Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns
Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns
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DOI：
10.1074/mcp.t500007-mcp200
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发表时间：
2005-07-01
影响因子：
7
通讯作者：
Jorgensen, TJD
中科院分区：
文献类型：
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作者：
Larsen, MR;Thingholm, TE;Jorgensen, TJD
蛋白质的可逆磷酸化调节大多数细胞过程，如增殖、分化和凋亡。在分子水平上对这些生物过程的基本理解需要对磷酸化蛋白进行表征。磷酸化通常是亚化学计量的，磷酸化蛋白衍生的磷酸化肽的富集过程是用现代质谱方法表征这些肽的必要先决条件。我们报道了一种基于TiO2微柱和肽在2,5-二羟基苯甲酸（DHB）中负载的高度选择性富集磷酸化肽的方法。DHB的作用是非常有效地减少非磷酸化肽与TiO2的结合，同时保持其对磷酸化肽的高结合亲和力。因此，DHB的加入极大地提高了TiO2富集磷酸化肽的选择性。我们用MALDI质谱法证明了这种新方法比IMAC更有选择性地结合磷酸化肽。此外，我们发现LC-ESI-MSMS偏向于单磷酸化肽，而MALDI MS则不是。其他取代的芳香族羧酸也能够特异性地减少非磷酸化肽的结合，而磷酸可以减少磷酸化和非磷酸化肽的结合。提出了这一有趣效应的推测机制。
Reversible phosphorylation of proteins regulates the majority of all cellular processes, e. g. proliferation, differentiation, and apoptosis. A fundamental understanding of these biological processes at the molecular level requires characterization of the phosphorylated proteins. Phosphorylation is often substoichiometric, and an enrichment procedure of phosphorylated peptides derived from phosphorylated proteins is a necessary prerequisite for the characterization of such peptides by modern mass spectrometric methods. We report a highly selective enrichment procedure for phosphorylated peptides based on TiO2 microcolumns and peptide loading in 2,5-dihydroxybenzoic acid (DHB). The effect of DHB was a very efficient reduction in the binding of nonphosphorylated peptides to TiO2 while retaining its high binding affinity for phosphorylated peptides. Thus, inclusion of DHB dramatically increased the selectivity of the enrichment of phosphorylated peptides by TiO2. We demonstrated that this new procedure was more selective for binding phosphorylated peptides than IMAC using MALDI mass spectrometry. In addition, we showed that LC-ESI-MSMS was biased toward monophosphorylated peptides, whereas MALDI MS was not. Other substituted aromatic carboxylic acids were also capable of specifically reducing binding of nonphosphorylated peptides, whereas phosphoric acid reduced binding of both phosphorylated and nonphosphorylated peptides. A putative mechanism for this intriguing effect is presented.