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Trypsin Immobilization On Hybrid Particles For Ultra Fast, Highly Efficient Proteome Digestion, Facile18O Labeling And Absolute Protein Puantification

Posted on:2013-07-13Degree:MasterType:Thesis
Country:ChinaCandidate:Z F SongFull Text:PDF
GTID:2181330371974694Subject:Biochemistry and Molecular Biology
Abstract/Summary:PDF Full Text Request
In recent years, quantitative proteomic research attracts great attention due to the urgent needs in biological and clinical research, such as the discovery and verification of key functional proteins, biomarkers and protein drug target. Currently, mass spectrometry (MS) based bottom up proteomic strategy has often be chosen for proteome quantification. In this strategy, the amount of proteins is determined by firstly quantifying the corresponding proteolytic peptides of the proteins, and then their amounts are calculated by the stoichiometry between proteins and their corresponding peptides digested with trypsin, therefore highly efficient and complete protein digestion is crucial for achieving accurate quantification results. However, the digestion efficiency and completeness obtained using conventional free protease digestion is not satisfactory for highly complex proteomic samples. In this work, we developed new types of immobilized trypsin using hairy non-crosslinked polymer chains hybrid magnetic nanoparticle(PHMN) and squamous polymer modified silica beads (SPSMB) separately as the matrix for preparing immobilized enzyme reactors aiming at ultra fast, highly efficient proteome digestion and facile18O labeling for absolution protein quantification. The hybrid particles are first synthesized in situ with the structure of hairy or squamous polymer chains from the particles surface using surface initiated atom transfer radical polymerization technique. The flexible non-crosslinked polymer chains not only provide large amount of binding sites but also work as three dimensional scaffolds to support trypsin immobilization which leads to increased loading amount and improved accessibility of the immobilized trypsin to proteins. The digestion efficiencies of the obtained SPSMB-Trypsin and PHMN-Trypsin were evaluated using both standard proteins and complex protein extracts obtained from E-coli and thermoanaerobacter tengcongensis, respectively. Highly efficient digestion was achieved in only1-2minutes. Furthermore, the SPSMB-Trypsin exhibits good stability, and therefore can be applied in discovery proteomic research in the future. And for complex proteomic samples digested by PHMN-Trypsin, obviously increased digestion efficiency and completeness was demonstrated by27.2%and40.8%increase in the number of identified unique proteins and peptides as well as remarkably reduced undigested proteins residues compared with that obtained using conventional free trypsin digestion. The successful application in absolute protein quantification of enolase from Thermoanaerobacter tengcongensis protein extracts using18O labeling and MRM strategy further demonstrated the potential of this hybrid nanoparticle immobilized trypsin for th absolute quantification of proteomes In the future.
Keywords/Search Tags:Atom transfer radical polymerization (ATRP), trypsin immobilization, magneticnanoparticle, silica beads, 18O labeling, quantitative proteomics, multiplereaction monitor (MRM)
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