Preparation Of Immobilized Metal Affinity Material For Enrichment Of Phosphopeptides

Phosphorylation is a crucial post-translational modification,which plays cardinal roles in regulating many different processes in eukaryotes organisms,including cell proliferation,development,differentiation,and communication.The abnormal phosphorylation is a key step to the development of tumor.Abnormal variations in the protein phosphorylation may lead to various types of diseases.Therefore,it brings a lot of benefits in analysis of phosphorylation of proteins.Recently,strategies involving mass spectrometry(MS)is becoming a foremost analytical technique in identification of protein phosphorylation due to its high sensitivity,high-throughput,and accuracy.Unfortunately,a number of difficulties challenge direct phosphopeptide profiling by MS,such as highly dynamic nature,low phosphorylation stoichiometry in biological samples,and reduced mass spectrometric signal.As a result,a dedicated isolation and enrichment of phosphopeptides from complex biological samples prior to MS analysis is requisite for in-depth phosphoproteome study.The detection of phosphopeptides has been improved by many enrichment approaches,but there are still requirements for developing novel methods,techniques,and affinity materials for large scale phosphoproteome analysis.In this dissertation,based on the principle of immobilized metal affinity chromatography(IMAC),novel phosphopeptide enrichment materials and approaches have been developed for phosphoprotein analysis.In Chapter 1,firstly,we summarized the background of protein phosphorylation.The physiological functions and basic research methods of protein phosphorylation were introduced.Secondly,an overview was given over the most frequently used methods in detection of phosphopeptides.Mass spectrometry is the most powerful method in study of protein phosphorylation.At last,we emphasizes on the enrichment methods of phosphopeptides and the application of novel technology in these methods.In Chapter 2,we designed and synthesized an IMAC material with Ti4+immobilized on polydopamine(PDA)-coated cotton and applied it to phosphopeptides enrichment successfully.The prepared material Cotton@PDA-Ti4+,exhibited excellent fiber strength,good stability in aqueous or nonaqueous solutions,and great biocompatibility.Besides the material can be obtained by a simple synthesis method.To shorten the extraction time,simplify the operation,in-pipet-tip SPE format was employed in this study.Our results showed that Cotton@PDA-Ti4+ adsorbent exhibited high selectivity in the enrichment of phosphopeptides from simple digestions and real biological samples such as human serum and tryptic digested non-fat milk.In Chapter 3,we employed pyridoxal 5'-phosphate(PLP),as an immobilized metal affinity chromate and for the enrichment of phosphopeptides.PLP was grafted onto several substrates such as silica(SiO2),oxidized carbon nanotube(OCNT)and silica coated magnetic nanoparticles(Fe3O4@SiO2).Then the metal ion Fe3+,Ga3+ and Ti4+were incorporated for the selective enrichment of phosphopeptides.It is indicated that Fe3O4@SiO2-PLP-Ti4+ has a superior selectivity towards phosphopeptides.Further Fe3O4@SiO2-PLP-Ti4+ was successfully employed in the sample pretreatment for profiling phosphopeptides from complex biological samples,including human serum,tryptic digested non-fat milk and rat brain proteins.