Sensitivity and mass accuracy for proteins analyzed directly from polyacrylamide gels: Implications for proteome mapping
Sensitivity and mass accuracy for proteins analyzed directly from polyacrylamide gels: Implications for proteome mapping
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DOI：
10.1002/elps.1150180312
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发表时间：
1997-03-01
期刊：
影响因子：
2.9
通讯作者：
Andrews, PC
中科院分区：
文献类型：
--
作者：
Loo, RRO;Mitchell, C;Andrews, PC
基质辅助激光解吸电离（MALDI）质谱已获得直接从薄层等电聚焦（IEF）凝胶与低至700毫微微摩尔的α-和β-链牛血红蛋白和牛碳酸酐酶，和2皮摩尔的牛胰蛋白酶原，大豆胰蛋白酶抑制剂，和牛血清白蛋白都加载到一个单一的车道。通过将凝胶浸泡在基质溶液中，基质沉积在整个凝胶表面上，允许MALDI向下扫描一维凝胶的完整泳道。只要基质晶体沉积精细的凝胶表面上，时滞聚焦技术能够改善一些质量精度的限制，固有的解吸从不均匀的绝缘体表面与外部校准。在1小时的过程中测量的扁豆凝集素的5 kDa的α-亚基蛋白质的11个测量值，并参考一个单一的校准产生了0.025%的标准偏差。胶体金染色被认为是兼容的解吸直接从IEF和十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶。这种直接的方法大大简化了凝胶电泳和质谱之间的接口，使该过程更易于自动化。
Matrix-assisted laser desorption ionization (MALDI) mass spectra have been obtained directly from thin-layer isoelectric focusing (IEF) gels with as little as 700 femtomoles of alpha- and beta-chain bovine hemoglobin and bovine carbonic anhydrase, and 2 picomoles of bovine trypsinogen, soybean trypsin inhibitor, and bovine serum albumin all loaded onto a single lane. By soaking the gel in a matrix solution, matrix was deposited over the entire gel surface, allowing MALDI scanning down complete lanes of the one-dimensional gel. As long as matrix crystals were deposited finely on the surface of the gel, time-lag focusing techniques were capable of ameliorating some of the mass accuracy limitations inherent in desorbing from uneven insulator surfaces with external calibration. Eleven measurements on the 5 kDa alpha-subunit proteins of lentil lectin measured over the course of 1 h and referenced to a single calibration yielded a standard deviation of 0.025%. Colloidal gold staining was found to be compatible with desorption directly from IEF and sodium dodecyl sulfate (SDS)-polyacrylamide gels. This direct approach simplifies the interface between gel electrophoresis and mass spectrometry dramatically, making the process more amenable to automation.