She Yi-Min, Wang Gu-Qi, Loboda Alexander, Ens Werner, Standing Kenneth G, Burczynski Frank J
Department of Physics and Astronomy, Faculty of Science, University of Manitoba, Man, R3T 2N2, Winnipeg, Canada.
Anal Biochem. 2002 Nov 15;310(2):137-47. doi: 10.1016/s0003-2697(02)00321-4.
A new technique is described that enables the direct determination of the complete or partial amino acid sequence of cytosolic proteins separated by gel electrophoresis and allows for the further observation of disease- or drug-induced posttranslational modifications. The procedure uses a two-phase extraction strategy (ethyl acetate/ammonium bicarbonate) for the efficient separation of proteins/peptides from an electrophoretic matrix and subsequent sequence analysis by matrix-assisted laser desorption ionization-quadrupole time-of-flight mass spectrometry. The method was tested using hepatocyte cytosolic proteins and compared to a complementary approach using direct solvent extraction from in-gel digests. Although the latter procedure identified the proteins, it did not enable complete amino acid sequence determination. In contrast, high sequence coverage was obtained using the peptide extraction procedure, without any apparent dependence on protein size. The technique minimized the chemically inconsistent modifications generated from in-gel digestion, thus aiding mass spectrometric interpretation and valid protein sequence identification.
本文描述了一种新技术，该技术能够直接测定经凝胶电泳分离的胞质蛋白的完整或部分氨基酸序列，并可进一步观察疾病或药物诱导的翻译后修饰。该方法采用两相萃取策略（乙酸乙酯/碳酸氢铵），从电泳基质中高效分离蛋白质/肽段，随后通过基质辅助激光解吸电离-四极杆飞行时间质谱进行序列分析。使用肝细胞胞质蛋白对该方法进行了测试，并与从胶内消化物中直接进行溶剂萃取的补充方法进行了比较。尽管后一种方法能够鉴定蛋白质，但无法完成氨基酸序列测定。相比之下，使用肽段萃取方法可获得较高的序列覆盖率，且对蛋白质大小没有明显依赖性。该技术将胶内消化产生的化学不一致修饰降至最低，从而有助于质谱解析和有效的蛋白质序列鉴定。
