A novel chromatographic method allows online reanalysis of the proteome
MCP Papers in Press. Published on April 29, 2008 as Manuscript M800141-MCP200
Waanders LF, Almeida R, Prosser S, Cox J, Eikel D, Allen MH, Schultz GA, Mann M
Liquid chromatography combined with electrospray ionization is widely used for direct analysis of polar and labile molecules by mass spectrometry (LCMS). The online coupling in LCMS is a major strength but also causes a principal limitation - that each eluting analyte has to be analyzed immediately and is not available for detailed interrogation after the LCMS run. Here we develop a new chromatographic strategy, which removes this limitation. After column separation the flow is split, one portion is analyzed directly and the other is diverted to a capture capillary. After the direct LCMS run, the flow is switched and the portion stored in the capillary is analyzed ('replay run'). We describe a set up consisting of an analytical column, a splitting valve and a focusing column, which performs at full sensitivity and undiminished chromatographic resolution. We demonstrate three principal advantages of this system; nearly continuous MS utilization, duplicate analysis without requirement for additional sample and targeting of important but under-sampled features in the replay run.
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