碩士 / 國立陽明大學 / 生化暨分子生物研究所 / 95 / Post-translational modifications (PTMs) are covalent groups that change the properties of a protein by proteolytic cleavage of the backbone or by alterations of R groups of a polypeptide. In combination with amino acid sequence, post-translational modifications help to define the primary structures of proteins. PTM of a protein can regulate activity state, localization, turnover, and interactions with other proteins. The various post-translational modifications of proteins may participate in many of the critical signaling pathway during neoplastic transformation.
In this report, we document how native proteins were prepared from human liver tissue and then subjected to native gel filtration chromatography. The collected GFC fractions were analyzed in a SDS polyacrylamide gel. The entire gel was subjected to in-gel tryptic digestion and subsequent liquid chromatography-tandem mass spectrometry. These data were analyzed using TurboSequest program, which, in total, revealed 1300+ different proteins in our samples.
Among these GFC fractions, the proteins in 6700kDa fraction were further analyzed using our comprehensive PTM mapping procedure to identify the PTMs present in these ~6700kDa protein complexes. Among ~510 proteins, only a few proteins had adequate protein coverages and these were suitable for screening of their modified peptides. We have identified 31 modifications in 23 proteins. Almost all of them (29 out of 31) were not previously reported in the literature. These protein modifications include phosphorylation, hydroxylation, methyaltion, glycosylation, acetylation and so on. These results indicate that our procedure is indeed capable of identifying diffent types of modifications in our experiment.
However, we also recognized several critical issues for the current approach, for example, low protein coverage percentages and poor acquisition rates for modified peptides. Based on these problems, we need to address critical issues about how to prepare sample with higher molar amounts but simpler compostions of the protein samples.
| Identifer | oai:union.ndltd.org:TW/095YM005107041 |
| Date | January 2007 |
| Creators | Yi-Shuan Pan, 潘奕璿 |
| Contributors | Yeou-Guang Tsay, 蔡有光 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 188 |
Page generated in 0.0085 seconds