Large-Scale Analysis of Apolipoprotein CIII Glycosylation by Ultrahigh Resolution Mass Spectrometry.

Daniel Demus,Viktoria Dotz,Bas C Jansen,Eric J G Sijbrands,Marco R Bladergroen,Jan Nouta,Simone Nicolardi,Annemieke Naber,Manfred Wuhrer,Mandy Van Hoek

doi:10.3389/fchem.2021.678883

Abstract

Apolipoprotein-CIII (apo-CIII) is a glycoprotein involved in lipid metabolism and its levels are associated with cardiovascular disease risk. Apo-CIII sialylation is associated with improved plasma triglyceride levels and its glycosylation may have an effect on the clearance of triglyceride-rich lipoproteins by directing these particles to different metabolic pathways. Large-scale sample cohort studies are required to fully elucidate the role of apo-CIII glycosylation in lipid metabolism and associated cardiovascular disease. In this study, we revisited a high-throughput workflow for the analysis of intact apo-CIII by ultrahigh-resolution MALDI FT-ICR MS. The workflow includes a chemical oxidation step to reduce methionine oxidation heterogeneity and spectrum complexity. Sinapinic acid matrix was used to minimize the loss of sialic acids upon MALDI. MassyTools software was used to standardize and automate MS data processing and quality control. This method was applied on 771 plasma samples from individuals without diabetes allowing for an evaluation of the expression levels of apo-CIII glycoforms against a panel of lipid biomarkers demonstrating the validity of the method. Our study supports the hypothesis that triglyceride clearance may be regulated, or at least strongly influenced by apo-CIII sialylation. Interestingly, the association of apo-CIII glycoforms with triglyceride levels was found to be largely independent of body mass index. Due to its precision and throughput, the new workflow will allow studying the role of apo-CIII in the regulation of lipid metabolism in various disease settings.

Highlights

Lipid metabolism is regulated by complex biological mechanisms in which apolipoproteins – proteins embedded in lipoprotein particles – modulate the transport and availability of blood lipids (Mahley et al, 1984)
We have developed a high-throughput method based on magnetic-bead extraction and matrix-assisted laser desorption/ ionization (MALDI) and ultrahigh-resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) for the analysis of serum apo-CIII proteoforms (Nicolardi et al, 2013a; Nicolardi et al, 2013b)
A common event observed in proteomics studies is the oxidation of methionine residues due to biological and pathological processes occurring in vivo (Stadtman et al, 2003), sample storage and processing (Borges et al, 2014)

Summary

Introduction

Lipid metabolism is regulated by complex biological mechanisms in which apolipoproteins – proteins embedded in lipoprotein particles – modulate the transport and availability of blood lipids (Mahley et al, 1984). It has been shown that the levels of apo-CIII and the specific glycoforms and their relative expression control triglyceride metabolism (Yassine et al, 2015; Koska et al, 2016). It has been shown that sialylation modulates the apo-CIII affinity for hepatic receptors that clear lipoprotein particles (Kegulian et al, 2019) and that different proteoforms of apoCIII may affect the inhibition of LPL (Holleboom et al, 2011) and the interaction of LDL with the vascular wall (Hiukka et al, 2009). Since the association of different apo-CIII proteoforms with specific cardiometabolic endpoints has not been fully elucidated, further research in large sample cohorts is warranted

Methods

Results

Conclusion