Smit Nico P M, Romijn Fred P H T M, van den Broek Irene, Drijfhout Jan W, Haex Martin, van der Laarse Arnoud, van der Burgt Yuri E M, Cobbaert Christa M
Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center, Albinusdreef 2, 2333 ZA, Leiden, The Netherlands.
Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center, Albinusdreef 2, 2333 ZA, Leiden, The Netherlands.
J Proteomics. 2014 Sep 23;109:143-61. doi: 10.1016/j.jprot.2014.06.015. Epub 2014 Jun 25.
In this study, we have followed up on previous liquid chromatography (LC) multiple reaction monitoring (MRM) mass spectrometry (MS) approaches for measurement of apolipoprotein (apo) A-I and apo B100 in serum aiming for implementation of a multiplexed assay in a clinical chemistry laboratory with full metrological traceability. Signature peptides were selected and detected by dynamic MRM, and stable isotope labeled (SIL)-peptides were used as internal standards. Five apo A-I and four apo B100 peptides were measured in serum digests with linearity (R(2)>0.992) in the physiologically relevant concentration ranges. Linearity with regard to protein concentration was ascertained at five concentration levels (R(2)>0.926 and R(2)>0.965, for the apo A-I and apo B100 peptides, respectively). Three native value-assigned sera were used as external calibrators for further method verification. Imprecision values on sample preparation and LC-MS/MS acquisition were below the established minimal specifications for apo A-I and apo B100 (5.0% and 5.3%, respectively). Correlation of LC-MS/MS results with immunoturbidimetric assay results, for normo- and hypertriglyceridemic samples, showed R(2)>0.944 for apo A-I and R(2)>0.964 for apo B100. This LC-MS/MS method has potential for clinical application in normo- and dyslipidemic patients.
Measurement of apo A-I and apo B100 may offer an alternative to high and low density lipoprotein cholesterol (HDL-c and LDL-c) methods for cardiovascular disease risk assessment in dyslipidemic patients [1]. An LC-MS/MS method for apo A-I and apo B100 has the advantage of antibody independent and specific detection of protein signature peptides. The introduction of an LC-MS/MS method for apo A-I and apo B100 can serve as an example for many existing and newly developed (multiplex) biomarker methods in quantitative clinical chemistry proteomics (qCCP). Such LC-MS/MS methods should meet basic clinical chemistry principles with regard to test evaluation [2]. Criteria for imprecision should be pre-defined, e.g., based on biological variation. The use of commutable and traceable serum-based calibrators will improve inter-laboratory reproducibility of LC-MS/MS methods and may contribute to a more rapid transition of biomarker discovery to clinical utility with benefit for the patient treatment and improvement of general health care.
在本研究中，我们对先前用于测量血清中载脂蛋白（apo）A-I和apo B100的液相色谱（LC）多反应监测（MRM）质谱（MS）方法进行了跟进，旨在在临床化学实验室中实施具有完全计量溯源性的多重检测。通过动态MRM选择并检测特征肽，并使用稳定同位素标记（SIL）肽作为内标。在血清消化物中测量了5种apo A-I肽和4种apo B100肽，在生理相关浓度范围内具有线性关系（R²>0.992）。在五个浓度水平确定了与蛋白质浓度的线性关系（apo A-I肽和apo B100肽的R²分别>0.926和R²>0.965）。使用三种具有指定天然值的血清作为外部校准物进行进一步的方法验证。样品制备和LC-MS/MS采集的不精密度值低于为apo A-I和apo B100确定的最低规格（分别为5.0%和5.3%）。对于正常和高甘油三酯血症样本，LC-MS/MS结果与免疫比浊法结果的相关性显示，apo A-I的R²>0.944，apo B100的R²>0.964。这种LC-MS/MS方法在正常血脂和血脂异常患者中具有临床应用潜力。
测量apo A-I和apo B100可能为血脂异常患者心血管疾病风险评估的高密度和低密度脂蛋白胆固醇（HDL-c和LDL-c）方法提供替代方案[1]。一种用于apo A-I和apo B100的LC-MS/MS方法具有独立于抗体且特异性检测蛋白质特征肽的优点。引入用于apo A-I和apo B100的LC-MS/MS方法可以作为定量临床化学生物标志物组学（qCCP）中许多现有和新开发的（多重）生物标志物方法的示例。此类LC-MS/MS方法在测试评估方面应符合基本的临床化学原理[2]。应预先定义不精密度标准，例如基于生物学变异。使用可互换且可溯源的基于血清的校准物将提高LC-MS/MS方法的实验室间再现性，并可能有助于生物标志物发现更快地转化为临床应用，从而有利于患者治疗和改善总体医疗保健。
