Proteomic analysis optimization: Selective protein sample on-column retention in reverse-phase liquid chromatography

作者：Winnik Witold M^*; Ortiz Pedro A

来源：Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences, 2008, 875(2): 478-486.

DOI：10.1016/j.jchromb.2008.09.029

• Proteomic profiling
• Microsomal proteins
• Cytochrome P450
• Reverse-phase liquid chromatography
• Fractionation
• Sample injection
• Flow-through
• Selectivity
• DMSO
• 1D gel electrophoresis
• SDS PAGE
• Nanospray LC-MS/MS
• Mass spectrometry

In an effort to optimize reverse-phase liquid chromatography (RPLC) for proteomics, we studied the impact of composition of the sample injection solution on protein on-column selection and retention. All the proteins studied were retained on-column when injections were made in 50% formic acid, 0.1% TFA or 8.3 M urea. When formic acid was increased to 80%, the superoxide dismutase standard (MW 26.159) and 58 mouse microsomal proteins that possessed low-range molecular weights, high pls or basic amino acid clusters were non-retained, resulting in retention selectivity during sample injection. Introducing to the 80% formic acid injection solution an organic solvent such as acetonitrile or acetonitrile-DMSO induced further retention selectivity, and increasing levels of organic solvents reduced on-column retention. The proteome was split into the proteins that were retained on-column which eluted at higher retention times (RTs), vs the proteins that collected in the injection flow-through which normally eluted at lower RTs. This protein selectivity was confirmed after fraction collection, 1D-GE and nano-LC-MS/MS. The significance of this procedure is that it can be exploited for fast extraction of small basic proteins from the bulk of the proteome and for on-column enrichment of hydrophobic proteins. Published by Elsevier B.V.

• 出版日期2008-11-15