Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China.
Anal Chem. 2010 Apr 1;82(7):2907-15. doi: 10.1021/ac902907w.
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.
蛋白质磷酸化是最具生物学相关性和普遍性的翻译后修饰之一。基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）是分析蛋白质磷酸化的有力工具，可通过检测磷酸化蛋白消化物中的磷酸肽来实现。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）对磷酸肽进行富集，然后进行 MALDI 分析，是常用的方法。然而，磷酸肽的加载和洗脱 pH 值与蛋白质消化以及 MALDI 基质溶液的制备不兼容。因此，需要进行一些预处理步骤，如 pH 值调整和脱盐，这使得该方法繁琐且不灵敏。在本研究中，制备了一种强阴离子交换（SAX）毛细管整体柱，用于从蛋白质消化物中富集磷酸肽，用于 MALDI-TOF MS 分析。结果表明，磷酸肽可以在高 pH 值（约 8）下在 SAX 柱上特异性保留，并可以用 5%甲酸洗脱。因此，未经任何预处理的蛋白质消化物可以在碱性 pH 值条件下加载到 SAX 柱上；用洗涤液去除非磷酸肽后，结合的磷酸肽可以直接洗脱到 MALDI 靶上，并通过 MALDI-TOF MS 进行分析。这种方法显著简化了分析步骤，减少了样品损失。由于具有优异的 MALDI MS 兼容程序和微尺度 SAX 柱，从β-酪蛋白消化物中分析磷酸肽的检测限低至 50 amol。为了避免样品加载的不便，提出了一种基于毛细作用的新的简单样品引入方法，将检测限进一步降低至 10 amol。
