Ottawa Institute of Systems Biology, University of Ottawa, Health Sciences Campus, 451 Smyth Road, Ottawa, ON K1H 8M5, Canada.
Anal Bioanal Chem. 2010 Aug;397(8):3421-30. doi: 10.1007/s00216-010-3791-8. Epub 2010 Jun 3.
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.
在“无胶”蛋白质组学中，蛋白质组样品在多维色谱之前的富集和处理仍然是一个挑战。我们之前报道了一种微流控装置的开发，称为“蛋白质组学反应器”，它依赖于通过使用强阳离子交换（SCX）来富集蛋白质，然后在 50 nL 小的间隙体积中进行胰蛋白酶消化。在这里，我们报告了一种基于聚合物强阴离子交换（SAX）材料的新型蛋白质组学反应器，用于分析蛋白质组样品。我们还将 SAX 蛋白质组学反应器的性能与我们之前报道的用于分析复杂酵母蛋白质组的 SCX 蛋白质组学反应器进行了比较。我们的结果表明，与 SCX 反应器相比，SAX 蛋白质组学反应器优先鉴定出更多的酸性肽和蛋白质。我们表明，SAX 和 SCX 反应器是互补的，它们的组合将鉴定的独特肽和蛋白质的数量增加了 50%。此外，我们表明，通过在蛋白质组学反应器上使用不同的蛋白酶，可将鉴定的蛋白质数量进一步增加多达 40%。
