Coverage and Recovery of Upstream Protein Fractionation Methods in LC-MS/MS Workflows

• Published in: Journal of biomolecular techniques Vol. 22; no. Suppl; p. S8
• Main Author: Foster, L.J.
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Association of Biomolecular Resource Facilities 01-10-2011
• Subjects: Scientific Session Abstracts
• ISSN: 1524-0215; 1943-4731

The proteome of any cell or even any subcellular fraction remains too complex for complete analysis by one dimension of liquid chromatography-tandem mass spectrometry (LC-MS/MS). Hence, to achieve greater depth of coverage for a proteome of interest, most groups routinely subfractionate the sample prior to LC-MS/MS so that the material entering LC-MS/MS is less complex than the original sample. Protein and/or peptide fractionation methods that biochemists have used for decades, such as strong cation exchange chromatography (SCX), isoelectric focusing (IEF) and SDS-PAGE, are the most common prefractionation methods used currently. There has, as yet, been no comprehensive, controlled evaluation of the relative merits of the various methods, although some binary comparisons have been made. We will discuss the most popular methods for fractionating samples at both the protein and peptide level, demonstrating quantitatively which are the best methods for optimal recovery and proteome coverage. A novel approach for fractionating samples at the level of protein complexes will also be discussed.