Coupling Strong Anion-Exchange Monolithic Capillary with MALDI-TOF MS for Sensitive Detection of Phosphopeptides in Protein Digest
Coupling Strong Anion-Exchange Monolithic Capillary with MALDI-TOF MS for Sensitive Detection of Phosphopeptides in Protein Digest
复制标题
将强阴离子交换整体毛细管与 MALDI-TOF MS 耦合用于蛋白质消化物中磷酸肽的灵敏检测
DOI：
10.1021/ac902907w
复制
发表时间：
2010-04-01
影响因子：
7.4
通讯作者：
Zou, Hanfa
中科院分区：
文献类型：
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作者：
Dong, Mingming;Wu, Minghuo;Zou, Hanfa
蛋白质磷酸化是最具生物学意义和普遍存在的翻译后修饰之一。基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）是一种通过检测磷蛋白消化物中的磷酸肽来分析蛋白质磷酸化的有力工具。通过固定化金属离子亲和色谱（IMAC）或金属氧化物亲和色谱（MOAC）富集磷酸肽，然后进行MALDI分析是常用的方法。然而，磷酸肽的加载和洗脱的pH与蛋白质消化以及MALDI基质溶液的制备不相容。因此，需要一些预处理步骤，例如pH调节和沉淀，这使得该方法繁琐且不敏感。本研究制备了一种强阴离子交换（SAX）毛细管整体柱，用于富集蛋白酶解产物中的磷酸肽，并用于MALDI-TOF MS分析。结果表明，在pH值为8左右的高pH条件下，磷酸肽能被特异性地保留在SAX柱上，并能被5%甲酸洗脱。因此，蛋白质酶不经任何预处理，在碱性条件下上样到SAX柱上，洗涤除去非磷酸肽后，结合的磷酸肽可以直接洗脱到MALDI靶上，并进行MALDI-TOF MS分析。由于良好的MALDI MS兼容程序和微型SAX柱，用于分析β-酪蛋白消化物中的磷酸肽的检测限低至50阿莫尔。为了克服上样的不便，提出了一种新的基于毛细管作用的简单进样方法，使检测限进一步降低到10阿莫尔。
Protein phosphorylation is one of the most biologically relevant and ubiquitous post-translational modifications. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a powerful tool for the analysis of protein phosphorylation by detection of phosphopeptides in phosphoprotein digest. Enrichment of phosphopeptides by immobilized metal ion affinity chromatography (IMAC) or metal oxide affinity chromatography (MOAC) followed with MALDI analysis is the common approach. However, the pH for loading and elution of phosphopeptides is incompatible with protein digestion as well as the preparation of the MALDI matrix solution. Therefore, some pretreatment steps, such as pH adjustment and desalting, are required, which make the approach tedious and insensitive. In this study, a strong anion-exchange (SAX) capillary monolith was prepared to enrich phosphopeptides from protein digest for MALDI-TOF MS analysis. It was found that phosphopeptides could be specifically retained on the SAX column at high pH around 8 and could be eluted by 5% formic acid. Thus, the protein digests without any pretreatment could be loaded onto the SAX column under basic pH condition; after removing nonphosphopeptides by washing, the bound phosphopeptides could be eluted directly onto a MALDI target and analyzed by MALDI-TOF MS. This approach significantly simplified the analytical procedures and reduced the sample loss. Because of the excellent MALDI MS compatible procedure and the microscale SAX column, a detection limit as low as 50 amol for the analysis of phosphopeptides from beta-casein digest was achieved. To circumvent the inconvenience of the sample loading, a new simple sample introducing method based on capillary action was proposed, which further reduced the detection limit to 10 amol.