Comparison of three methods for fractionation and enrichment of low molecular weight proteins for SELDI-TOF-MS differential analysis
Comparison of three methods for fractionation and enrichment of low molecular weight proteins for SELDI-TOF-MS differential analysis
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DOI：
10.1016/j.talanta.2010.04.029
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发表时间：
2010-06-30
期刊：
影响因子：
6.1
通讯作者：
Fillet, Marianne
中科院分区：
文献类型：
--
作者：
De Bock, Muriel;de Seny, Dominique;Fillet, Marianne
在大多数疾病中，临床对血清/血浆标志物的需求从未如此重要，不仅是为了诊断，也是为了选择最有效的治疗方法，以及排除无效或有毒的治疗方法。由于样品的高复杂性，预分离对于探索深层蛋白质组和寻找特定标记物是必不可少的。在本研究中，研究了三种不同的样品制备方法（即高丰蛋白沉淀，限制性进入材料（RAM）结合IMAC色谱和肽配体亲和珠），以选择最佳的分离步骤，进一步进行以LMW蛋白质组（MW小于40000 Da）为重点的差异蛋白质组学实验。事实上，目的不是覆盖整个血浆/血清蛋白质组，而是丰富潜在的有趣的组织渗漏蛋白。对这三种方法的重复性、分离后产生的SELDI-TOF-MS肽/蛋白峰以及所提供的信息进行了评价。所研究的方法似乎提供了互补的信息，并具有良好的再现性（低于20%）。发现肽配体亲和珠可以有效地去除HMW蛋白，并在蛋白/肽谱中达到峰值富集。(C) 2010 Elsevier B.V.版权所有
In most diseases, the clinical need for serum/plasma markers has never been so crucial, not only for diagnosis, but also for the selection of the most efficient therapies, as well as exclusion of ineffective or toxic treatment. Due to the high sample complexity, prefractionation is essential for exploring the deep proteome and finding specific markers.In this study, three different sample preparation methods (i.e., highly abundant protein precipitation, restricted access materials (RAM) combined with IMAC chromatography and peptide ligand affinity beads) were investigated in order to select the best fractionation step for further differential proteomic experiments focusing on the LMW proteome (MW inferior to 40,0000 Da). Indeed, the aim was not to cover the entire plasma/serum proteome, but to enrich potentially interesting tissue leakage proteins. These three methods were evaluated on their reproducibility, on the SELDI-TOF-MS peptide/protein peaks generated after fractionation and on the information supplied.The studied methods appeared to give complementary information and presented good reproducibility (below 20%). Peptide ligand affinity beads were found to provide efficient depletion of HMW proteins and peak enrichment in protein/peptide profiles. (C) 2010 Elsevier B.V. All rights reserved.