ID-LC-MS/MS候选参考方法测定血清载脂蛋白A-Ⅰ和B的优化

Optimization of ID-LC-MS/MS candidate reference method for determining apolipoprotein A-Ⅰand apolipoprotein B in serum

目的优化检测载脂蛋白A-Ⅰ(apoA-Ⅰ)和载脂蛋白B(apoB)的同位素稀释液相色谱串联质谱(ID-LC-MS/MS)候选参考方法。方法选5个正常三酰甘油(TG)含量(TG≤2.3 mmol/L)的样品为校准品,2个高三酰甘油血症患者和1个正常三酰甘油患者的血清为待测样品。载脂蛋白经过变性、烷基化、胰蛋白酶酶解后,经LC-MS/MS进行分离和定量。对apoA-Ⅰ的特征肽AKPALEDLR、apoB的特征肽TGISPLALIK和TEVIPPLIENR进行母子离子对的优化。结果优化后与优化前的峰面积比范围在1.5至67.2之间。优化后,定性/定量离子对的比值的不精密度降低,最高降低3.1%(AKPALEDLR 338.2/403.2),最低降低0.6%(AKPALEDLR 338. 2/532. 4)。AKPALEDLR肽段和VQPYLDDFQK肽段定量apoA-Ⅰ结果一致;TEVIPPLIENR肽段和TGISPLALIK肽段定量apoB结果除HTG2外一致。结论选择合适的离子对可以显著提高载脂蛋白相应特征肽的峰面积,降低定性/定量离子对的比值的不精密度,使定量结果更准确。

Objective To optimize isotope-diluted( ID)-LC-MS/MS candidate reference method for determining apolipoprotein A-Ⅰand apolipoprotein B. Methods Five samples with normal levels of triglycerides( TG ≤2.3 mmol/L) were selected as calibrators,and 2 samples with high levels and 1 sample with normal level of triglycerides were used as the test samples. After apolipoproteins in the samples were treated with denaturation,alkylation and trypsin digestion,the samples were separated and quantified by LC-MS/MS systems. The target peptides AKPALEDLR of apo A-Ⅰ,TGISPLALIK and TEVIPPLIENR of apo B were optimized in terms of their precursor and product transitions. Results The peak area ratios after optimization was ranged from 1.5 to 67.2 compared with those before optimization. After optimization the imprecision of qualitative/quantitative transition ratios decreased with a percentage of up to 3.1% for AKPALEDLR 338.2/403.2 and 0.6% in the minimum for AKPALEDLR 338. 2/532. 4. The quantitative results of apo A-Ⅰ by using AKPALEDLR and VQPYLDDFQK showed consistent on value,which was similar to the quantification of apo B by using TEVIPPLIENR and TGISPLALIK except for the sample HTG2. Conclusion The appropriate selection of precursor-product transitions may obviously increase the peak areas of corresponding target peptides for apolipoprotein and lower the imprecision of qualitative/quantitative transition ratio,and consequently acquire more accurate quantitative results.