| Quantitative proteomics is one of the most popular and challenging field in currentproteomic research. Quantitative proteomics consists of absolute quantitation andrelative quantitation, and the methods of absolute quantitation of proteins are the mainobstacles that restrict the development of quantitative proteomics. Problems such asquantitative kit with exorbitant price, need of high throughout-put of proteomics,difficulties in preparing internal standard peptides and incomplete enzymatic digestionof biological samples limit the establishment of absolute quantitation methods.To address these issues in absolute quantitation methods mentioned above, mythesis focuses on solving the problems of preparing internal standard peptides andincomplete enzymatic digestion of biological samples. A novel Trypsin-immobilizdreactor on silver wire is first prepared by atom transfer radical polymerization in orderto solve problems in traditional enzymatic digestion in solution, such as overlongdigestion time, disruption of enzyme self-digestion, the disposable character of enzymeand incomplete digestion of biological samples. Then a novel absolute quantitationmethod is developed using metal element chelated tags coupled with selected ionmonitoring mass spectrometry (SIM), which is applied to absolute quantitative analysisof proteins extracted from Thermoanaerobacter tengcongensis.This paper consists of three chapters. The current research trends in quantitativeproteomics, application of chemical label coupled with mass spectrometry in quantitative proteomics, the current research trends in metal element chelated tags andthe influence of digestion on quantitative proteomics are reviewed in the first chapter.The research contents based on the current research background are proposed at theend of the first chapter.In the second chapter, preparation of a silver as the carrier of a new type ofimmobilized enzyme reactor was discribed. we here demonstrated that trypsin could beimmobilized on silver wire modified by atom transfer radical polymerization to preparea new kind of enzyme immobilized reactor. The digestion efficiency, repeatability andrecovery of the silver wire-trypsin reactor (SW-Trypsin) were evaluated by usingstandard proteins and subsequent mass spectrometry (MS) analysis. Highly efficientdigestion was achieved by using SW-Trypsin within only20minutes. Standard proteinscould be almost completely digested with sequence coverage up to93%, which ishigher than that of79%sequence coverage obtained by in-solution digestion for16h.BSA was digested8times within a month by using the SW-Trypsin. The result ofsequence coverage was between89%to97%, with an average sequence coverage of94%, which showed that SW-Trypsin has good stability. In addition, The recovery testby using MYO showed that the recovery rate was87.67%. At last, the extract fromTengchong thermophilic bacteria was digested by SW-Trypsin in20min and in-solutiontrypsin in16h, respectively. The results of sequence coverage and the number ofidentified proteins were similar. Moreover, the ratio of the number of peptides with zeromissed cleavages to the number of all identified peptides by using SW-Trypsin is higherthan that by in-solution digestion. Also, the SW-Trypsin is easily removed fromdigestion solution.A novel method is established based on metal element chelated tags coupled with SIM. We have examined the labeling efficiency and stability of metal element chelatedtags, and also the chromatographic retention behavior, mass-spectrometry behavior,linear range and accuracy of labeled peptides. The result revealed that the metal elementchelated tags method has the advantages of high label efficiency, high stability andconsistent chromatographic retention behavior. This method has a good linearitybetween1fmol and500fmol are of (R2>0.99). Moreover, this method is of highaccuracy. All results showed that this method is feasible and reliable in quantitativeproteomic research. |
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