Targeted Proteomics Using Stable Isotope Labeled Protein Fragments Enables Precise and Robust Determination of Total Apolipoprotein(a) in Human Plasma

Lipoprotein(a), also known as Lp(a), is an LDL-like particle composed of apolipoprotein(a) (apo(a)) bound covalently to apolipoprotein B100. Plasma concentrations of Lp(a) are highly heritable and vary widely between individuals. Elevated plasma concentration of Lp(a) is considered as an independent, causal risk factor of cardiovascular disease (CVD). Targeted mass spectrometry (LC-SRM/MS) combined with stable isotope-labeled recombinant proteins provides robust and precise quantification of proteins in the blood, making LC-SRM/MS assays appealing for monitoring plasma proteins for clinical implications. This study presents a novel quantitative approach, based on prototypic peptides, to determine the absolute concentration of apo(a) from two microliters of plasma and qualified according to guideline requirements for targeted proteomics assays. After optimization, assay parameters such as linearity, lower limits of quantification (LLOQ), intra-assay variability (CV: 4.72%) and inter-assay repeatability (CV: 7.76%) were determined and the LC-SRM/MS results were benchmarked against a commercially available immunoassay. In summary, the measurements of an apo(a) single copy specific peptide and a K4 specific peptide allows for the determination of molar concentration and relative size of apo(a) in individuals.

脂蛋白(a)（Lipoprotein(a)，简称Lp(a)）是一种类似低密度脂蛋白（LDL）的颗粒，由载脂蛋白(a)（apo(a)）与载脂蛋白B100共价结合而成。血浆中Lp(a)浓度具有高度遗传性，且个体间差异显著。血浆Lp(a)浓度升高被认为是心血管疾病（cardiovascular disease，简称CVD）的独立、因果性风险因素。靶向质谱（targeted mass spectrometry）结合稳定同位素标记的重组蛋白（stable isotope-labeled recombinant proteins），可对血液中的蛋白质进行可靠且精准的定量分析，这使得液相色谱-选择反应监测/质谱法（LC-SRM/MS）检测在临床血浆蛋白监测方面具有吸引力。本研究提出了一种基于原型肽（prototypic peptides）的新型定量方法，可从2微升血浆中测定apo(a)的绝对浓度，并符合靶向蛋白质组学检测（targeted proteomics assays）的指南要求。优化后，测定了线性、定量下限（lower limits of quantification，简称LLOQ）、批内变异系数（intra-assay variability，CV：4.72%）和批间重复性（inter-assay repeatability，CV：7.76%）等检测参数，并将LC-SRM/MS结果与市售免疫测定法进行了基准比较。综上，通过测定apo(a)单拷贝特异性肽（apo(a) single copy specific peptide）和K4特异性肽（K4 specific peptide），可确定个体中apo(a)的摩尔浓度和相对大小。