采用超高效液相色谱串联四极杆飞行时间质谱法测定8条肉类特征肽,探讨色谱分离条件、净化方法和质谱参数对特征肽含量测定的影响。采用C18柱分离,以体积分数0.1%的甲酸水溶液和乙腈(含体积分数0.1%甲酸)为流动相梯度洗脱,电喷雾正离子化检测、采用飞行时间质谱的准多反应监测模式(time-of-flight mass spectrometry-pseudo-multiple response monitoring,TOF-MRM)采集方法定量测定。结果表明,目标特征肽的母离子带2个或3个电荷的信号强度高、稳定性好,二级质谱图中各肽段的碎片离子主要是y离子,且母离子带3个电荷的肽段中也有带2个电荷的碎片离子,使用TOF-MRM采集模式和目标增益采集数据可增加目标离子的灵敏度。优化后的梯度洗脱条件分离效果非常好,质谱信号强,8条合成肽标准溶液的质量浓度和检测响应的线性关系良好,其相关系数均大于0.98;检出限在0.19~1.50 μg/L,TOF-MRM法准确度高,精密度和重复性均可满足要求。对牛肉丸制品中特征肽含量测定及鉴别发现,七类样品中共检测出5条特征肽,其种属来源分别为牛肉2条、猪肉1条、鸡肉2条,这表明测定的牛肉丸样品中不同程度地混有鸡肉或猪肉,且其混入量不等,与牛肉丸价格高低无关,而种属来源为鸭肉的2条特征肽未检出,这表明测定的牛肉丸样品中未混入鸭肉。该文建立了一种肉类特征肽的检测方法,为肉类蛋白质来源鉴定提供了有力的技术支持。
Eight marker peptides in meat were identified and quantitatively determined by ultra-high pressure liquid chromatography coupled with quadrupole time-of-flight mass spectrometry,and the effects of chromatographic separation conditions and mass spectrometry parameters on the determination of marker peptides in meat were investigated. The samples were separated by a C18 column, and gradient elution was performed with 0.1% formic acid in water and acetonitrile (containing 0.1% formic acid) as mobile phase. Then, the electrospray positive ion mode (ESI+) was used to detect the marker peptides, and quantitative analysis and data collection were performed by pseudo-multiple reaction monitoring by time-of-flight mass spectrometry (TOF-MRM). Results showed that the parent ions of the targeted marker peptides had a 2+ or 3+ charge, which showed high signal intensity and good stability. And the fragment ions of each marker peptide in the mass spectrometry was mainly y ion. Moreover, the fragment ions with 3+ charge were accompanied by the formation of 2+ charges. The TOF-MRM acquisition mode and target enhancement increased the detection sensitivity of target ions. Under the optimized conditions, the separation of 8 marker peptides was good and the mass signals were strong. There was a good linear relationship between the concentration of 8 marker peptides standards and detection response, and the correlation coefficients were greater than 0.98. The limit of detection (LOD) for analysts was in the range of 0.19 - 1.50 μg/L, and the optimized TOF-MRM method was successfully applied to the analysis of marker peptides in meat samples due to the high accuracy, precision and repeatability. The application of marker peptides on 7 class beef meatball products showed that five marker peptides could be detected including 2 beef, 1 pork and 2 chicken meat, which indicated that cheaper chicken or pork was spiked in beef meatball samples. The abundance of marker peptides had no correlation with the price. However, no duck-specific marker peptides were found. The established species-specific peptide detection method provided powerful technical support for the identification of protein species origin.
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