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/ddMS2)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/ddMS2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0.04~0.19 μg/L、0.01~0.16 μg/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/ddMS2) 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/ddMS2 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 0.04-0.19 μg/L, and 0.01-0.16 μg/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.
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