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J Sep Sci. 2012 Jul;35(14):1755-63. doi: 10.1002/jssc.201200054.

Fully automatable two-dimensional hydrophilic interaction liquid chromatography-reversed phase liquid chromatography with online tandem mass spectrometry for shotgun proteomics.

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  • 1Department of Chemistry, The University of Hong Kong, Hong Kong, China.

Abstract

We have developed a fully automatable two-dimensional liquid chromatography platform for shotgun proteomics analyses based on the online coupling of hydrophilic interaction liquid chromatography (HILIC) - using a nonionic type of TSKgel Amide 80 at either pH 6.8 (neutral) or 2.7 (acidic) - with conventional low-pH reversed-phase chromatography. Online coupling of the neutral-pH HILIC and reversed phase chromatography systems outperformed the acidic HILIC-reversed phase chromatography combination, resulting in 18.4% (1914 versus 1617 nonredundant proteins) and 41.6% (12,989 versus 9172 unique peptides) increases in the number of identified peptides and proteins from duplicate analyses of Rat pheochromocytoma lysates. Armed with this optimized HILIC-reversed phase liquid chromatography platform, we identified 2554 nonredundant proteins from duplicate analyses of a Saccharomyces cerevisiae lysate, with the detected protein abundances spanning from approximately 41 to 10(6) copies per cell, which contained up to approximately 2092 different validated protein species with a dynamic range of concentrations of up to approximately 10(4) . This present study establishes a fully automated platform as a promising methodology to enable online coupling of different hydrophilic HILIC and reversed phase chromatography systems, thereby expanding the repertoire of multidimensional liquid chromatography for shotgun proteomics.

© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

PMID:
22807358
[PubMed - indexed for MEDLINE]
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