基于四极杆-飞行时间质谱的非标记蛋白定量检测方法学研究

Label-free Quantitation of Protein Based on Quadrupole-time of Flight Tandem Mass Spectrometry

以四极杆飞行时间质谱检测的蛋白肽段的质谱信息为基础,开发了一种简单、准确的蛋白定性、定量检测方法,并用于牛血清白蛋白的定性、定量检测。对酶解肽段的梯度洗脱条件和流速等液相色谱条件进行了优化;进行蛋白样品的酶解及质谱检测,利用MassHunter定量软件对数据进行分析;为减少假阳性结果的出现,实验优化了数据处理软件中的设置参数,并对方法的重现性进行了考察。结果显示,重复检测的8个肽段峰面积的RSD均在3.0%以下。该方法对牛血清白蛋白的检出限为100 ng。实验发现,样品中蛋白的含量与检测到的肽段匹配率呈对数关系,并从检测到的丰度较高的8个肽段中确定可用于BSA蛋白定性检测的肽段为:HLVDEPQNLIK,定量检测的肽段为:ATEEQLK,方法的回收率为106%。该方法操作简便,检测快速,成本较低,专一性强,可用于蛋白特异性肽段的筛选及复杂生物样品中蛋白的检测。

A label-free approach to qualify and quantify bovine serum albumin（BSA） based on the information of mass spectrum was reported.The conditions of high-performance liquid chromatography（HPLC） were optimized,including program of gradient elution and flow rate.The content of BSA was determined in optimal conditions by HPLC-quadrupole-time of flight tandem mass spectrometry（Q-TOF）after digested with trypsin in-solution.The data was processed with MassHunter Qualitative Analysis software.The setting parameters of software were optimized to decrease the probability of false positive results.The reproducibility of the experiments was investigated.According to the peak area results of eight peptides,the RSDs（n=4） were below 3.0%.The detection limit for BSA was achieved as 100 ng.The results also indicated that there was a logarithmic manner between concentration of protein and sequence coverage of peptides.Among the eight peptides of BSA with higher abundance,one unique peptide：HLVDEPQNLIK was selected to qualify the BSA,and another unique peptide：ATEEQLK was used to quantify the BSA.The recovery of this method was calculated to be 106%.This method is simple,rapid,low cost and specific,and could be employed for screening characteristic peptides of specific proteins and detecting of complex biological sample.