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Mol Cell Proteomics. 2011 Feb;10(2):M110.001479. doi: 10.1074/mcp.M110.001479. Epub 2010 Jul 12.

Pressurized pepsin digestion in proteomics: an automatable alternative to trypsin for integrated top-down bottom-up proteomics.

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  • 1Biological Science Division, Pacific Northwest National Laboratory, Richland, Washington 99354, USA. daniel.lopez-ferrer@pnl.gov

Abstract

Integrated top-down bottom-up proteomics combined with on-line digestion has great potential to improve the characterization of protein isoforms in biological systems and is amendable to high throughput proteomics experiments. Bottom-up proteomics ultimately provides the peptide sequences derived from the tandem MS analyses of peptides after the proteome has been digested. Top-down proteomics conversely entails the MS analyses of intact proteins for more effective characterization of genetic variations and/or post-translational modifications. Herein, we describe recent efforts toward efficient integration of bottom-up and top-down LC-MS-based proteomics strategies. Since most proteomics separations utilize acidic conditions, we exploited the compatibility of pepsin (where the optimal digestion conditions are at low pH) for integration into bottom-up and top-down proteomics work flows. Pressure-enhanced pepsin digestions were successfully performed and characterized with several standard proteins in either an off-line mode using a Barocycler or an on-line mode using a modified high pressure LC system referred to as a fast on-line digestion system (FOLDS). FOLDS was tested using pepsin and a whole microbial proteome, and the results were compared against traditional trypsin digestions on the same platform. Additionally, FOLDS was integrated with a RePlay configuration to demonstrate an ultrarapid integrated bottom-up top-down proteomics strategy using a standard mixture of proteins and a monkey pox virus proteome.

PMID:
20627868
[PubMed - indexed for MEDLINE]
PMCID:
PMC3033671
Free PMC Article
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