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.

Keywords: Apolipoprotein C-I, C-II, C-III; Biomarkers; Mass spectrometric immunoassay; Proteoforms; Quantification.

Figure 1

Mass spectra resulting from multiplex MSIA of apoC-I, apoC-II and apoC-III utilizing lysozyme as an IRS. Labeled on the mass spectra are the major C apolipoprotein proteoforms. The inlets represent mass spectra obtained from samples where the specific proteoforms were more prominent.

Figure 2

Quantitative apoC-I, apoC-II and apoC-III mass spectrometric immunoassay (MSIA). a) Representative MSIA mass spectra for the six standard apoCs samples containing lysozyme as an IRS; b) Representative standard curves generated with the multiplex apoCs MSIA, with an r²= 0.9988 and SEE = 0.0928 for apoC-I; r² = 0.9940 and SEE = 0.0262 for apoC-II; and r² = 0.9996 and SEE = 0.1605 for apoC-III.

Figure 3

Individual concentrations of apolipoprotein C-I, C-II and C-III and their proteoforms in a cohort of 82 human EDTA-plasma samples. Box - 25–75^th percentile; Solid line – median concentration; Short dash line – mean concentration; Error bars – 10^th and 90^th percentile; Symbols – outlying points.