纳升级电喷雾离子源-四极杆-飞行时间质谱中磷酸化肽的离子抑制作用

目前磷酸化蛋白质组学研究中的主要技术是蛋白质酶解产生的磷酸化肽的质谱检测。但是实际样品中的磷酸化肽(特别是多磷酸化肽)很难被检测到。其原因普遍认为是由于质谱检测时,非磷酸化肽抑制磷酸化肽。但也有认为非磷酸化肽对磷酸化肽没有抑制作用。另外磷酸化肽之间是否存在离子抑制作用还没有报道。本文采用相同氨基酸序列的标准磷酸化肽和非磷酸化肽,将其单独和混合进行质谱检测,通过对比混合前后磷酸化肽的信号强度,证明了非磷酸化肽对磷酸化肽有离子抑制作用；单磷酸化肽对二磷酸化肽有一定的抑制作用,但不太显著;单磷酸化肽对三磷酸化肽、二磷酸化肽对三磷酸化肽均有显著的离子抑制作用。该研究为今后单磷酸化肽和多磷酸化肽的分段富集和检测提供了有力的证明。

Ionization suppression effect of phosphopeptides in nano-electrospray ionization quadrupole time-of-flight mass spectrometry

Protein phosphorylation is an important post-translational modification (PTM), and involves in many cell activities and disease processes. Nowadays, mass spectrometry (MS) is a powerful tool in peptide-based proteomics. But phosphopeptides, especially multiply phosphorylated peptides, are difficult to be detected by MS. One opinion is that the presence of unphosphorylated peptides lead to the ionization suppression of phosphopeptides. But contrary opinion exists. Moreover, the suppression effect caused by different types of phosphopeptides hasn’t been studied yet. In this study, a set of synthetic standard phosphopeptides (singly, doubly and triply phosphorylated peptides) with the same amino acid sequence, and the unphosphorylated peptides were injected into mass spectrometer with or without mixing. Through the ratios of signal intensities of the phosphopeptides before and after mixing, we confirmed that the signals of phosphopeptides were suppressed by those of the unphosphorylated peptides. In addition, the signals of the doubly phosphorylated peptides were slightly suppressed by those of the singly phosphorylated peptides with the same amino acid sequence. The signals of the triply phosphorylated peptides were notably suppressed by the singly and doubly phosphorylated peptides with the same amino acid sequence. Therefore, the singly and multiply phosphorylated peptides should be fractionated and enriched before mass spectrometry analysis.