Characterization of SDS-PAGE-separated proteins by matrix-assisted laser desorption ionization mass spectrometry
Characterization of SDS-PAGE-separated proteins by matrix-assisted laser desorption ionization mass spectrometry
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DOI：
10.1021/ac950685z
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发表时间：
1996-03-15
影响因子：
7.4
通讯作者：
Lubman, DM
中科院分区：
文献类型：
--
作者：
Liang, XL;Bai, J;Lubman, DM
描述了一种使用 MALDI MS 表征 SDS-PAGE 分离蛋白质的新策略。在 SDS-PAGE 分离后，将蛋白质电印迹到硝酸纤维素上，并用可逆丽春红 S 染料染色，通过将膜溶解在用丙酮制备的基质溶液中，可以轻松回收蛋白质。所得混合物适合直接 MALDI MS 分析，这提供了一种快速、准确的方法来测量 SDS-PAGE 分离的蛋白质和硝酸纤维素膜上蛋白质 CNBr 消化产生的肽的分子量。与传统的洗脱方法相比，该程序可以更有效地检测蛋白质和肽，特别是来自膜的较高分子量的蛋白质。使用此方法可以检测凝胶上加载的低至 3.5 pmol 的溶菌酶和 15 pmol 的牛白蛋白。检测灵敏度高于或相当于传统考马斯亮蓝染色程序。
A new strategy to characterize SDS-PAGE-separated proteins with MALDI MS is described. The proteins, electroblotted onto nitrocellulose after SDS-PAGE separation and stained with reversible Ponceau S dye, are readily recovered by dissolving the membrane in matrix solutions prepared with acetone. The resulting mixtures are amenable to direct MALDI MS analysis, which provides a rapid and accurate means of measuring the molecular weights of SDS-PAGE-separated proteins and of peptides that result from CNBr digestion of proteins on the nitrocellulose membrane. Compared with the traditional elution method, this procedure provides more efficient detection of proteins and peptides, especially the higher molecular weight proteins from the membrane. As little as 3.5 pmol of lysozyme and 15 pmol of bovine albumin loaded onto a gel can be detected using this method. The detection sensitivity is higher than or comparable to that of the traditional Coomassie Brilliant Blue staining procedure.