Based On Stable Isotope Labeling Derivatization Combined With Liquid Chromatography-mass Spectrometry For Polypeptide Analysis

Stable isotope labeling derivatization coupled with liquid chromatography-mass spectrometry(LC-MS)has become an important technique for the analysis of proteins and polypeptides.At present,the most widely used technique is the first-order mass spectrometry relative quantitative method based on stable isotope labeling.This article includes a brief introduction of proteomics research techniques and relative quantitative analysis methods of proteomes,and a comparison of several domestic and foreign literatures on proteomics research status.1-(5-fluoro-2,4-dinitrophenyl)-4-methylpiperazine(PPZ)and iodomethane synthesis d0-MPPZ,d4-MPPZ and d8-MPPZ.We developed a derivative reagent with more than four labels for quantitative analysis of various types of polypeptides using liquid chromatography.tandem mass spectrometry(LC-MS).The mass difference of the derivative reagent for each two adjacent light/heavy labels is 4 Da,which is derived from different 13C and D isotopes.The derivative reagent carries+1 charge,the labeled analyte can alleviate the inherent quantitative obstacle of electrospray ionization,especially for the analyte with anti-charge or low abundance.Three derivative reagents were designed and synthesized,their structures were characterize by means of NMR hydrogen/carbon spectrum and liquid chromatography-mass spectrometry.Because the derivative reagent has the advantages of high reactivity,strong ionization ability and lowering the detection limit of the substance to be measured,the quantitative analysis of polypeptide was carried out by high perfornance liquid chromatography-mass spectrometry,and the optimum derivative conditions and mass spectrometry parameters were optimized.After derivatization labeling,the physical properties of the analyte were changed,the chromatographic performance of polar substances was significantly improved,and the detection sensitivity of quantitative target substances was enhanced.Relative quantitative analysis of polypeptides by first-order mass spectrometry ensures that'isotope labeling has a good linear relationship in the dynamic range of 1:10 to 10:1.At the same time,it was found from the mass spectrometry data after derivatization reaction that the order of peaks of derivative products and the peaks of derivative'reagents could deternine whether tyrosine existed in the substance.The derivative reagent also reduces the minimum detection limit of the substance to be measured by more than two orders of magnitude.