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Originally published In Press as doi:10.1074/mcp.M800141-MCP200 on April 29, 2008.
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Molecular & Cellular Proteomics 7:1452-1459, 2008.
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

A Novel Chromatographic Method Allows On-line Reanalysis of the Proteome*,S

Leonie F. Waanders{ddagger},§, Reinaldo Almeida§,¶, Simon Prosser||, Jürgen Cox{ddagger}, Daniel Eikel||, Mark H. Allen, Gary A. Schultz||,** and Matthias Mann{ddagger},{ddagger}{ddagger}

From the {ddagger} Proteomics and Signal Transduction, Max Planck Institute for Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany, Advion BioSciences Limited, 26 Queens Road, Hethersett NR9 3DB, United Kingdom, and || Advion BioSystems, Ithaca, New York 14850

Liquid chromatography combined with electrospray ionization is widely used for direct analysis of polar and labile molecules by LCMS. The on-line 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 developed 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 setup 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 undersampled features in the replay run.

** To whom correspondence may be addressed. Tel.: 607-266-0665; Fax: 607-266-0749; E-mail: schultzg{at}advion.com

{ddagger}{ddagger} To whom correspondence may be addressed. Tel.: 49-89-8578-2557; Fax: 49-89-8578-3209; E-mail: mmann{at}biochem.mpg.de


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