Ruprecht Benjamin, Koch Heiner, Domasinska Petra, Frejno Martin, Kuster Bernhard, Lemeer Simone
Chair of Proteomics and Bioanalytics, Technische Universität München, Emil Erlenmeyer Forum 5, 85354, Freising, Germany.
Center for Integrated Protein Science Munich (CIPSM), Freising, Germany.
Methods Mol Biol. 2017;1550:47-60. doi: 10.1007/978-1-4939-6747-6_5.
Phosphorylation is among the most important post-translational modifications of proteins and has numerous regulatory functions across all domains of life. However, phosphorylation is often substoichiometric, requiring selective and sensitive methods to enrich phosphorylated peptides from complex cellular digests. Various methods have been devised for this purpose and we have recently described a Fe-IMAC HPLC column chromatography setup which is capable of comprehensive, reproducible, and selective enrichment of phosphopeptides out of complex peptide mixtures. In contrast to other formats such as StageTips or batch incubations using TiO or Ti-IMAC beads, Fe-IMAC HPLC columns do not suffer from issues regarding incomplete phosphopeptide binding or elution and enrichment efficiency scales linearly with the amount of starting material. Here, we provide a step-by-step protocol for the entire phosphopeptide enrichment procedure including sample preparation (lysis, digestion, desalting), Fe-IMAC column chromatography (column setup, operation, charging), measurement by LC-MS/MS (nHPLC gradient, MS parameters) and data analysis (MaxQuant). To increase throughput, we have optimized several key steps such as the gradient time of the Fe-IMAC separation (15 min per enrichment), the number of consecutive enrichments possible between two chargings (>20) and the column recharging itself (<1 h). We show that the application of this protocol enables the selective (>90 %) identification of more than 10,000 unique phosphopeptides from 1 mg of HeLa digest within 2 h of measurement time (Q Exactive Plus).
磷酸化是蛋白质最重要的翻译后修饰之一，在生命的所有领域都具有众多调节功能。然而，磷酸化通常是亚化学计量的，需要选择性和灵敏的方法从复杂的细胞消化物中富集磷酸化肽段。为此已设计出各种方法，我们最近描述了一种铁离子固定金属亲和色谱（Fe-IMAC）高效液相色谱柱层析装置，它能够从复杂的肽混合物中全面、可重复且选择性地富集磷酸肽段。与其他形式如StageTips或使用二氧化钛（TiO）或钛离子固定金属亲和色谱（Ti-IMAC）磁珠的批量孵育不同，Fe-IMAC高效液相色谱柱不存在磷酸肽段结合或洗脱不完全以及富集效率与起始材料量不成线性关系的问题。在这里，我们提供了整个磷酸肽段富集过程的详细步骤方案，包括样品制备（裂解、消化、脱盐）、Fe-IMAC柱层析（柱设置、操作、充电）、液相色谱-串联质谱（LC-MS/MS）测量（纳升高效液相色谱梯度、质谱参数）和数据分析（MaxQuant）。为了提高通量，我们优化了几个关键步骤，如Fe-IMAC分离的梯度时间（每次富集15分钟）、两次充电之间可连续富集的次数（>20次）以及柱再充电本身的时间（<1小时）。我们表明，应用该方案能够在2小时测量时间内（Q Exactive Plus）从1毫克HeLa细胞消化物中选择性（>90%）鉴定出超过10,000个独特的磷酸肽段。
