Research For Peptide-based Enrichment Approach And Analysis Of Specific Protein Identification And Quantification

Many active substances are present in the form of polypeptides,which are involved in various physiological regulation processes,such as hormone regulation,neural regulation,cell growth and reproduction.Peptidomics has become a hot topic of scientific research in view of its important functions.Most polypeptides are complex and low abundance and easy to lose and degrade during enrichment process,the optimization of polypeptide analysis and enrichment has become a key step.Graphene has a large specific surface area,which adsorbs object more closely,and it also can simplify handling process significantly.In this study,graphene and RP-C18 matrix were used to enrich peptides,and the two methods were evaluated by RP-HPLC and MS analysis.The results showed that graphene had the characteristic of adsorption,and we got more hydrophilic peptides than hydrophobic peptides from graphene,while RP-C18 matrix preferred to enrich hydrophobic peptides.Moreover,18proteins were identified only by the combination of graphene and RP-C18matrix.Some proteins could be identified by the two methods respectively,but the coverage rate of matching peptides from identified proteins which were enriched by the combination of two methods was increased,we found 44 such proteins in pituitary sample and 25 proteins in hippocampus sample.So the combination of graphene and RP-C18matrix not only could identify more proteins,but also increase the credibility of proteins identified.Accurate measurement of different protein expressions and the dynamic changes of post-translational modification has become one of the means to explore biological function of proteins.In this part,specific protein during liver regeneration was analyzed by using ¹⁸O stable isotope labeling combined with MRM-MS technology.Peptide sequences selected from specific protein were synthesized as standard,which were labeled by ¹⁸O stable isotope and mixed with complex sample for MRM detection.This strategy verified the existence of target protein and obtained its precise concentration,which could provide factual basis for interpretation of some pathological processes.In this study,methods of peptide enrichment and specific protein analysis were improved,providing a reference for the optimization of methods of peptidomics research and preliminary analysis of the polypeptides from pituitary and hippocampus of mouse.Besides,this method validated the different expression of specific protein in rat liver regeneration and provided thoughts for the subsequent search on different expression in biological samples under physiological and pathological conditions.