Sample preparation and digestion for proteomic analyses using spin filters
Sample preparation and digestion for proteomic analyses using spin filters
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DOI：
10.1002/pmic.200401063
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发表时间：
2005-05-01
期刊：
影响因子：
3.4
通讯作者：
Liebler, DC
中科院分区：
文献类型：
--
作者：
Manza, LL;Stamer, SL;Liebler, DC
我们描述了使用市售的微量离心设备（旋转过滤器）来净化和消化蛋白质样品以进行质谱分析。将蛋白质样品添加到具有 >= 3000 分子量截止膜的旋转过滤器的上室，然后在重悬于碳酸氢铵中之前进行洗涤。然后蛋白质在上室中被还原、烷基化和胰蛋白酶消化，并通过膜离心回收肽。该方法提供的消化效率与标准溶液内消化相当，避免了冗长的透析步骤，并允许快速净化含有盐、一些去污剂以及酸性或碱性缓冲液的样品。
We describe the use of commercially available microcentrifugation devices (spin filters) for cleanup and digestion of protein samples for mass spectrometry analyses. The protein sample is added to the upper chamber of a spin filter with a >= 3000 molecular weight cutoff membrane and then washed prior to resuspension in ammonium bicarbonate. The protein is then reduced, alkylated, and digested with trypsin in the upper chamber and the peptides are recovered by centrifugation through the membrane. The method provides digestion efficiencies comparable to standard in-solution digests, avoids lengthy dialysis steps, and allows rapid cleanup of samples containing salts, some detergents, and acidic or basic buffers.