Synthesis Of Stable Isotope Labeling Reagents D0/d8-MPPZ And Optimizing The Derivatization Conditions

Since the human genome sequence determination is completed,all about protein study of proteomics and genomics become one of the most cutting-edge technology research in the 21st century.Because of the differences expression of protein in biological body can reflect the life state of the object,so the application of the research scope is very broad.But different from genomics,proteomics is a global,dynamic and complex.So its research method development very quickly.The method based on mass spectrometry of stable isotope labeling proteomic quantitative method is the most popular,providing important technical support for life science research.The method carry out by using the weight of the stable isotope labeling reagent reaction with normal and diseased samples and use mass spectrometry to the relative quantitative analysis.Nowadays we developed various chemical markers,and applied to the quantitative analysis of different protein group.However,due to the level of mass spectrum quantitative method has the defect of low abundance sample quantitative results is not accurate,low dynamic range,it is difficult to achieve more than four flux tag at the same time.This study aimed at the difficult to achieve more than four flux tag at the same time,Five flux tag reagents are designed.synthesis part of the five reagent and optimized the derivatization reaction conditions.In this paper,the first chapter,i sketched the existing protein quantitative research methods refer to the relevant studies of protein quantitative methods in recent years in all kinds of documents.In chapter two,we use 1,5-difluoro-2,4-dinitrobenzene reaction with N-methylpiperazine and N-methylpiperazine,2,3,3,5,5,6,6-d8 respectively at room temperature for 30 min,then purified the product to give 1-(2,4-Dinitro-5-fluoprophrnyl)-4-methylpiperazine(d0/d8-PPZ).And then d0/d8-PPZ reacts with methyl iodide at 60 ? for 30 min to synthesis the d0/d8-1-(2,4-Dinitro-5-fluorophenyl)-4,4-dimethylpiperazinium(d0/d8-MPPZ),and attested them.The third chapter mainly discusses the MPPZ and peptide derivatization reaction conditions.Because the derivatization reaction relate to the reaction temperature,reaction time,reaction pH,the molar ratio of reactants;so i explored the best conditions of derivatization reaction,confirming the best reaction conditions are temperature 60 ?,reaction time 120 min,reaction pH 8.2,the reaction molar ratio 40-1(d0-MPPZ/d8-MPPZ:yg-5).With the 8 different mass of the isotope reagents tag labeling same amount of yg-5,and then mixt with same quality to detection with LC-MS,and the ratio of Mass spectrum peak intensity is 0.98,witch is approximately equal to 1,proving the derivatization reagent can be applied to the relative quantitative analysis in polypeptide.The tag derivatization process is simple,the reaction condition is mild,the reaction degree is relatively completely,with less by-products,stability product,inexpensive reagents etc.Lay the foundation of researching more complex structure protein.