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Open Access 01-12-2024 | Diabetes | Research

Multiplexed MRM-based proteomics for identification of circulating proteins as biomarkers of cardiovascular damage progression associated with diabetes mellitus

Authors: Francesco Piarulli, Cristina Banfi, Eugenio Ragazzi, Erica Gianazza, Marco Munno, Massimo Carollo, Pietro Traldi, Annunziata Lapolla, Giovanni Sartore

Published in: Cardiovascular Diabetology | Issue 1/2024

Abstract

Background

Type 2 diabetes mellitus (T2DM) increases the risk of coronary heart disease (CHD) by 2–4 fold, and is associated with endothelial dysfunction, dyslipidaemia, insulin resistance, and chronic hyperglycaemia. The aim of this investigation was to assess, by a multimarker mass spectrometry approach, the predictive role of circulating proteins as biomarkers of cardiovascular damage progression associated with diabetes mellitus.

Methods

The study considered 34 patients with both T2DM and CHD, 31 patients with T2DM and without CHD, and 30 patients without diabetes with a diagnosis of CHD. Plasma samples of subjects were analysed through a multiplexed targeted liquid chromatography mass spectrometry (LC-MS)-based assay, namely Multiple Reaction Monitoring (MRM), allowing the simultaneous detection of peptides derived from a protein of interest. Gene Ontology (GO) Analysis was employed to identify enriched GO terms in the biological process, molecular function, or cellular component categories. Non-parametric multivariate methods were used to classify samples from patients and evaluate the relevance of the analysed proteins’ panel.

Results

A total of 81 proteins were successfully quantified in the human plasma samples. Gene Ontology analysis assessed terms related to blood microparticles, extracellular exosomes and collagen-containing extracellular matrix. Preliminary evaluation using analysis of variance (ANOVA) of the differences in the proteomic profile among patient groups identified 13 out of the 81 proteins as significantly different. Multivariate analysis, including cluster analysis and principal component analysis, identified relevant grouping of the 13 proteins. The first main cluster comprises apolipoprotein C-III, apolipoprotein C-II, apolipoprotein A-IV, retinol-binding protein 4, lysozyme C and cystatin-C; the second one includes, albeit with sub-grouping, alpha 2 macroglobulin, afamin, kininogen 1, vitronectin, vitamin K-dependent protein S, complement factor B and mannan-binding lectin serine protease 2. Receiver operating characteristic (ROC) curves obtained with the 13 selected proteins using a nominal logistic regression indicated a significant overall distinction (p < 0.001) among the three groups of subjects, with area under the ROC curve (AUC) ranging 0.91–0.97, and sensitivity and specificity ranging from 85 to 100%.

Conclusions

Targeted mass spectrometry approach indicated 13 multiple circulating proteins as possible biomarkers of cardiovascular damage progression associated with T2DM, with excellent classification results in terms of sensitivity and specificity.

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Title: Multiplexed MRM-based proteomics for identification of circulating proteins as biomarkers of cardiovascular damage progression associated with diabetes mellitus
Authors: Francesco Piarulli
Cristina Banfi
Eugenio Ragazzi
Erica Gianazza
Marco Munno
Massimo Carollo
Pietro Traldi
Annunziata Lapolla
Giovanni Sartore
Publication date: 01-12-2024
Publisher: BioMed Central
Keywords: Diabetes
Coronary Heart Disease
Biomarkers
Published in: Cardiovascular Diabetology / Issue 1/2024
Electronic ISSN: 1475-2840
DOI: https://doi.org/10.1186/s12933-024-02125-1

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