【摘要】 该文建立了一种利用磷酰化修饰结合电喷雾质谱(ESI-Q-TOF)测定多肽氨基酸序列的有效方法。利用Atherton-Todd反应,以二丙基亚磷酰酯(DPP)为磷酰化试剂,应用生物质谱技术,对磷酰化修饰后的5种模型肽的磷酰化反应情况进行了系统研究,考察了磷酰化肽的二级质谱特征,并与未经磷酰化反应的肽的二级质谱特征对比。结果表明,经过磷酰化修饰后,肽的二级质谱中的a1离子信号强度明显增加,可以准确鉴定其N端氨基酸;b系列离子信息完整,信号强度增强,使得多肽C ID测序的谱图简单、清晰,有利于肽的氨基酸序列的测定;赖氨酸(K,128.10 u)和谷氨酰胺(Q,128.13 u)两种氨基酸质荷比相近,由于二者磷酰化修饰后的差异性,使其得到准确区分。经过5种已知氨基酸序列的模型肽的磷酰化后结合质谱技术进行氨基酸序列测定验证,结果表明该方法简单、快速、准确,提高了利用质谱技术进行多肽测序的准确度和灵敏度,可为蛋白质组学研究提供有效的技术手段。更多还原

【Abstract】 A simple and rapid method for determining peptide amino-acid sequence using ESI-Q-TOF-MS-based phosphorylation strategy was developed.Five model peptides were studied by ESI-Q-TOF-MS after modifying with dipropyl phosphonate(DPP) via the Atherton Todd reaction in the presence of tetrachloromethane and triethylamine.The characteristics of MS/MS spectra of phosphorylated peptide were studied and compared with those of non-phosphorylated peptides.Results showed that the signal intensity of a1 ion in the MS/MS spectra of phosphorylated peptides increased significantly and could be used to identify accurately the N-terminal amino acid;the signal intensities of b series ions also increased making the CID mass spectra of peptides simple,vivid and favorable for determining peptide amino-acid sequence.The mass/charge ratio of lysine(K,128.10 u) originally closed to that of glutamine(Q,128.13 u) was differentiated after phosphorylation.The feasibility of this method was validated by several synthetic peptides with known peptide sequence.Results showed that the proposed method was feasible,and reliable,and could be conveniently used for the de novo peptide sequencing in proteome research.更多还原