Larsen M R, Larsen P M, Fey S J, Roepstorff P
Department of Biochemistry Molecular Biology, University of Southern Denmark, Odense University.
Electrophoresis. 2001 Feb;22(3):566-75. doi: 10.1002/1522-2683(200102)22:3<566::AID-ELPS566>3.0.CO;2-T.
Two-dimensional gel electrophoresis, bioinformatics, and mass spectrometry are key analysis tools in proteome analysis. The further characterization of post-translational modifications in gel-separated proteins relies fully on data obtained by mass spectrometric analysis. In this study, stress-induced changes in protein expression in Saccharomyces serevisiae were investigated. A total of eleven spots on a silver-stained two-dimensional (2-D) gel were identified by matrix-assisted laser desorption/ionization (MALDI) peptide mass mapping to represent C and/or N-terminal processed forms of enolase 2. The processing sites were determined by MALDI peptide mass mapping using a variety of proteolytic enzymes, by optimizing the sample preparation procedure and by specific labeling of all C-termini derived from in-gel digestion using a buffer containing 16O:18O (1:1). Out of eleven processed forms of enolase 2, six were fully characterized and the approximate processing sites identified for the remaining five.
二维凝胶电泳、生物信息学和质谱分析是蛋白质组分析中的关键分析工具。凝胶分离蛋白质中翻译后修饰的进一步表征完全依赖于质谱分析获得的数据。在本研究中，对酿酒酵母中应激诱导的蛋白质表达变化进行了研究。通过基质辅助激光解吸/电离（MALDI）肽质量图谱鉴定了银染二维（2-D）凝胶上总共11个斑点，这些斑点代表烯醇化酶2的C端和/或N端加工形式。通过使用多种蛋白水解酶进行MALDI肽质量图谱分析、优化样品制备程序以及使用含有16O:18O（1:1）的缓冲液对凝胶内消化产生的所有C端进行特异性标记来确定加工位点。在烯醇化酶2的11种加工形式中，6种已得到充分表征，其余5种的大致加工位点也已确定。
