Beckers Gerold J M, Hoehenwarter Wolfgang, Röhrig Horst, Conrath Uwe, Weckwerth Wolfram
Department of Molecular Systems Biology, University of Vienna, Vienna, Austria.
Methods Mol Biol. 2014;1072:621-32. doi: 10.1007/978-1-62703-631-3_42.
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.
在真核细胞中，许多不同的细胞功能受可逆性蛋白质磷酸化作用调控。近年来，磷酸化蛋白质组学已成为研究蛋白质磷酸化的强大工具，因为它能在单次实验中对数百种蛋白质的体内磷酸化进行无偏差定位和位点特异性定量分析。从复杂生物样品中鉴定磷酸化蛋白质及其磷酸化位点的常用策略是，先从消化后的细胞裂解物中富集磷酸肽，然后进行质谱分析。然而，尽管现代质谱仪灵敏度很高，但蛋白质丰度的动态范围大以及蛋白质磷酸化的瞬时性，仍然是基于质谱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法（MOAC）是一种强大且高度选择性的方法，用于鉴定和位点特异性定量低丰度磷酸化蛋白质，该方法基于对磷酸化蛋白质和磷酸肽的连续富集。此策略结合了变性条件下的蛋白质提取、基于Al(OH)3的MOAC进行磷酸化蛋白质富集、对富集的磷酸化蛋白质进行胰蛋白酶消化，随后基于TiO2的MOAC对磷酸肽进行处理。因此，串联MOAC有效地靶向磷酸化蛋白质和磷酸肽的磷酸基团，从而能够以前所未有的深度探究磷酸化蛋白质组。
