A Novel Method For Relative Quantification And Absolute Quantitation Of Proteins By Using Metal Element Chelated Tags Mass Spectrometry

With the development of proteomic studies, the era of quantitation of proteins ona large scale has come. However, the current quantitation method is not perfectenough on large scale or high accuracy, which remain to be the biggest challenges inquantitation proteomics reseach. Metal labeling coupled with mass spectrometry hasdrawn great attention as it has higher labeling efficiency, higher accuracy and lowercost.In this thesis, based on metal element labeling, we have developed a novelquantitation method in the identification of N-terminal peptides using metal elementlabeling coupled with mass spectrometry and an absolute quantitation method using18O/metal labeling coupled with mass spectrometry. The method of metal elementlabeling has broadened its application in proteomic research.This thesis consists of three chapters. In chapter1, the current trend ofquantitation proteomics and the application of stable isotope labeling and metalelement labeling in quantitation proteomics is reviewed. Also, identification andrelative quantitation of N-terminal peptides based on mass spectrometry areintroduced, and the latest progresses in isobaric labeling coupled with massspectrometry for the absolute quantitation of proteins are summarized. The researchcontents is placed at the last part of chapter1in this thesis.In chapter2, we have developed a novel method for identification and relativequantification of N-terminal peptides using metal element chelated tags coupled withmass spectrometry. By means of the reaction between a DOTA-NHS-esterbifunctional reagent and N-terminal peptides of proteins, and then chelation of lanthanide metal ions as tags, we established a novel method for the identification ofN-terminal peptides of proteins and their relative quantification usingmetal-element-chelated tags coupled with mass spectrometry. The experimentalresults indicate that metal elements are able to completely label N-terminal peptides atprotein level. The N-terminal peptides are enriched as the peptides digested withtrypsin are selectively eliminated by isothiocyanate-coupled silica beads. Wesuccessfully identified the N-terminal peptides of158proteins ofThermoanaerobacter tengcongensis incubated at55C and75C, among whichN-terminal peptides of24proteins are partially acetylated. Moreover, metal-elementtags with high molecule weights make convenient identification of N-terminalpeptides consisting of less than6amino acids, which make up55percent of theidentified proteins. Finally, we developed a general approach for the relativequantification of proteins based on N-terminal peptides. We adopted lysozyme andribonuclease B as model proteins; the correlation coefficients (R2) of the standardcurves for the quantitative method were0.9994and0.9997, respectively, with eachconcentration ratio ranging from0.1to10and both relative standard derivations(RSD) measured at less than5%. In T. tengcongensis at two incubation temperatures,80proteins possess quantitative information. In addition, compared with the proteinsof T. tengcongensis incubated at55C, in T. tengcongensis incubated at75C,7proteins upregulate whereas16proteins downregulate, and most differential proteinsare related to protein synthesis.In chapter3, we have developed a novel absolute quantitation method of18O/metal isobaric labeling coupled with mass spectrometry. Double labeling forisobaric tags is achieved by labeling the metal elements at N-terminal and the18O atC-terminal. Double labeling coupled with multiple reaction monitoring (MRM) massspectrometry is used in absolute quantitation of proteins for the first time. The resultsshow that18O+Ho and Tm labeling on peptides are both highly efficient and stable in7days. Both labeled peptides have the same retention time, which is the basis formass spectrometry analysis. The18O+Ho and Tm labeled peptides are mixed in same amount before mass spectrometry analysis,, and when performing mass spectrometryanalysis the ratios of the peak areas of b-ion and y-ion pairs are1:1with4Da shiftbetween ion pairs, which indicates a high accuracy in the absolute quantitation ofproteins. With the addition of tryptic digested Thermoanaerobacter tengcongensis asthe matrix, the linear correlation coefficients of b-ions and y-ions are both0.999inliner range of two orders of amplitude, with the relative standard derivation less than10%and the limit of quantitation0.7fmol/μL. These results show that the approach isadequate for the absolute quantitation of target proteins in a real biological sample.