Chromatography Research Today is a free monthly online journal that collates and summarizes the latest research about Chromatography, including details on column chromatography, gas chromatography (gc), liquid chromatograpy, hplc.

Comprehensive two-dimensional separation in coupling of reversed-phase chromatography with capillary isoelectric focusing followed by MALDI-MS identification using on-target digestion for intact protein analysis.

Yu W, Li Y, Deng C, Zhang X

Department of Chemistry & Research Center of Proteome, Fudan University, Shanghai, PR China.

A coupling of capillary RP LC as the first dimension with CIEF as the second dimension followed by MALDI-MS identification was demonstrated. Based on 2-D separation system developed by our group (Electrophoresis 2003, 24, 3289-3295), this paper focused on incorporating tryptic digestion into the top-down proteomics methodology, retaining the benefits of the top-down method. Hydrophobic layer of packing-material C18 coated with SE-30 on the MALDI-target surface was used to permit the CIEF fractions to be easily concentrated and free of ampholytes using on-target washing. Following the removal of ampholytes, on-target tryptic digestion was performed to generate PMF for protein identification. Using the proteome analytical strategy, we could obtain not only intact protein pI value but also PMF for accurate protein identification. The feasibility of the strategy was first tested with a mixture of model proteins with different pIs and molecular masses. Proteome of rat liver tissue extracts was further analyzed using the system for verification. The results have shown that the system is effective for complex proteomic analysis.

Published 5 June 2006 in Electrophoresis, 27(11): 2100-10.
Full-text of this article is available online (may require subscription).

© 2005-2008 Chromatography Research Today. All Rights Reserved.