Proteomic and Biomolecular Mass Spectrometry Centre, Institute of Food Science CNR, Avellino, Italy.
Proteomics. 2010 Feb;10(3):380-93. doi: 10.1002/pmic.200800710.
The most challenging analytical task facing phosphoproteome determination requires the isolation of phosphorylated peptides from the myriad of unphosphorylated species. In the past, several strategies for phosphopeptide isolation have been proposed in combination with subsequent mass spectrometric investigations. Among these techniques, immobilized metal affinity chromatography and titanium dioxide have been recognized as the most effective. Here, we present an alternative method for the enrichment of phosphopeptides based on hydroxyapatite (HAP) chromatography. By taking advantage of the strong interaction of HAP with phosphate and calcium ions, we developed an efficient method for the selective separation and fractionation of phosphorylated peptides. The effectiveness and efficiency of recovery for this procedure was assayed using tryptic digests of standard phosphorylated protein mixtures. Based on the higher affinity of multi-phosphorylated peptides for HAP surfaces, the introduction of a phosphate buffer gradient for stepwise peptide elution resulted in the separation of mono-, di-, tri-, and multi-phosphorylated peptides. Thus, we demonstrated that this technique is highly selective and independent of the degree of peptide phosphorylation.
面对磷酸化蛋白质组测定最具挑战性的分析任务，需要从无数的未磷酸化物种中分离出磷酸化肽。过去，已经提出了几种与随后的质谱研究相结合的磷酸肽分离策略。在这些技术中，固定化金属亲和层析和二氧化钛已被认为是最有效的。在这里，我们提出了一种基于羟基磷灰石（HAP）层析的磷酸肽富集的替代方法。利用 HAP 与磷酸盐和钙离子的强相互作用，我们开发了一种有效且高效的方法，用于选择性分离和分级磷酸化肽。使用标准磷酸化蛋白混合物的胰蛋白酶消化来测定该方法的回收效果和效率。基于多磷酸化肽对 HAP 表面的更高亲和力，通过磷酸盐缓冲液梯度进行逐步肽洗脱，导致单、二、三、多磷酸化肽的分离。因此，我们证明该技术具有高度的选择性，并且与肽磷酸化程度无关。
