Two-dimensional SEC/RPLC coupled to mass spectrometry for the analysis of peptides
Two-dimensional SEC/RPLC coupled to mass spectrometry for the analysis of peptides
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DOI：
10.1021/ac961156d
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发表时间：
1997-07-01
影响因子：
7.4
通讯作者：
Anderegg, RJ
中科院分区：
文献类型：
--
作者：
Opiteck, GJ;Jorgenson, JW;Anderegg, RJ
本文描述了一种二维液相色谱系统，其使用尺寸排阻液相色谱（SEC），然后使用反相液相色谱（RPLC）来分离由蛋白质的酶消化产生的肽的混合物。一种新型的LC/LC接口，使用两个RPLC柱平行，而不是存储回路，加入了两个色谱尺寸。这种新的接口设计允许使用传统的分析直径的HPLC柱，7.8毫米的SEC和4.6毫米的RPLC，使该系统的建设和维护非常容易。反相色谱法使用直径为1.5 μ m的无孔C-18改性二氧化硅颗粒，可进行快速有效的分析。在高分辨率二维色谱分离之后，电喷雾质谱仪检测肽片段。质谱仪扫描2000 m/z范围，以根据分子量识别分析物。卵清蛋白和血清白蛋白的胰蛋白酶消化的分析分别描述。
A two-dimensional liquid chromatography system is described here which uses size exclusion liquid chromatography (SEC) followed by reversed phase liquid chromatography (RPLC) to separate the mixture of peptides resulting from the enzymatic digestion of a protein. A novel LC/LC interface, using two RPLC columns in parallel rather than storage loops, joins the two chromatographic dimensions. This new interface design permits the use of conventional analytical diameter HPLC columns, 7.8 mm for SEC and 4.6 mm for RPLC, making construction and maintenance of this system very easy. The reversed phase chromatography utilizes 1.5 mu m diameter, nonporous C-18 modified silica particles, which produce fast and efficient analyses. Following the high-resolution two-dimensional chromatographic separation, an electrospray mass spectrometer detects the peptide fragments. The mass spectrometer scans a 2000 m/z range to identify the analytes from their molecular weights. The analyses of tryptic digests of ovalbumin and serum albumin are each described.