New ammunition for the proteomic reactor: strong anion exchange beads and multiple enzymes enhance protein identification and sequence coverage |
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Authors: | Hu Zhou Weimin Hou Jean-Philippe Lambert Daniel Figeys |
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Institution: | (1) Ottawa Institute of Systems Biology, University of Ottawa, Health Sciences Campus, 451 Smyth Road, Ottawa, ON, K1H 8M5, Canada;(2) Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, ON, K1H 8M5, Canada;(3) Department of Chemistry, Faculty of Science, University of Ottawa, 10 Marie Curie, D’Iorio Hall, Ottawa, ON, K1N 6N5, Canada; |
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Abstract: | The enrichment and processing of proteomic samples prior to multi-dimensional chromatography remain a challenge in ‘gel-free’
proteomics. We previously reported the development of a microfluidic device called the “proteomic reactor” that relied on
enriching proteins by using strong cation exchange (SCX) followed by trypsin digestion in an interstitial volume as little as 50 nL. Here, we report a novel proteomic reactor
that is based on polymeric strong anion exchange (SAX) material to analyse proteomic samples. We also compare the performance of the SAX proteomic reactor to our previously
reported SCX proteomic reactor for analysing complex yeast proteomes. Our results indicate that the SAX protein reactor preferentially
identifies more acidic peptides and proteins compared to the SCX reactor. We show that the SAX and SCX reactors are complementary
and that their combination increases the number of unique peptides and proteins identified by 50%. Furthermore, we show that
the number of protein identified can be increased further by up to 40% using different proteolytic enzymes on the proteomic
reactor. |
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