ID—LC-MS检测血清载脂蛋白A-Ⅰ和B载脂蛋白的多肽释放研究

Peptides releasing study of serum apolipoprotein A-Ⅰ and apolipoprotein B by ID-LC-MS

目的研究质谱技术检测载脂蛋白A-Ⅰ和载脂蛋白B的多肽释放情况，为准确定量蛋白提供依据。方法方法学研究。确定载脂蛋白A-Ⅰ和B的目标肽段。以目标肽段为检测对象，用同位素稀释液相色谱串联质谱法测定5份市售人血清样本中的载脂蛋白A-Ⅰ和B，浓度范围分别为0.90～2.54 g/L和0.54～1.39 g/L。计算不同时间点的多个肽段释放量和速率，并绘制散点图。计算不同样本间，肽段释放量的相关系数R2。结果多数肽段在4 h内即达释放峰值。Apo A－Ⅰ的肽段VQ、DY和VS，以及Apo B的肽段TE和FP生成速度相对较慢。达到峰值后，TEV/SIL－TEV、AK/SIL－AK和VQ/SIL－VQ的比值相对稳定。对于Apo A-Ⅰ，不同肽段之间的相关性较高，R2在0.904～0.999之间；对于Apo B，部分肽段之间的相关性较低，如TG－SV的R2＝0.543（3 h）。结论Apo A-Ⅰ和Apo B不同肽段的酶解释放情况不同，合适的选择代表性肽段和酶解条件将有助于准确定量目标蛋白。

ObjectiveTo investigate the digestion kinetics of Apolipoprotein A-Ⅰ and B by ID-LC-MS method for accurate quantification of proteins.MethodsMethodological research. The target peptides of ApoA-Ⅰ and B were determined. The ApoA-Ⅰ and B from 5 human serum samples on market with levels from 0.90-2.54 g/L and 0.54-1.39 g/L separately, were measured in terms of target peptides by isotope dilution liquid chromatography mass spectrometry method. The releasing amount and rate of peptides were analyzed and plotted according to different time points. The correlation coefficient R2 was calculated among peptide releasing amount between samples.ResultsMost peptides reached their peaks within 4 hours. The peptides VQ, DY and VS from Apo A-Ⅰ, TR and FP from Apo B were released relatively slowly. After getting to their peak stage, the ratio between TEV and SIL-TEV, AK and SIL-AK, VQ and SIL-VQ presented stable state. As for Apo A-Ⅰ the correlations among peptides are high, from 0.904 to 0.999. Some peptides from Apo B show lower correlations, such as TG-SV with R2 0.543 （3 h）.ConclusionsPeptides from Apo A-Ⅰ and Apo B present different releasing properties after trypsin digestion. Proper selection of representative peptides and enzymatic conditions can benefit accurate quantification of target proteins.