Research And Application Of Ion-pair Reversed-phase × Reversed-phase Two-dimensional Liquid Chromatography In Proteomic

Proteomics is a subject that aims to study protein expression levels,post-translational modifications,and interactions between proteins and proteins and their molecules in a highthroughput and comprehensive manner.It has become an important branch in the field of analytical chemistry.With the development of sample preparation methods,mass spectrometry and separation technology,proteomics has been widely used in elucidating biological mechanisms,identifying drug targets,and discovering important related proteins in diseases.The identification of proteins and peptides by detection techniques relying on mass spectrometry largely depends on the separation efficiency before mass spectrometry.Although many multidimensional methods have been developed to improve chromatographic separation capabilities,the separation method with simple operation and higher separation needs to be developed urgently under completely acidic condition capacity.In this paper,a novel fractionation method based on low p H reversed-phase liquid chromatography using TFA as ion-pairing reagent was developed,and the method was studied as follows: first,the complex protein digestion of mouse liver was used to confirm that TFA IPRP and the low p H RP had good orthogonality.Secondly,comparing the IPRP fractionation method with the traditional high p H RP fractionation method,the results show that the IPRP × low p H RPLC 2D system is able to separate peptides at approximately 1.6times the rate of high p H RP × low p H,the number of identified peptides increased by 21%,confirming the high separation performance of the IPRP × low p H RPLC 2D system,and then by performing IPRP × low p H RPLC analysis on common sample serum in clinical analysis,from 2169 proteins and 8540 peptides were confidently identified in crude serum samples,and many disease-related proteins were found,which shows that this method has great potential in the future clinical proteomics.Finally based on "bottom-up" good results of the proteomics experiment,the method was applied to the "top-down" proteomics strategy,and the whole protein extracted from Hela cells was subjected to two fragmentation methods,HCD and UVPD,which proved that UVPD excellent performance in the overall protein identification,and compared with the previous method,the overall protein identification number has been greatly improved(35.95%).The regulatory mechanism provides a new perspective and more potential possibilities for the discovery of potential cancer biomarkers and drug targets.In this study,TFA was used as the ion-pairing reagent,and the high-throughput and high-efficiency separation of complex proteomic samples under simple and completely acidic conditions was achieved by 2D IPRP × low p H RPLC 2D system fractionation,which is very important for the realization of accurate proteomic analysis.Better applications in fields such as medicine and biomedicine have positive implications.