Li G, Waltham M, Anderson N L, Unsworth E, Treston A, Weinstein J N
Laboratory of Molecular Pharmacology, National Cancer Institute (NCI), NIH, Bethesda, MD 20892-4255, USA.
Electrophoresis. 1997 Mar-Apr;18(3-4):391-402. doi: 10.1002/elps.1150180313.
We report a rapid method for identifying proteins resolved by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) using matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). In-gel digestion was performed in a way such that the volume ratio of trypsin solution to gel plug was quantitatively controlled to promote reproducible digestion and to maximize the digestion yield. To make the digestion samples more compatible with MALDI-MS, the volatile salt ammonium bicarbonate in the digestion buffer was largely removed prior to peptide extraction. Samples of mixed tryptic peptides from in-gel digestion were used without purification to obtain molecular weights by MALDI-MS with alpha-cyano, 4-hydroxy-cinnamic acid as the matrix. Modifications of MALDI sample loading procedures improved the detection sensitivity by one half to one order of magnitude. The peptide mass peaks in MALDI-MS spectra were distinguished from those of impurities by using several types of controls, and masses were corrected by using trypsin autodigestion fragments as internal calibration standards. Two different peptide-matching computer programs were used to interrogate sequence databases and identify proteins. Identification was enhanced by generation of orthogonal data sets (by using different proteases) and by including experimental values of isoelectric point (pI) and molecular weight to exclude false entries in the candidate lists. Approximately 1% of the material from a spot was used in each sample loading, and nine protein spots from rat liver 2-D PAGE gels were identified correctly, as judged by comparison with identification results previously obtained from Edman sequencing. A previously identified low-abundance spot was not identified by MALDI-MS, presumably because there was insufficient material in a single gel. The sample handling procedure reported here should permit us to identify many 2-D PAGE protein spots of medium abundance.
我们报道了一种利用基质辅助激光解吸电离质谱（MALDI-MS）快速鉴定二维聚丙烯酰胺凝胶电泳（2-D PAGE）分离出的蛋白质的方法。凝胶内消化以定量控制胰蛋白酶溶液与凝胶块体积比的方式进行，以促进可重复的消化并使消化产率最大化。为使消化样品更适合MALDI-MS，在肽提取之前，很大程度上去除了消化缓冲液中的挥发性盐碳酸氢铵。凝胶内消化得到的混合胰蛋白酶肽样品未经纯化，以α-氰基-4-羟基肉桂酸为基质，通过MALDI-MS获得分子量。MALDI样品加载程序的改进将检测灵敏度提高了一半到一个数量级。通过使用几种类型的对照，将MALDI-MS谱图中的肽质量峰与杂质峰区分开来，并以胰蛋白酶自消化片段作为内标对质量进行校正。使用两种不同的肽匹配计算机程序查询序列数据库并鉴定蛋白质。通过生成正交数据集（使用不同的蛋白酶）以及纳入等电点（pI）和分子量的实验值以排除候选列表中的错误条目，增强了鉴定效果。每次样品加载使用约1%的斑点材料，通过与先前从埃德曼测序获得的鉴定结果比较判断，大鼠肝脏2-D PAGE凝胶中的9个蛋白质斑点被正确鉴定。一个先前鉴定的低丰度斑点未被MALDI-MS鉴定出来，可能是因为单个凝胶中的材料不足。本文报道的样品处理程序应能使我们鉴定出许多中等丰度的2-D PAGE蛋白质斑点。
