Improvement of in-gel digestion protocol for peptide mass fingerprinting by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
Improvement of in-gel digestion protocol for peptide mass fingerprinting by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
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DOI：
10.1002/rcm.379
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发表时间：
2001-01-01
影响因子：
2
通讯作者：
Oda, Y
中科院分区：
文献类型：
--
作者：
Katayama, H;Nagasu, T;Oda, Y
蛋白质组学研究中的高灵敏度、高通量蛋白质分析通常通过聚丙烯酰胺凝胶电泳结合质谱进行。然而，所获得的数据的质量取决于所采用的凝胶内消化程序。这项工作描述了基质辅助激光解吸/电离飞行时间质谱（MALDI-TOFMS）分析的凝胶内消化效率的提高。当正辛基葡糖苷加入到缓冲液中时，观察到胰蛋白酶肽的覆盖率的显著改善。从S.通过二维凝胶电泳分离酿酒酵母，并用银染色。采用改进的胶内酶切法和MALDI-TOFMS对蛋白质点进行鉴定。此外，将使用基质α-氰基-4-羟基肉桂酸（CHCA）获得的质谱与使用2，5-二羟基苯甲酸（DHB）获得的质谱进行了比较。DHB矩阵通常给出更多的峰，这导致更高的序列覆盖度，因此，在蛋白质鉴定中具有更高的置信度。这种改进的胶内酶切方法简单，适用于MALDI-TOFMS蛋白质鉴定。版权所有（C）2001约翰威利父子有限公司
High-sensitivity, high-throughput analysis of proteins for proteomics studies is usually performed by polyacrylamide gel electrophoresis in combination with mass spectrometry. However, the quality of the data obtained depends on the in-gel digestion procedure employed. This work describes an improvement in the in-gel digestion efficiency for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) analysis. A dramatic improvement in the coverage of tryptic peptides was observed when n-octyl glucoside was added to the buffer. Whole cell extracted proteins from S. cerevisiae were separated by two-dimensional gel electrophoresis and stained with silver. Protein spots were identified using our improved in-gel digestion method and MALDI-TOFMS. In addition, the mass spectra obtained by using the matrix alpha -cyano-4-hydroxycinnamic acid (CHCA) were compared with those obtained using 2,5-dihydroxybenzoic acid (DHB). The DHB matrix usually gave more peaks, which led to higher sequence coverage and, consequently, to higher confidence in protein identification. This improved in-gel digestion protocol is simple and useful for protein identification by MALDI-TOFMS. Copyright (C) 2001 John Wiley & Sons, Ltd.