Towards an SI-Traceable Reference Measurement System for Seven Serum Apolipoproteins Using Bottom-Up Quantitative Proteomics: Conceptual Approach Enabled by Cross-Disciplinary/Cross-Sector Collaboration

Abstract

Current dyslipidemia management in patients with atherosclerotic cardiovascular disease (ASCVD) is based on traditional serum lipids. Yet, there is some indication from basic research that serum apolipoproteins A-I, (a), B, C-I, C-II, C-III, and E may give better pathophysiological insight into the root causes of dyslipidemia. To facilitate the future adoption of clinical serum apolipoprotein (apo) profiling for precision medicine, strategies for accurate testing should be developed in advance. Recent discoveries in basic science and translational medicine set the stage for the IFCC Working Group on Apolipoproteins by Mass Spectrometry. Main drivers were the convergence of unmet clinical needs in cardiovascular disease (CVD) patients with enabling technology and metrology. First, the residual cardiovascular risk after accounting for established risk factors demonstrates that the current lipid panel is too limited to capture the full complexity of lipid metabolism in patients. Second, there is a need for accurate test results in highly polymorphic and atherogenic apolipoproteins such as apo(a). Third, sufficient robustness of mass spectrometry technology allows reproducible protein quantification at the molecular level. Fourth, several calibration hierarchies in the revised ISO 17511:2020 guideline facilitate metrological traceability of test results, the highest achievable standard being traceability to SI. This article outlines the conceptual approach aimed at achieving a novel, multiplexed Reference Measurement System (RMS) for seven apolipoproteins based on isotope dilution mass spectrometry and peptide-based calibration. This RMS should enable standardization of existing and emerging apolipoprotein assays to SI, within allowable limits of measurement uncertainty, through a sustainable network of Reference Laboratories.

Simultaneous Mass Spectrometry-Based Apolipoprotein Profiling and Apolipoprotein E Phenotyping in Patients with ASCVD and Mild Cognitive Impairment

“…To obtain the same information, it would be required to perform PCR-based genotyping plus eight single apolipoprotein immunoassays. The advantages of such a multiplexed method as a candidate reference method for apolipoprotein quantitation and phenotyping using LC-MS/MS have also been recognized [28]. In our study, 98% agreement of apoE phenotype identified by LC-MS/MS and APOE genotyping was achieved which is comparable to previously published results [15].…”

Apolipoprotein B and Cardiovascular Disease: Biomarker and Potential Therapeutic Target

“…With mass spectrometry, detection of individual peptide components and comparison of molar ratios may improve risk prediction. Data are supplied on individual subfractions within apoB-containing particles and the extent of oxidation of phospholipids on apoB particles can be determined [ 192 , 272 , 273 ]. The method is antibody-independent and can be automated.…”

The Time Has Come for Quantitative Protein Mass Spectrometry Tests That Target Unmet Clinical Needs

“…Therefore, calibrators should be used that guarantee traceability to secondary and finally primary reference materials and behave in a common way. An example of a traceability chain to SI is depicted in Figure (source: ISO 17511:2020), recently discussed in more detail …”

“…Traceability of apo­(a) was secondary through immunoassay measurement to SRM 2B . Currently, there are no reference materials available for apoC-I, C-II, C-III and E, but a working group of the International Federation for Clinical Chemistry (IFCC WG APO-MS, ) has developed a complete reference measurement system for global standardization of seven serum apolipoproteins . Long-term stability in our laboratory is exemplified for three apolipoproteins in two QC samples (Figure , with Levey-Jennings plots of apoA-I; (1.32 g/L) ± 0.07 (1SD 5.3%), apoB; (1.02 g/L) ± 0.04 (1SD 4.2%) and apo­(a); (102.0 nmol/L) ± 7.4 (1SD 7.3%)).…”