Thingholm Tine E, Jørgensen Thomas J D, Jensen Ole N, Larsen Martin R
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense, Denmark.
Nat Protoc. 2006;1(4):1929-35. doi: 10.1038/nprot.2006.185.
The characterization of phosphorylated proteins is a challenging analytical task since many of the proteins targeted for phosphorylation are low in abundance and phosphorylation is typically substoichiometric. Highly efficient enrichment procedures are therefore required. Here we describe a protocol for selective phosphopeptide enrichment using titanium dioxide (TiO2) chromatography. The selectivity toward phosphopeptides is obtained by loading the sample in a 2,5-dihydroxybenzoic acid (DHB) or phthalic acid solution containing acetonitrile and trifluoroacetic acid (TFA) onto a TiO2 micro-column. Although phosphopeptide enrichment can be achieved by using TFA and acetonitrile alone, the selectivity is dramatically enhanced by adding DHB or phthalic acid since these compounds, in conjunction with the low pH caused by TFA, prevent binding of nonphosphorylated peptides to TiO2. Using an alkaline solution (pH > or = 10.5) both monophosphorylated and multiphosphorylated peptides are eluted from the TiO2 beads. This highly efficient method for purification of phosphopeptides is well suited for the characterization of phosphoproteins from both in vitro and in vivo studies in combination with mass spectrometry (MS). It is a very easy and fast method. The entire protocol requires less than 15 min per sample if the buffers have been prepared in advance (not including lyophilization).
磷酸化蛋白质的表征是一项具有挑战性的分析任务，因为许多被磷酸化的蛋白质丰度较低，且磷酸化通常是亚化学计量的。因此需要高效的富集方法。在此，我们描述了一种使用二氧化钛（TiO₂）色谱法选择性富集磷酸肽的方案。通过将样品加载到含有乙腈和三氟乙酸（TFA）的2,5 - 二羟基苯甲酸（DHB）或邻苯二甲酸溶液中的TiO₂微柱上，可实现对磷酸肽的选择性。虽然仅使用TFA和乙腈就能实现磷酸肽的富集，但加入DHB或邻苯二甲酸可显著提高选择性，因为这些化合物与TFA引起的低pH值共同作用，可防止非磷酸化肽与TiO₂结合。使用碱性溶液（pH≥10.5）可从TiO₂微珠上洗脱单磷酸化和多磷酸化肽。这种高效的磷酸肽纯化方法非常适合结合质谱（MS）对体外和体内研究中的磷酸化蛋白质进行表征。它是一种非常简便快捷的方法。如果提前制备好缓冲液（不包括冻干），每个样品的整个操作流程耗时不到15分钟。
