Department of Chemistry, Oklahoma State University, Stillwater, Oklahoma 74078-3071, USA.
J Proteome Res. 2009 Oct;8(10):4592-603. doi: 10.1021/pr900399q.
In this report, we describe an integrated fluidic platform composed of tandem affinity columns for the depletion of high-abundance proteins from human serum and on-line fractionation/concentration of medium- and low-abundance proteins by tandem immobilized metal-ion affinity chromatography (IMAC) columns and reversed phase (RP) column for in-depth proteomics analysis. The depletion columns were based on monolithic polymethacrylate with surface immobilized protein A, protein G', and antibodies for depleting the top 8 high-abundance proteins. The IMAC fractionation/concentration columns consisted of monolithic stationary phases with surface bound iminodiacetic acid (IDA) chelated with Zn2+, Ni2+ and Cu2+, while the RP column was packed with nonpolar polymer beads. The integrated multicolumn fluidic platform was very effective in reducing simultaneously both the dynamic range differences among the protein constituents of serum and the complexity of the proteomics samples, thus, facilitating the in-depth proteomics analysis by 2-DE followed by MALDI-TOF and LC-MS/MS. In fact, the number of detected spots was approximately 1450 using SYPRO fluorescent stain from which 384 spots were subsequently detected by Coomassie Blue. Since the investigation was simply a proof of concept, 295 proteins were readily identified in some selected spots by MALDI-TOF and LC-MS/MS.
在本报告中，我们描述了一种集成的流体制备平台，它由串联亲和柱组成，用于从人血清中去除高丰度蛋白质，并通过串联固定化金属离子亲和色谱（IMAC）柱和反相（RP）柱在线分离/浓缩中低丰度蛋白质，用于深入的蛋白质组学分析。耗尽柱基于表面固定有蛋白 A、蛋白 G'和抗体的整体型聚甲基丙烯酸酯，用于去除前 8 种高丰度蛋白质。IMAC 分级/浓缩柱由表面结合有亚氨基二乙酸（IDA）的整体固定相组成，IDA 与 Zn2+、Ni2+和 Cu2+螯合，而 RP 柱则填充有非极性聚合物珠。集成的多柱流体制备平台非常有效地同时减少血清中蛋白质成分的动态范围差异和蛋白质组学样品的复杂性，从而通过 2-DE 后进行 MALDI-TOF 和 LC-MS/MS 来促进深入的蛋白质组学分析。事实上，使用 SYPRO 荧光染料可检测到约 1450 个斑点，其中 384 个斑点随后通过考马斯亮蓝染色检测到。由于这项研究仅仅是一个概念验证，通过 MALDI-TOF 和 LC-MS/MS 可以在一些选定的斑点中轻松鉴定出 295 种蛋白质。
