Parks Bryan A, Schieltz David M, Andrews Michael L, Gardner Michael S, Rees Jon C, Toth Christopher A, Jones Jeffrey I, McWilliams Lisa G, Kuklenyik Zsuzsanna, Pirkle James L, Barr John R
Division of Laboratory Sciences, Centers for Disease Control and Prevention, Atlanta, GA, USA.
Proteomics Clin Appl. 2017 Jul;11(7-8). doi: 10.1002/prca.201600128. Epub 2017 Apr 3.
Apolipoprotein A-I (ApoA-I) and apolipoprotein B-100 (ApoB-100) are amphipathic proteins that are strong predictors of cardiovascular disease risk. The traceable calibration of apolipoprotein assays is a persistent challenge, especially for ApoB-100, which cannot be solubilized in purified form.
A simultaneous quantitation method for ApoA-I and ApoB-100 was developed using tryptic digestion without predigestion reduction and alkylation, followed by LC separation coupled with isotope dilution MS analysis. The accuracy of the method was assured by selecting structurally exposed signature peptides, optimal choice of detergent, protein:enzyme ratio, and incubation time. Peptide calibrators were value assigned by isobaric tagging isotope dilution MS amino acid analysis.
The method reproducibility was validated in technical repeats of three serum samples, giving 2-3% intraday CVs (N = 5) and <7% interday CVs (N = 21). The repeated analysis of interlaboratory harmonization standards showed -1% difference for ApoA-I and -12% for ApoB-100 relative to the assigned value. The applicability of the method was demonstrated by repeated analysis of 24 patient samples with a wide range of total cholesterol and triglyceride levels.
The method is applicable for simultaneous analysis of ApoA-I and ApoB-100 in patient samples, and for characterization of serum pool calibrators for other analytical platforms.
载脂蛋白A-I（ApoA-I）和载脂蛋白B-100（ApoB-100）是两亲性蛋白质，是心血管疾病风险的强预测指标。载脂蛋白检测的可溯源校准一直是一项挑战，尤其是对于无法以纯化形式溶解的ApoB-100。
开发了一种同时定量ApoA-I和ApoB-100的方法，该方法采用胰蛋白酶消化，无需预消化还原和烷基化，然后进行液相色谱分离并结合同位素稀释质谱分析。通过选择结构暴露的特征肽、洗涤剂的最佳选择、蛋白质与酶的比例以及孵育时间来确保该方法的准确性。肽校准物通过等压标记同位素稀释质谱氨基酸分析进行赋值。
该方法的重现性在三个血清样品的技术重复中得到验证，日内变异系数（CV）为2%-3%（N = 5），日间CV<7%（N = 21）。实验室间比对标准品的重复分析显示，相对于指定值，ApoA-I的差异为-1%，ApoB-100的差异为-12%。通过对24例总胆固醇和甘油三酯水平范围广泛的患者样品进行重复分析，证明了该方法的适用性。
该方法适用于患者样品中ApoA-I和ApoB-100的同时分析，以及其他分析平台血清库校准物的表征。
