基于LC-MS/MS技术同时定量分析片仔癀中特征胰酶水解肽

Simultaneous Quantitative Analysis of Characteristic Tryptic Peptides in Pien Tze Huang Using Liquid Chromatography Tandem Mass Spectrometry(LC-MS/MS)

目的:阐明片仔癀中的蛋白组,定量分析其中的特征胰酶水解肽,以提高片仔癀的质量控制水平。方法:优化总蛋白提取条件,采用二辛可宁酸(BCA)法测定总蛋白含量,利用鸟枪法蛋白组学表征片仔癀的蛋白组,并进一步选择含量较高、稳定性好、长度适宜的特征多肽作为目标分析物,采用在线能量分辨质谱法(online ER-MS)优化特征肽的离子对及最佳碰撞能,建立反相液相色谱-选择反应监测(RPLC-SRM)定量分析方法,测定11批片仔癀中目标多肽的含量。结果:利用UniProt数据库从片仔癀中共鉴定了来源于200个蛋白的343个肽段,以酶和骨架蛋白为主;筛选得到ALSSALHER、TLLEGEESR和VAPLGEEFR 3个特征肽,以其作为目标分析物进行定量分析,online ER-MS优化3个特征肽的定量参数离子对分别为m/z 492.3→799.4、517.3→819.4和509.3→637.3,碰撞能分别为25.0、24.4、28.6 eV。方法学验证符合定量要求,3个多肽类成分在各批片仔癀样品中均被检出(ALSSALHER:0.06~0.44μg·g^(–1);TLLEGEESR:0.08~0.26μg·g^(–1);VAPLGEEFR:0.27~1.09μg·g^(–1)),含量差异较小,其中特征肽VAPLGEEFR的含量较高。结论:建立的方法可靠,可为富含蛋白类成分的中药制剂的质量评价提供思路与方法。

Objective:To carry out proteomic profiling for Pien Tze Huang(PTH)and quantitate its characteristic tryptic peptides to improve the quality control level of PTH.Methods:The conditions for extracting total proteins was optimized and bicinchoninic acid(BCA)assay was adopted to determine the content of total proteins.The proteome of PTH was characterized by shotgun method.Moreover,some characteristic tryptic peptides with relatively high abundance,high stability and appropriate lengths were selected as the targets for analysis,while the ion pairs and collision energies were optimized using online energy-resolved mass spectrometry(online ER-MS).Therefore,the quantitative analysis method based on Reversed-phase Liquid Chromatography-Selective Reaction Monitoring(RPLC-SRM)was established to determine the contents of target peptides in 11 batches of PTH.Results:A total of 343 peptides from 200 proteins were identified from PTH using UniProt database,which were mainly enzymes and cytoskeletal proteins.Three characteristic tryptic peptides,namely ALSSALHER,TLLEGEESR and VAPLGEEFR,were chosen as the targets for quantitative analysis.The quantitative parameters for the three characteristic peptides were optimized using online ER-MS as follows:m/z of ion pairs 492.3→799.4,517.3→819.4 and 509.3→637.3,collision energy 25.0,24.4,28.6 eV,respectively.The methodological validation met with the requirement for quantitative analysis.The three peptides could be detected in all batches without significant difference in their contents(ALSSALHER:0.06-0.44μg·g^(–1);TLLEGEESR:0.08-0.26μg·g^(–1);VAPLGEEFR:0.27-1.09μg·g^(–1)),among which the content of VAPLGEEFR was relatively higher.Conclusion:The method in this study is reliable,which may provide ideas and methods for quality evaluation of traditional Chinese medical preparations with abundant protein compositions.