Matsui N M, Smith D M, Clauser K R, Fichmann J, Andrews L E, Sullivan C M, Burlingame A L, Epstein L B
Department of Pediatrics, University of California, San Francisco 94143, USA.
Electrophoresis. 1997 Mar-Apr;18(3-4):409-17. doi: 10.1002/elps.1150180315.
Two-dimensional (2-D) polyacrylamide gel electrophoresis combined with mass spectrometry is a powerful combination of technologies that allows high resolution separation of proteins and their rapid identification. Immobilized pH gradient (IPG) first-dimensional gels have several advantages over carrier ampholyte isoelectric focusing, including a high degree of reproducibility, good protein spot resolution, and a selection of pH range. Here we demonstrate the utility and efficacy of combining IPG 2-D gel electrophoresis with mass spectrometry to identify interferon-gamma- (IFN) and tumor necrosis factor (TNF)-regulated proteins in ME-180 cervical carcinoma cells. Three cytokine-regulated proteins have been identified, using imidazole-zinc-stained preparative IPG 2-D gels and in-gel tryptic digestion followed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry for determination of peptide masses and sequences: 1) triosephosphate isomerase, a glycolytic pathway enzyme, 2) proteasome subunit C3, which is important in protein degradation, and 3) Ran, a GTP-binding protein important in cell cycle regulation, protein import into the nucleus, and RNA export from the nucleus.
二维（2-D）聚丙烯酰胺凝胶电泳与质谱联用是一种强大的技术组合，能够实现蛋白质的高分辨率分离及其快速鉴定。固定化pH梯度（IPG）第一维凝胶相对于载体两性电解质等电聚焦具有多个优势，包括高度的可重复性、良好的蛋白质斑点分辨率以及pH范围选择。在此，我们展示了将IPG二维凝胶电泳与质谱联用，以鉴定ME-180宫颈癌细胞中γ-干扰素（IFN）和肿瘤坏死因子（TNF）调节蛋白的实用性和有效性。使用咪唑锌染色的制备性IPG二维凝胶以及胶内胰蛋白酶消化，随后通过基质辅助激光解吸电离飞行时间（MALDI-TOF）质谱法测定肽质量和序列，已鉴定出三种细胞因子调节蛋白：1）磷酸丙糖异构酶，一种糖酵解途径酶；2）蛋白酶体亚基C3，在蛋白质降解中起重要作用；3）Ran，一种在细胞周期调节、蛋白质导入细胞核以及RNA从细胞核输出中起重要作用的GTP结合蛋白。
