Tandem metal-oxide affinity chromatography for enhanced depth of phosphoproteome analysis.
Tandem metal-oxide affinity chromatography for enhanced depth of phosphoproteome analysis.
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串联金属氧化物亲和色谱可增强磷酸化蛋白质组分析的深度。
DOI：
10.1007/978-1-62703-631-3_42
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发表时间：
2014
影响因子：
--
通讯作者：
W. Weckwerth
中科院分区：
文献类型：
--
作者：
G. Beckers;W. Hoehenwarter;H. Röhrig;U. Conrath;W. Weckwerth
在真核细胞中，许多不同的细胞功能是由可逆的蛋白质磷酸化调节的。近年来，磷酸化蛋白质组学已成为研究蛋白质磷酸化的有力工具，因为它允许在一个单一的实验中无偏定位和位点特异性定量数百种蛋白质的体内磷酸化。从复杂的生物样品中鉴定磷蛋白及其磷酸化位点的常见策略是从消化的细胞裂解物中富集磷酸肽，然后进行质谱分析。然而，尽管现代质谱仪的高灵敏度，蛋白质丰度的大动态范围和蛋白质磷酸化的瞬时性质仍然是基于MS的磷酸化蛋白质组学的主要缺陷。串联金属氧化物亲和色谱（MOAC）是一种基于磷蛋白和肽的连续富集的低丰度磷蛋白的鉴定和位点特异性定量的稳健和高选择性的方法。该策略结合了在变性条件下的蛋白质提取，使用Al（OH）3-基MOAC的磷蛋白富集，富集的磷蛋白的胰蛋白酶消化，然后用基于TiO 2的MOAC的磷酸肽。因此，串联MOAC有效地靶向磷酸蛋白和磷酸肽的磷酸部分，因此，允许探测磷酸蛋白质组到前所未有的深度。
In eukaryotic cells many diverse cellular functions are regulated by reversible protein phosphorylation. In recent years, phosphoproteomics has become a powerful tool to study protein phosphorylation because it allows unbiased localization, and site-specific quantification, of in vivo phosphorylation of hundreds of proteins in a single experiment. A common strategy to identify phosphoproteins and their phosphorylation sites from complex biological samples is the enrichment of phosphopeptides from digested cellular lysates followed by mass spectrometry. However, despite the high sensitivity of modern mass spectrometers the large dynamic range of protein abundance and the transient nature of protein phosphorylation remained major pitfalls in MS-based phosphoproteomics. Tandem metal-oxide affinity chromatography (MOAC) represents a robust and highly selective approach for the identification and site-specific quantification of low abundant phosphoproteins that is based on the successive enrichment of phosphoproteins and -peptides. This strategy combines protein extraction under denaturing conditions, phosphoprotein enrichment using Al(OH)3-based MOAC, tryptic digestion of enriched phosphoproteins followed by TiO2-based MOAC of phosphopeptides. Thus, tandem MOAC effectively targets the phosphate moiety of phosphoproteins and phosphopeptides and, thus, allows probing of the phosphoproteome to unprecedented depth.