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Analysis of low-abundance proteins using the proteomic reactor with pH fractionation |
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| Authors: | Hu Zhou Weimin Hou Jean-Philippe Lambert Ruijun Tian Daniel Figeys |
| Affiliation: | a Ottawa Institute of Systems Biology, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, Canada K1H 8M5 b Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, Canada K1H 8M5 c Department of Chemistry, Faculty of Science, University of Ottawa, 10 Marie Curie, D’Iorio Hall, Ottawa, ON, Canada K1N 6N5 |
| Abstract: | We developed a new method consisting of the proteomic reactor coupled with step pH fractionation for the analysis of low-abundance proteins from minute amount of sample. These new reactors were implemented using both SAX and SCX materials. The pH fractions from the SAX reactor provided higher peptide and protein identification than SCX reactor and conventional solution digestion. Interestingly, the physical characteristics (pI, molecular weight, missed cleavage site and grand average hydrophobicity (GRAVY) index, and number of acid and basic amino acid) of the peptides obtained from the SAX and SCX proteomic reactors are drastically different. Furthermore, nearly half of the peptides observed from the pH fractionations from the SAX reactor are of low abundance while only 22% low-abundance proteins are observed with conventional in-solution digestion following 2D LC-MS/MS analysis. |
| Keywords: | SAX, strong anion exchange SCX, strong cation exchange GRAVY, grand average hydrophobicity IEF, isoelectric focusing FFE, free flow electrophoresis MudPIT, multidimensional protein identification technology DTT, dithiothreitol IAA, iodoacetamide CAI, codon adaptation index LC-MS/MS, liquid chromatography-tandem mass spectrometry |
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