Phosphopeptide enrichment using offline titanium dioxide columns for phosphoproteomics.
Phosphopeptide enrichment using offline titanium dioxide columns for phosphoproteomics.
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DOI：
10.1007/978-1-62703-360-2_8
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发表时间：
2013
期刊：
影响因子：
--
通讯作者：
Veenstra T
中科院分区：
文献类型：
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作者：
Yu LR;Veenstra T
鉴定磷蛋白或磷肽作为癌症生物标志物是磷蛋白质组学的一个新兴领域。由于蛋白质磷酸化的低化学计量性质，磷蛋白或磷酸肽必须在下游质谱分析之前富集。二氧化钛（TiO2）由于其对磷酸肽的高亲和性和方法简单，已被广泛用于从复杂蛋白质组样品中富集磷酸肽。在该协议中，离线磷酸肽富集程序使用TiO 2柱进行描述。将来自蛋白质组裂解物的肽在酸性环境中加载到TiO 2柱上，然后在酸性条件下用水性、有机和谷氨酸铵（NH4Glu）缓冲液洗涤柱。磷酸肽洗脱使用氨溶液在高pH值。使用NH4Glu显着减少非特异性结合，而高回收率（84%）的磷酸肽保留。该方法针对大规模磷酸化蛋白质组学分析和从亚毫克或毫克蛋白质组样品开始的磷酸化蛋白质生物标志物发现进行了优化。
Identification of phosphoproteins or phosphopeptides as cancer biomarkers is an emerging field in phosphoproteomics. Owing to the low stoichiometric nature of protein phosphorylation, phosphoproteins or phosphopeptides must be enriched prior to downstream mass spectrometry analysis. Titanium dioxide (TiO2) has been prevalently used to enrich phosphopeptides from complex proteome samples due to its high affinity for phosphopeptides and the method is straightforward. In this protocol, an offline phosphopeptide enrichment procedure using TiO2 columns is described. Peptides from a proteome lysate are loaded onto a TiO2 column in an acidic environment, followed by column washing with aqueous, organic, and ammonium glutamate (NH4Glu) buffers at acidic conditions. Phosphopeptides are eluted using an ammonia solution at high pH. Use of NH4Glu significantly reduces non-specific bindings while a high recovery rate (84%) of phosphopeptides is retained. The method is optimized for large-scale phosphoproteomic analysis and phosphoprotein biomarker discovery starting from sub-milligram or milligrams of proteome samples.