超高效液相色谱-高分辨质谱法定量测定肉类特征肽的质谱采集模式对比

Comparison of acquisition modes for the quantitative determination of meat marker peptides using ultra-performance liquid chromatography-high resolution mass spectrometry

采用超高效液相色谱-高分辨质谱法测定肉类特征肽,探讨液相色谱洗脱参数、质谱采集模式和参数等对肉类特征肽定量测定的影响,以建立肉类特征肽鉴别和定量测定的方法。样品中蛋白质经超声辅助法提取,胰蛋白酶酶解后经固相柱净化去除基质中的盐类等干扰物,以体积分数0.1%的甲酸水溶液和乙腈为流动相,通过超高效液相色谱梯度洗脱分离,采用四极杆静电场轨道阱高分辨质谱的平行反应监测(parallel reaction monitoring,PRM)模式和靶标单一离子监测/数据依赖扫描(targeted single ion monitoring/data-dependent MS/MS scans,tSIM/ddMS 2)模式采集,外标法定量测定。结果表明,色谱梯度洗脱条件、碰撞能量、采集模式等对特征肽的测定影响较大,梯度洗脱流速增加至0.3 mL/min可有效减少峰展宽与拖尾,PRM采集模式不需优化碰撞能可直接采用归一化碰撞能为28的值,tSIM/ddMS 2采集模式可采用Skyline软件优化出的最优碰撞能值,从而简化优化碰撞能的过程。不同采集模式下各特征肽标准溶液质量浓度的线性关系良好(相关系数≥0.99),检出限分别为0.04~0.19μg/L、0.01~0.16μg/L,在牛肉丸提取液中的基质效应为81.3%~114.7%,3个添加浓度下的加标回收率为89%~117%,且相对标准偏差≤10%(n=5)。2种采集模式均可利用二级碎片离子鉴别目标物以提高检测的准确性,12批次牛肉丸样品检测中共检出含有猪肉源或鸡肉源特征肽的比例为58.3%,其中有明确标识的为42.8%。

Ultra performance liquid chromatography-high resolution mass spectrometry was used to determine meat marker peptides,and the effects of liquid chromatography gradient elution parameters and acquisition parameters of mass spectrometry were investigated to establish a method for the quantitative determination of meat marker peptides.Proteins were extracted by ultrasonic-assisted method from samples,and salts and other interfering substances were removed by solid phase extraction column after enzymatic hydrolysis by trypsin.The marker peptides were eluted with 0.1%(volume ratio)formic acid aqueous solution and acetonitrile(containing 0.1%formic acid,volume ratio)as the mobile phase in ultra-performance liquid chromatography-high resolution mass spectrometry.Parallel reaction monitoring(PRM)mode and targeted single ion monitoring followed by data-dependent MS/MS scans(tSIM/ddMS 2)mode were acquired for quantitative determination by an external standard.The results showed that gradient elution parameters,collision energy and acquisition modes showed a significant effect on the determination of marker peptides,and 0.3 mL/min of flow rate effectively reduced the peak broadening and trailing.In PRM acquisition mode,the normalized collision energy value of 28 was directly used without optimization of collision energy,while in tSIM/ddMS 2 acquisition mode,the optimal collision energy values optimized by Skyline software were used to simplify the process of collision energy optimization.The linear relationship of the standard solution of 8 marker peptides was good(correlation coefficient≥0.99).The detection limits of two acquisition modes were 0.04-0.19μg/L,and 0.01-0.16μg/L,respectively.The matrix effect of eight marker peptides in beef meatball extract ranged from 81.3%to 114.7%,and the recoveries were in the range of 89%to 117%with a relative standard deviation≤15%(n=5)at three spiked concentrations.The target products were identified by fragment ions in both acquisition modes to improve the detection accuracy,and 58.3%of 12 batches of beef meatball samples were detected to contain pork-or chicken-specific peptides,among which only 42.8%was clearly stated.