Depletion of high-abundance proteins from serum by immunoaffinity chromatography: A MALDI-FT-MS study
Depletion of high-abundance proteins from serum by immunoaffinity chromatography: A MALDI-FT-MS study
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DOI：
10.1016/j.jchromb.2006.09.038
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发表时间：
2007-02-15
影响因子：
3
通讯作者：
Bischoff, Rainer
中科院分区：
文献类型：
--
作者：
Dekker, Lennard J.;Bosman, Jan;Bischoff, Rainer
免疫去除血清中的高丰度蛋白是生物标记物发现研究中广泛使用的第一步。在目前的工作中，我们通过比较250例前列腺癌患者的血清样本来研究去脂步骤的重复性。所有样品在6周的时间段内在单个免疫亲和色谱柱上被耗尽，并自动进行峰检测和组分收集。在280 nm处耗尽的血清蛋白质峰的表面积的重复性低于7%的相对标准偏差(R.S.D.)。相关部位的收集体积为0.97mL(4.5%相对标准偏差)。然后用胰酶消化耗尽的血清部分中的蛋白质，并用MALDI-Fr-MS进行分析。白蛋白、转铁蛋白和α-1-抗胰蛋白酶的耗竭程度是通过比较从250个耗尽的随机样品中以固定时间间隔采集的11个样品耗尽前后的多肽峰的强度来确定的。作为阳性对照，我们评估了载脂蛋白A1(耗竭后剩余的最丰富的血清蛋白)的峰，显示在耗尽的样本中这些峰的强度明显增加。从这项研究中，我们得出结论，250份血清样本的耗尽是完全的，并且在6周内是可重复性的。(C)2006年，爱思唯尔出版。
Immunodepletion of high-abundance proteins from serum is a widely used initial step in biomarker discovery studies. In the present work we have investigated the reproducibility of the depletion step by comparing 250 serum samples from prostate cancer patients. All samples were depleted on a single immunoaffinity column over a time period of 6 weeks with automated peak detection and fraction collection. Reproducibility in terms of surface area of the depleted serum protein peak at 280 nm was below 7% relative standard deviation (R.S.D.) and the collected volume of the relevant fraction was 0.97 mL (4.5% R.S.D.). Proteins in the depleted serum fraction were subsequently digested with trypsin and analyzed by MALDI-Fr-MS. The degree of the depletion of albumin, transferrin and alpha-1-antitrypsin was determined by comparing the intensity of peptide peaks before and after depletion of 11 samples taken at regular time intervals from amongst the 250 depleted, randomized samples. As a positive control we evaluated peaks of apolipoprotein A1 (the most abundant serum protein remaining after depleteion) showing a clear increase in intensity of these peaks in the depleted samples. From this study we conclude that the depletion of the 250 serum samples was complete and reproducible over a period of 6 weeks. (c) 2006 Published by Elsevier B.V.