分離工作利用垂直式等電點聚焦電泳，將其受質蛋白質依照等電點做分離後，再以SDS –PAGE做第二維分離，並將其萃取與水解後以質譜做鑑定分析。 In Saccharomyces cerevisiae, proteins are subject to post-translation modification, including methylation, ubiquitination, phosphorylation, glycosylation. This research was performed to explore ubiquitination and methylation, respectively: I： The core transcription factor TFIIH of Saccharomyces cerevisiae is essential for both transcription and nucleotide excision repair (NER). Core TFIIH purified from yeast possesses an E3 ubiquitin ligase activity, which resides in a RING finger domain of the SSL1 subunit and is involved in the base excision repair (BER) DNA repair pathway. The ubiquitin conjugating enzyme (UBC) that is specifically linked to the SSL1 ubiquitination pathway remained to unknown. The SSL1C403A mutant strain was first constructed by site-directed mutagenesis and plasmid shuffle. The haploid deletion strains of individual UBC gene △UBCs, were subject to MMS response test. For the heterozygous diploid deletion strains, tetrad dissection was carried out to obtain the haploid deletion strains prior to the MMS test. Thus, we isolated a UBC deletion strain which exhibits similar MMS response with the SSL1C403A mutant strain. II： The ORF of YJR129C of S. cerevisiae has a close match with the methyltransferase on database and has proved to be active with protein substrates. Using the purified recombinant YJR129Cp fused with His-tag, the protein extract from △YJR129C strain was methylated with the cosubstrate [C3H3]S-adenosyl-L- methionine prior to separation. The labeled proteins were first separated by isoelectric focusing (IEF) on a vertical slab gel. The labeled spot was further separated on SDS-PAGE. Finally the labeled spot was extracted from SDS-PAGE and digested for identification by mass spectrometry.
