Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid, Plaza de Ramón y Cajal s/n, 28040 Madrid, Spain.
J Proteomics. 2010 Apr 18;73(6):1183-95. doi: 10.1016/j.jprot.2010.02.005. Epub 2010 Feb 20.
The study of Saccharomyces cerevisiae cell surface proteins was performed because of their important role in cell wall biogenesis and in the physiology of the yeast. Two different proteomic approaches were carried out. First, proteins loosely associated or S-S linked to structural wall components were released by treatment of whole intact cells with dithiothreitol, separated by 2D-PAGE and identified by mass spectrometry. Second, cell surface-exposed proteins (surfome) were digested with trypsin and DTT from whole intact cells, and analyzed by LC-MS/MS. Ninety-nine different proteins were identified: 67 with DTT treatment and 52 with DTT and trypsin digestion. These proteins were classified in different cellular processes: control of cell wall organization, cell rescue, defence, and virulence, protein fate, protein synthesis and metabolism. Most of the proteins have already been reported as present on the cell surface showing that the yeast cell surface is composed not only by typical but also by atypical cell wall proteins. "Bona fide" cell wall proteins were identified by both protocols but a higher number with the non-gel strategy. However, only 20% of the proteins identified were common to both protocols, thus, for a complete knowledge of the cell surface proteome, several strategies have to be used.
本研究旨在探索酿酒酵母细胞表面蛋白，因为它们在细胞壁生物发生和酵母生理学中具有重要作用。我们采用了两种不同的蛋白质组学方法。首先，通过用二硫苏糖醇（DTT）处理完整的细胞，释放与结构细胞壁成分松散或 S-S 连接的蛋白质，然后通过 2D-PAGE 分离并通过质谱鉴定。其次，用胰蛋白酶和 DTT 从完整的细胞中消化细胞表面暴露的蛋白质（表面组），并通过 LC-MS/MS 进行分析。鉴定出了 99 种不同的蛋白质：67 种经 DTT 处理，52 种经 DTT 和胰蛋白酶消化。这些蛋白质被分类为不同的细胞过程：细胞壁组织的控制、细胞抢救、防御和毒力、蛋白质命运、蛋白质合成和代谢。大多数蛋白质已被报道存在于细胞表面，表明酵母细胞表面不仅由典型的细胞壁蛋白组成，还由非典型的细胞壁蛋白组成。两种方案都鉴定了“真正的”细胞壁蛋白，但非凝胶策略的数量更多。然而，两种方案鉴定的蛋白质只有 20%是共同的，因此，为了全面了解细胞表面蛋白质组，必须使用多种策略。
