Loo J A, Brown J, Critchley G, Mitchell C, Andrews P C, Ogorzalek Loo R R
Chemistry Department, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, Ann Arbor, MI 48105, USA.
Electrophoresis. 1999 Apr-May;20(4-5):743-8. doi: 10.1002/(SICI)1522-2683(19990101)20:4/5<743::AID-ELPS743>3.0.CO;2-I.
The molecular weight measurement of intact Escherichia coli proteins separated by isoelectric focusing-immobilized pH gradient (IEF-IPG) gels and analyzed by mass spectrometry is presented. Two methods are discussed: (i) electrospray ionization (ESI) mass spectrometry (MS) of extracted proteins, and (ii) matrix-assisted laser desorption/ionization (MALDI)-MS analysis directly from IEF-IPG gels. Both ESI and MALDI methods yield sub-picomole sensitivity and good mass measurement accuracy. The use of an array detector for ESI-MS was essential to discriminate against contaminating background ions and to selectively detect high mass protein ions. MALDI-MS offers high-throughput analysis of one- and potentially two-dimensional (2-D) gels. The "virtual 2-D" gel method with first-dimensional IEF separation and the second dimension as molecular mass determination by MS, is a particularly promising method for protein analysis due to its ultra high sensitivity and correspondence to classical 2-D gels. Further sensitivity enhancements for the MALDI-MS method are provided by post acceleration detection optimized for high mass time-of-flight analysis.
本文介绍了通过等电聚焦 - 固定化pH梯度（IEF - IPG）凝胶分离并经质谱分析的完整大肠杆菌蛋白质的分子量测量方法。讨论了两种方法：（i）提取蛋白质的电喷雾电离（ESI）质谱（MS），以及（ii）直接从IEF - IPG凝胶进行基质辅助激光解吸/电离（MALDI） - MS分析。ESI和MALDI方法均具有亚皮摩尔灵敏度和良好的质量测量准确性。使用阵列检测器进行ESI - MS对于区分污染背景离子和选择性检测高质量蛋白质离子至关重要。MALDI - MS可对一维和潜在的二维（2 - D）凝胶进行高通量分析。“虚拟二维”凝胶方法，即第一维采用IEF分离，第二维通过MS测定分子量，由于其超高灵敏度以及与经典二维凝胶的对应性，是一种特别有前景的蛋白质分析方法。通过针对高质量飞行时间分析优化的后加速检测，可进一步提高MALDI - MS方法的灵敏度。
