Hagiwara Tatsuo, Saito Yumi, Nakamura Yukiko, Tomonaga Takeshi, Murakami Yasufumi, Kondo Tadashi
Division of Pharmacoproteomics, National Cancer Center Research Institute, Chuo-ku, Tokyo 104-0045, Japan.
Int J Proteomics. 2011;2011:739615. doi: 10.1155/2011/739615. Epub 2011 Sep 8.
The intact plasma proteome is of great interest in biomarker studies because intact proteins reflect posttranslational protein processing such as phosphorylation that may correspond to disease status. We examined the utility of a solid-phase hexapeptide ligand library in combination with conventional plasma proteomics modalities for comprehensive profiling of intact plasma proteins. Plasma proteins were sequentially fractionated using depletion columns for albumin and immunoglobulin, and separated using an anion-exchange column. Proteins in each fraction were treated with a solid-phase hexapeptide ligand library and compared to those without treatment. Two-dimensional difference gel electrophoresis demonstrated an increased number of protein spots in the treated samples. Mass spectrometric studies of these protein spots with unique intensity in the treated samples resulted in the identification of high- and medium-abundance proteins. Our results demonstrated the possible utility of a solid-phase hexapeptide ligand library to reveal greater number of intact plasma proteins. The characteristics of proteins with unique affinity to the library remain to be clarified by more extensive mass spectrometric protein identification, and optimized protocols should be established for large-scale plasma biomarker studies.
完整血浆蛋白质组在生物标志物研究中备受关注，因为完整蛋白质反映了翻译后蛋白质加工过程，如磷酸化，而这可能与疾病状态相关。我们研究了固相六肽配体文库与传统血浆蛋白质组学方法相结合用于完整血浆蛋白质全面分析的效用。使用去除白蛋白和免疫球蛋白的耗尽柱对血浆蛋白质进行顺序分级分离，并使用阴离子交换柱进行分离。对每个级分中的蛋白质用固相六肽配体文库进行处理，并与未处理的蛋白质进行比较。二维差异凝胶电泳显示处理后样品中的蛋白质斑点数量增加。对处理后样品中具有独特强度的这些蛋白质斑点进行质谱研究，从而鉴定出高丰度和中等丰度蛋白质。我们的结果证明了固相六肽配体文库在揭示更多完整血浆蛋白质方面的潜在效用。对文库具有独特亲和力的蛋白质的特性仍有待通过更广泛的质谱蛋白质鉴定来阐明，并且应为大规模血浆生物标志物研究建立优化方案。
