Mazanek Michael, Mituloviae Goran, Herzog Franz, Stingl Christoph, Hutchins James R A, Peters Jan-Michael, Mechtler Karl
Research Institute of Molecular Pathology (IMP), Dr Bohr-Gasse 7, A-1030 Vienna, Austria.
Nat Protoc. 2007;2(5):1059-69. doi: 10.1038/nprot.2006.280. Epub 2006 Dec 14.
We have developed a new offline chromatographic approach for the selective enrichment of phosphorylated peptides that is directly compatible with subsequent analysis by online nano electrospray ionization tandem mass spectrometry. In this technique, a titanium dioxide (TiO2)-packed pipette tip is used as a phosphopeptide trap that acts as an offline first-dimension separation step in a two-dimensional chromatography system. This is followed by online nano reversed-phase high-performance liquid chromatography. Here, we present suitable methods for enrichment, optimized separately for each step: sample loading, washing and elution from the TiO2-filled tips. To increase the trapping selectivity of the TiO2 column, we used the sodium salt of 1-octanesulfonic acid combined with 2,5-dihydroxybenzoic acid as ion-pairing agents and displacers for acidic peptides. These agents also improve the binding of phosphorylated peptides and block the binding of non-phosphorylated ones. This enrichment procedure takes 30 min, followed by a 100-min HPLC program, including washing and an elution gradient.
我们开发了一种新的离线色谱方法，用于选择性富集磷酸化肽段，该方法可直接与随后的在线纳升电喷雾电离串联质谱分析兼容。在这项技术中，填充有二氧化钛（TiO₂）的移液器吸头用作磷酸化肽段捕集器，在二维色谱系统中作为离线的一维分离步骤。接下来是在线纳升反相高效液相色谱。在此，我们介绍了适合每个步骤的富集方法，分别针对样品加载、从TiO₂填充吸头的洗涤和洗脱进行了优化。为了提高TiO₂柱的捕集选择性，我们使用1-辛烷磺酸的钠盐与2,5-二羟基苯甲酸作为离子对试剂和酸性肽的置换剂。这些试剂还改善了磷酸化肽段的结合，并阻断了非磷酸化肽段的结合。这种富集过程需要30分钟，随后是100分钟的HPLC程序，包括洗涤和洗脱梯度。
