Developemnt Of New Enrichment And Analytical Method For Post-translational Modification Proteins And Peptides Depend On New Materials

Post-translational modifications （PTMs） of proteins have been viewed as the thirdimportant life information storage following nucleic acids and proteinsand are involvedin the control ofalmost all life processes, such as proliferation, growth, differentiation,cellular signaling pathways. Among the common PTMs of proteins, phosphorylationand glycosylation are the most widely distributed and affected almost all biologicalprocess. As one of the core technologies in post genomics era, bio-mass spectrometry iswidely applied in the PTM research.However, the low stoichiometric level, strongbackground interference and tedious sample processing procedure makes it particularlydifficult for the identification of protein phosphorylation and glycosylation by massspectrometry. Therefore, the primary task of phosphorylation and glycosylation researchis to selectively and efficiently isolatethe phosphopeptides/glycopeptides from complexproteolytic peptide mixture. In this study, we developed a few highly efficient methodsfor phosphopeptides and glycans enrichment and in-situ imaging technique for proteinglycosylation using novel functional materials.In the first chapter, we developed a new type of capillary column using thesurface-initiatedatomic transfer free radical polymerization （SI-ATRP） and successfullyapplied it in on linelarge scale phosphopeptides enrichment and identification.There is afew key features of the newly developed capillary. First, three-dimensional wavelikepolymer structures are grown on the inner wall of capillary columnsfor the firsttime, results in largely increased surface area. Second, the three-dimensionalwavelikepolymerstructureon the inner wallof capillary columns carries a large numberof densely packed enrichment functional groups, and thus, increased enrichmentefficiency of phosphopeptides is achieved. Finally, compared with conventional opentubular capillarycolumn, the loading capacity of SI-ATRP column is increased by nearlyone order of magnitude.The SI-ATRP modified capillarycolumn was successful appliedin the online phosphopeptides enrichment and identification of HepG2cell lysates andresulted in obviously increased number of identifiedphosphopeptides compared withreported on line method using TiO₂packing column. This work was publishedinAnalytical Chemistry2010,82,9461-9468. To further increase the number ofphosphorylation identification, we also developed a new off-line phosphopeptidesenrichment strategy by combining TiO₂packed columns enrichment and high pHreversed-phase chromatography separations. The new strategy was successfully appliedin mouse liver phosphorylation identification.Unique phosphopeptides （11291） andphosphoprotein （2610） were identified which are12%higher than that reported in StevenP. Gygi et al. PNAS.2007.104（5）:1488-93using SCX combined IMAC technology. Thenew strategy has two advantages. First, the large loading capacity ofTiO₂packedcolumnssignificantlyincrease the initial sample loading amount.Second, the high pHreversed-phase separation technique was first applied to the separation of thephosphopeptides mixture after enrichment. The high orthogonality of this2D LCseparation efficiently reduce samplecomplexity and increase the phosphorylationidentification in complex samples. The establishment of these new enrichment methodswill provide powerful technical support for phosphoproteomic research.In the second chapter, we reported a rapid, highly efficient, and visualized approachfor glycans enrichment using1-pyrenebutyryl chloride functionalized free grapheneoxide （PCGO）. Combined with bio-mass spectrometry, this new method, wassuccessfully applied in the enrichment and identification of glycans fromHepG2cellmembrane protein.Compared with conventional glycans enrichment methods, the newstrategy has exhibits the following features and advantages. First, Improved enrichmentefficiency is achieved by the large specific surface area of freePCGO and heavyfunctionalization of highly active1-pyrenebutyryl chloride. Second, reversible covalentbond between the hydroxyl groups of glycans and the acyl chloride groups on grapheneoxide （GO） increased the enrichment specificity comparing with HILIC orlectins based enrichment. Third, the multiple hydroxyl groups of glycans lead tocross-linking andself-assembly of free PCGO sheets into visible aggregation, therefore visualmonitoringof the enrichment process is achieved. Finally, largely reduced the time required forenrichment and simplification of the operation. This work was published in AnalyticalChemistry2013,852703-2709.To realize real time and in-situ investigationon therelationship between cell membrane glycans and disease progression,we prepared highlywater-soluble and lectin functionalized upconversionnanoparticles（UCNPs） usingSI-ATRP technique for cell membrane glycansimaging.SI-ATRP modification resultsin in situ growth of hydrophilic polymer on UCNPs surface and well defined core-shellstructure which renders UCNPs largely improved biocompatibility with intactluminance property. Furthermore, the numerous functional groups on the polymer brushshell provided large number of binding site and3D support for lectin immobilization.The densely packed lectins with diversified orientation\facilitate multivalent bindingbetween the immobilized lectin and target glycans and leads to improved labelingspecificity. Finally, thelectin-ATRP-UCNP is successfully applied in in vitro and in vivoimaging of glycans on hepatocellular carcinoma cells （HCC） and distinct difference inglycan profile was found betweenHCC with and normal liver cells. The establishment ofthis new imaging method will provide powerful technical support for investigation therelationship between cell surface glycans patterns and cancer development.