Enrichment of low molecular weight serum proteins using acetonitrile precipitation for mass spectrometry based proteomic analysis
Enrichment of low molecular weight serum proteins using acetonitrile precipitation for mass spectrometry based proteomic analysis
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DOI：
10.1002/rcm.3729
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发表时间：
2008-10-01
影响因子：
2
通讯作者：
Creaser, Colin
中科院分区：
文献类型：
--
作者：
Kay, Richard;Barton, Chris;Creaser, Colin
我们开发了一种基于乙腈 (ACN) 的快速提取方法，可在质谱分析之前可重复地消耗血清中的高丰度和高分子量蛋白质。使用纳流液相色谱/串联质谱 (nano-LC/MS/MS) 多反应监测 (MRM) 方法对 57 种高至中等丰度血清蛋白进行表征，以表征胰蛋白酶消化后的 ACN 耗尽级分。在 57 种目标蛋白中，检测到 29 种，而白蛋白（血清和血浆中最丰富的蛋白质）被确定为提取物中第 20 位最丰富的蛋白质。 ACN 去除和一维纳米 LC/MS/MS 相结合，能够检测低丰度血清蛋白、胰岛素样生长因子-I (IGF-I)，其血清浓度约为 100ng/mL。对去除血清进行一维十二烷基硫酸钠/聚丙烯酰胺凝胶电泳 (SDS-PAGE) 分析显示，提取后没有与分子量超过 75 kDa 的蛋白质相对应的条带，证明了该方法去除高分子量蛋白质的效率。 ACN 提取物的总蛋白分析表明，大约 99.6% 的蛋白已从血清中去除。 ACN 去除策略为基于免疫化学的蛋白质去除技术提供了一种可行的替代方案，该技术通常用于在基于 MS 的蛋白质组分析之前从血清中去除高丰度蛋白质。版权所有 (C) 2008 约翰·威利父子有限公司
A rapid acetonitrile (ACN)-based extraction method has been developed that reproducibly depletes high abundance and high molecular weight proteins from serum prior to mass spectrometric analysis. A nanoflow liquid chromatography/tandem mass spectrometry (nano-LC/MS/MS) multiple reaction monitoring (MRM) method for 57 high to medium abundance serum proteins was used to characterise the ACN-depleted fraction after tryptic digestion. Of the 57 targeted proteins 29 were detected and albumin, the most abundant protein in serum and plasma, was identified as the 20th most abundant protein in the extract. The combination of ACN depletion and one-dimensional nano-LC/ MS/MS enabled the detection of the low abundance serum protein, insulin-like growth factor-I (IGF-I), which has a serum concentration in the region of 100ng/mL. One-dimensional sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the depleted serum showed no bands corresponding to proteins of molecular mass over 75 kDa after extraction, demonstrating the efficiency of the method for the depletion of high molecular weight proteins. Total protein analysis of the ACN extracts showed that approximately 99.6% of all protein is removed from the serum. The ACN-depletion strategy offers a viable alternative to the immunochemistry-based protein-depletion techniques commonly used for removing high abundance proteins from serum prior to MS-based proteomic analyses. Copyright (C) 2008 John Wiley & Sons, Ltd.