Freiburg Institute for Advanced Studies (FRIAS), School of Life Sciences-LifeNet, University of Freiburg, Albertstr. 19, 79104 Freiburg, Germany.
J Proteome Res. 2011 Aug 5;10(8):3474-83. doi: 10.1021/pr200092z. Epub 2011 Jul 8.
Reversible phosphorylations play a critical role in most biological pathways. Hence, in signaling studies great effort has been put into identification of a maximum number of phosphosites per experiment. Mass spectrometry (MS)-based phosphoproteomics approaches have been proven to be an ideal analytical method for mapping of phosphosites. However, because of sample complexity, fractionation of phosphopeptides prior to MS analysis is a crucial step. In the current study, we compare the chromatographic strategies electrostatic repulsion-hydrophilic interaction chromatography (ERLIC), hydrophilic interaction liquid chromatography (HILIC), and strong cation exchange chromatography (SCX) for their fractionation behavior of phosphopeptides. In addition, we investigate the use of repetitive TiO(2)-based enrichment steps for a maximum identification of phosphopeptides. On the basis of our results, SCX yields the highest number of identified phosphopeptides, whereas ERLIC is optimal for the identification of multiphosphorylated peptides. Consecutive incubations of fractions and flow-through by TiO(2) beads enrich qualitatively different sets of phosphopeptides, increasing the number of identified phosphopeptides per analysis.
可逆磷酸化在大多数生物途径中起着关键作用。因此，在信号研究中，人们付出了巨大的努力来确定每个实验中磷酸化位点的最大数量。基于质谱（MS）的磷酸蛋白质组学方法已被证明是一种用于绘制磷酸化位点的理想分析方法。然而，由于样品的复杂性，在 MS 分析之前对磷酸肽进行分级是至关重要的一步。在本研究中，我们比较了色谱策略——静电排斥-亲水相互作用色谱（ERLIC）、亲水相互作用液相色谱（HILIC）和强阳离子交换色谱（SCX）在磷酸肽分级行为方面的表现。此外，我们还研究了重复使用 TiO2 基富集步骤以最大程度地鉴定磷酸肽。根据我们的结果，SCX 产生的鉴定磷酸肽数量最多，而 ERLIC 则最适合鉴定多磷酸化肽。TiO2 珠连续孵育和流经各个馏分，可富集定性不同的磷酸肽，从而增加每次分析中鉴定的磷酸肽数量。
