Mass Spectrometry-Based Investigation Of Protein Post-translational Modifications

Protein post-translational modifications play key roles in almost all biological processes of life and are closely related to the occurrence of a wide range of diseases,including cancer,diabetes and neurodegenerative diseases.There are currently more than 300 kinds of known post-translationally modified proteins occuring on different amino acids.In this PhD dissertation,I mainly investigated the methylation and phosphorylation of proteins.As two important post-translational modifications,methylation and phosphorylation share some common features of protein PTMs,such as low abundance,sub-stoichiometry,and large dynamic range.Therefore,the use of conventional biological technology cannot achieve a global large-scale study with high throughput and deep coverage.The rapid development of proteomics,especially the sensitivity,speed and automation of mass spectrometers,provides an indispensable tool for global-scale studies of protein post-translational modifications.The"shotgun"proteomics strategy is widely used in the study of protein post-translational modifications.In this dissertation,I explored three aspects by combining the mass spectrometry-based"shotgun"proteomics technology,including the qualitative and quantitative methods of methylation and the phosphorylation substrate screening.Firstly,by removing interference of glycosylation,when enrichment of methylation peptides,we combined with multiple enzymes and multidimensional separation means.DOMAIN(De-glycO-assisted MethylAtion site IdeNtification)strategy,an antibody-free methylation enrichment method with high reproducibility,low cost was developed.Furthermore,the method could be used for large-scale identification of whole proteome methylation.In addition,the DOMAIN strategy is a convenient and effective approach that can be applied in the clinical study.Secondly,combining MS-based shotgun proteomics technology with stable isotope labeling quantitative proteomics and RNA interference,we determined the differential regulation of several methylated proteins that are related to protein arginine N-methyltransferase 3(PRMT3).Furthermore,we developed a qualitative and quantitative method for the specific identification of arginine dimethylation,which called CoNeucode.In addition,we also developed relevant software for processing CoNeuocde data,which can be applied in processing other Neucode data.Thirdly,using the"shotgun"mass spectrometry based proteomics technique,the Ackl phosphokinase substrate was studied in detail.From histone proteins to non-histone proteins,and finally to global protein levels.We aim to identify molecular signatures of phosphokinase in signaling pathway and signaling cascades.In summary,the application of advanced mass spectrometry technology not only enables the global-scale study of protein post-translational modifications,but also provides new methodology for expanded applications.The novel MS tools developed in this thesis provides great potential for the discovery of new post-translational modification sites and holds significant promise for the future of biomarker discovery in clinical proteomics.