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

• Proteoforms play important role in physiological and pathological processes.
• Top-down MS-based approaches are ideally suited for proteoforms detection.
• We developed multiplex MS- immunoassay for apoC-I, C-II and C-III and their proteoforms.
• The multiplexed assay was fast (40 min) and high-throughput (96 samples at once).
• The concentrations of each proteoform were determined and compared in a cohort of 82 human plasma samples.

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.

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