Development of multiplex mass spectrometric immunoassay for detection and quantification of apolipoproteins C-I, C-II, C-III and their proteoforms

Abstract

The impetus for discovery and evaluation of protein biomarkers has been accelerated by recent development of advanced technologies for rapid and broad proteome analyses. Mass spectrometry (MS)-based protein assays hold great potential for in vitro biomarker studies. Described here is the development of a multiplex Mass Spectrometric Immunoassay (MSIA) for quantification of apolipoprotein C-I (apoC-I), apolipoprotein C-II (apoC-II), apolipoprotein C-III (apoC-III) and their proteoforms. The multiplex MSIA assay was fast (~40 min) and high-throughput (96 samples at a time). The assay was applied to a small cohort of human plasma samples, revealing the existence of multiple proteoforms for each apolipoprotein C. The quantitative aspect of the assay enabled determination of the concentration for each proteoform individually. Low-abundance proteoforms, such as fucosylated apoC-III, were detected in less than 20% of the samples. The distribution of apoC-III proteoforms varied among samples with similar total apoC-III concentrations. The multiplex analysis of the three apolipoproteins C and their proteoforms using quantitative MSIA represents a significant step forward toward better understanding of their physiological roles in health and disease.

Disialylated apolipoprotein C-III proteoform is associated with improved lipids in prediabetes and type 2 diabetes

“…Samples for these analyses were stored for several years at 80°C before MSIA was performed. Previous investigation of the effects of storage, time, and freeze/thaw cycles on these assays has indicated that the measurements are relatively stable (27). Moreover, despite differences in the storage time of the samples in the two different cohorts, there was a striking consistency in the relationships between apoC-III proteoforms and plasma lipids.…”

“…apoC-III proteoforms were measured by MSIA, as previously described (27). After rapid thawing on ice, samples were centrifuged for 5 min at 3,000 rpm and then aliquoted into 96-well microplates and stored at 80°C until measured.…”

“…We recently developed a high throughput apoC mass spectrometric immunoassay (MSIA) (27) that can simultaneously measure both major and minor proteoforms of apoC-III, and applied it to a small group of obese youth participants without type 2 diabetes (28). Our results revealed a strong inverse association between the ratio of apoC-III 2 to the most abundant apoC-III 1 and fasting plasma triglycerides.…”

Automated Multiplex LC-MS/MS Assay for Quantifying Serum Apolipoproteins A-I, B, C-I, C-II, C-III, and E with Qualitative Apolipoprotein E Phenotyping

“…In addition, the lower concentrations on the basis of the quantification peptide TPDVSSALDK for apoC-I could be the result of the occurrence of des-TP apoC-I, a cleavage product of dipeptidyl-peptidase IV, resulting in the peptide DVSSALDK. Trenchevska et al reported that the des-TP apoC-I proteoform is present in human plasma at approximately 25% of the total apoC-I concentration (37 ). For future clinical studies, samples with between-peptide inconsistencies should be reevaluated or reanalyzed to confirm the most representative peptide for quantification.…”

The Analysis of Human Serum Albumin Proteoforms Using Compositional Framework

“…When a convenient reference standard exists, it may be included in the MSIA assay [19,27]. The reference standard is used to determine the absolute concentrations of the proteoforms from a calibration curve.…”