
Peter James
Professor

Critical Comparison of Multidimensional Separation Methods for Increasing Protein Expression Coverage
Author
Summary, in English
We present a comparison of two-dimensional separation methods and how they affect the degree of coverage of protein expression in complex mixtures. We investigated the relative merits of various protein and peptide separations prior to acidic reversed-phase chromatography directly coupled to an ion trap mass spectrometer. The first dimensions investigated were density gradient organelle fractionation of cell extracts, 1D SDS-PAGE protein separation followed by digestion by trypsin or GluC proteases, strong cation exchange chromatography, and off-gel isoelectric focusing of tryptic peptides. The number of fractions from each first dimension and the total data accumulation RP-HPLC-MS/MS time was kept constant and the experiments were run in triplicate. We find that the most critical parameters are the data accumulation time, which defines the level of under-sampling and the avoidance of peptides from high expression level proteins eluting over the entire gradient.
Department/s
- Department of Immunotechnology
Publishing year
2012
Language
English
Pages
2644-2652
Publication/Series
Journal of Proteome Research
Volume
11
Issue
5
Document type
Journal article
Publisher
The American Chemical Society (ACS)
Topic
- Immunology in the medical area
Keywords
- protein and peptide separation
- methods comparison
- off-gel isoelectric
- focusing
- GluC
- trypsin
- GelLC
- density gradient cell fractionation
- strong cation exchange chromatography
- reversed-phase chromatography
- proteome coverage
Status
Published
ISBN/ISSN/Other
- ISSN: 1535-3893