)-Enantiomers'activity is higher than (R)-Enantiomers, and most side effects by (R) configuration caused a clear optical selectivity. Atenolol (AT) is a selectiveβ1-blocker, clinical for the treatment of high blood pressure, angina and arrhythmia, but also for the treatment of glaucoma. Atenolol chemical is 2-[4-[2-Hydroxy-3-(isopropylamino) propoxy] phenyl] acetamide, its molecules have a chiral center, there are pairs of enantiomers (enantiomers ), and currently used in clinical preparations are on S and R enantiomers of the racemic mixture half body (racemate) the form of administration. However, these two enantiomers don't only in the type of pharmacological action, intensity and in vivo absorption, distribution, metabolism, etc. There are significant differences, the S-typeβblocking activity of the heart are 46 times more R-type. In recent years, the atenolol in the clinical treatment of many side effects has been reported, and the resultingβ-blockers on whether the first-line drugs should be removed from the lively discussion. Therefore, the study of atenolol in two optical isomers of the molecular mechanism of the differences have important theoretical and practical significance. Proteomics and medicine gradually form a new interdisciplinary field of study - Pharmaceutical proteomics. At its pre-clinical studies include: discovery of all possible drug targets, as well as against the target of all these possible compounds. Application of proteomics methods also includeed the role of study pharmaceutical mechanisms and toxicology; in clinical research include: specific protein the drugs effected as the basis for selection of effective drugs and clinical diagnostic marker, also can use the method similar with Pharmacogenetics , in accordance with the protein spectrum to classify patients, to give individual treatment, and prediction of drug efficacy. Two-dimensional electrophoresis and mass spectrometry in combination with sophisticated bioinformatics set up a set of powerful, general method for drug proteomics has provided technical support.In this article, we use multi- tandem mass spectrometry and two-dimensional electrophoresis to study changes in the protein spectrum of whole cell extracts isolated from R/S- atenolol-treated vascular smooth muscle cell. The whole cell protein was preliminary separated by two-dimensional electrophoresis .The peptide mixture peptides tryspined were enriched and fractioned and identified, using High-Performance Liquid Chromatographic Chip Mass Spectrometry (HPLC-Chip/MS). and about 5 kinds of different protein were identified. The interaction between target proteins andβ- receptor blocker-type mechanism of chiral drugs have a better understanding, thus the development of chiral drugs provide a new research strategy and experimental basis.Methods: Protein sample preparation: The VSMC is grown in DMEM containing fetal bovine serum (FBS) 10%, 37℃, 5% CO2, humidity of 90% of the environment. When the cells achieve logarithmic phase, switch the VSMC in to 96-well plates. The VSMC is grown in non-serum DMEM with the racemic atenolol 0,0.15,1.5,15,30,60,120,μmol / L for 4 hours. Culture the VSMC in medium with 5 mg / mL MTT of PBS solution for 4 hours. Aspirated all 100μL MTT of PBS solution, the DMSO solution dissolved the crystal, microplate was scanned in 590nm.Analysis of differently expressed Proteins in R/S- atenolol treated VSMC cells with 2-DE : later the VSMC is grown in DMEM with 120μmol / L of the R-atenolol and S-atenolol for 4 hours. The whole cell protein extracts of VSMC treated with R/S-atenolol were stored in -80℃.the protein sample were separated by two dimensional electrophoresis respectively. 200μg protein samples re-hang at re-expansion(7mol / L urea, 2mol / L thiourea, 4% CHAPS, 30mmol / L DTT, 0.5% IPG buffer, trace bromophenol blue),then passive hydrated overnight (12 ~ 16h). After the two-dimensional gel electrophoresis (First direction: IEF; second direction: 12% SDS-PAGE electrophoresis), under the Bio-Rad two-dimensional gel electrophoresis experiment guidelines. the results of 2-DE. Stained the gel using classical mass spectrometry-compatible silver staining method. The Staind gel scannd to PDQest7.4.0 gel image analysis software, then analysis and look for points of difference.HPLC-Chip/MS analysis : The elution conditions of enrichment column: mobile phase was 0.1% (v / v) formic acid - ultra-pure aqueous solution, flow rate: 4μL / min; gradient elution of separation column : mobile phase A for 0.1% (v / v) formic acid - ultra-pure aqueous solution, mobile phase B was 0.1% (v / v) formic acid - acetonitrile solution, the initial mobile phase was 3% (v / v) B, maintain 2min, 17min when the mobile phase B for 55% (v / v), 20 min, when B was 75% (v / v), to maintain 5min, flow rate: 0.3μL/min. CHIP cube as the ion source of Ultra XCT ion trap Mass Spectrometer, drying air flow rate 0.32 L / min, drying temperature 325℃, the quality of scanning range m / z: 300 ~ 1500 Da. pectrum Mill MS Proteomics Workbench (Rev A.03.03.078) automatic analysis of MS and MS / MS data, search in UniProtKB / SWISS-PORT, Homo Sapiens (Human) database. Database search parameters: Trypsin(Trypsin), Monoisotopic Mass values(quality of single-isotope), Peptide tol.(Parent ion mass Tolerance)±2.5 Da, MS/MS tol.(MS/MS quality-of-Tolerance)±0.7Da,Carbamidometholation(C)modification.Automatically to verify the validity of the following conditions: protein score greater than 11.0; peptide score greater than 6.0; SPI (Structure predictability index for protein sequences) is greater than 60%.Results: According to MTT experiment, we selected 120μmol / L as the experiment concentration. By optimizing conditions, eventually determine the appropriate sample volume for Samples on 200μg.After silver staining, we analyzed two piece of 2D gel and found six differences in protein spots.Digged the six differences spots for LC-CHIP-MS/MS analysis.Conclusion: In this study, we set up a new 2D and LC-CHIP- LC-CHIP-MS/MS analyze differently expressed Proteins in R/S- atenolol treated VSMC cells. Identified 6 kinds of different proteins. The 6 proteins may play an important role in the chiral selective of R-ATE and S-ATE.