| Summary: | 博士 === 國立陽明大學 === 生化暨分子生物研究所 === 101 === Primary structures of protein bioproducts, namely amino acid sequence and posttranslational modification, can be rapidly accessed by their accurate masses. Recent development in high-resolution mass spectrometry indeed enables direct evaluation of monoisotopic masses of smaller compounds, but the monoisotopic masses of macromolecules still cannot be determined directly. In this dissertation, we first report the development of a new approach to accurate measurement of monoisotopic masses of proteins based on their relationship with the most abundant mass observed with high-resolution mass spectrometry. 6,158 Escherichia coli proteins were used to empirically calculate their monoisotopic and most abundant masses using a new informatics method. Unexpectedly, this calculation uncovers a linear correlation between these two protein masses. Coupled with this intriguing relationship, liquid chromatography-mass spectrometry was employed to measure the monoisotopic masses of two standard proteins insulin and RNase A with remarkable sub-ppm accuracy. Furthermore, we demonstrates that our approach significantly improve mass accuracy for monoisotopic mass determination in comparison with those via previous approaches. We have next applied our approach to verify the monoisotopic masses of a recombinant protein, hEGF, with sub-ppm accuracy. In addition to rapid evaluation of protein sequences, we can detect variants with delicate structural changes, like truncation of protein ends occurring during preparation of these recombinant proteins. Hence, this method shows great promise in rapid characterization of protein primary structures, particularly posttranslational modifications like proteolytic processing.
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