A high-throughput mass spectrometry-based assay for large-scale profiling of circulating human apolipoproteins

Abstract

<jats:p>Apolipoproteins govern lipoprotein metabolism and are promising biomarkers of metabolic and cardiovascular diseases. Unlike immunoassays, MS enables the quantification and phenotyping of multiple apolipoproteins. Hence, here, we aimed to develop a LC-MS/MS assay that can simultaneously quantitate 18 human apolipoproteins [A-I, A-II, A-IV, A-V, B48, B100, C-I, C-II, C-III, C-IV, D, E, F, H, J, L1, M, and (a)] and determined apoE, apoL1, and apo(a) phenotypes in human plasma and serum samples. The plasma and serum apolipoproteins were trypsin digested through an optimized procedure and peptides were extracted and analyzed by LC-MS/MS. The method was validated according to standard guidelines in samples spiked with known peptide amounts. The LC-MS/MS results were compared with those obtained with other techniques, and reproducibility, dilution effects, and stabilities were also assessed. Peptide markers were successfully selected for targeted apolipoprotein quantification and phenotyping. After optimization, the assay was validated for linearity, lower limits of quantification, accuracy (biases: –14.8% to 12.1%), intra-assay variability [coefficients of variation (CVs): 1.5–14.2%], and inter-assay repeatability (CVs: 4.1–14.3%). Bland-Altman plots indicated no major statistically significant differences between LC-MS/MS and other techniques. The LC-MS/MS results were reproducible over five repeated experiments (CVs: 1.8–13.7%), and we identified marked differences among the plasma and serum samples. The LC-MS/MS assay developed here is rapid, requires only small sampling volumes, and incurs reasonable costs, thus making it amenable for a wide range of studies of apolipoprotein metabolism. We also highlight how this assay can be implemented in laboratories.</jats:p>

Multiplatform Approach for Plasma Proteomics: Complementarity of Olink Proximity Extension Assay Technology to Mass Spectrometry-Based Protein Profiling

“…In line with current findings in MS-based plasma proteome profiling, we mainly observe abundant functional proteins such as serum albumin, apolipoproteins, immunoglobulins, acute phase proteins and coagulation cascade components. This large fraction of high abundant proteins involved in the inflammation and lipid metabolism has enormous value for the profiling of cardiovascular and metabolic diseases (37,38). However, we faced the limit of MS to dig into the low -abundant and -molecular weight fraction (which was not certified by peer review) is the author/funder.…”

Changes in Key Mitochondrial Lipids Accompany Mitochondrial Dysfunction and Oxidative Stress in NAFLD

“…Measurements. ApoE was measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as described previously [24].…”

Apolipoprotein Proteomic Profiling for the Prediction of Cardiovascular Death in Patients with Heart Failure

“…[13] High-throughput mass spectrometry based assay for large scale profiling of human circulating Apo, requiring small sampling has shown reproducibility and comparable results with those obtained with other techniques. [14] Here, we therefore performed a proteomic profiling of 15 Apo using the LC-MRM-MS with the aim to select new biomarkers that are associated with cardiovascular mortality at three years in patients with systolic HF and that may be involved in HF physiopathology.…”

“…[ 13 ] High‐throughput mass spectrometry based assay for large scale profiling of human circulating Apo, requiring small sampling has shown reproducibility and comparable results with those obtained with other techniques. [ 14 ]…”