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Multiplexed MRM-based protein quantification of putative prognostic biomarkers for chronic kidney disease progression in plasma.

ABSTRACT: Current diagnostic measures for Chronic Kidney Disease (CKD) include detection of reduced estimated glomerular filtration rate (eGFR) and albuminuria, which have suboptimal accuracies in predicting disease progression. The disease complexity and heterogeneity underscore the need for multiplex quantification of different markers. The goal of this study was to determine the association of six previously reported CKD-associated plasma proteins [B2M (Beta-2-microglobulin), SERPINF1 (Pigment epithelium-derived factor), AMBP (Protein AMBP), LYZ (Lysozyme C), HBB (Hemoglobin subunit beta) and IGHA1 (Immunoglobulin heavy constant alpha 1)], as measured in a multiplex format, with kidney function, and outcome. Antibody-free, multiple reaction monitoring mass spectrometry (MRM) assays were developed, characterized for their analytical performance, and used for the analysis of 72 plasma samples from a patient cohort with longitudinal follow-up. The MRM significantly correlated (Rho?=?0.5-0.9) with results from respective ELISA. Five proteins [AMBP, B2M, LYZ, HBB and SERPINF1] were significantly associated with eGFR, with the three former also associated with unfavorable outcome. The combination of these markers provided stronger associations with outcome (p?

SUBMITTER: Makridakis M

PROVIDER: S-EPMC7076027 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Multiplexed MRM-based protein quantification of putative prognostic biomarkers for chronic kidney disease progression in plasma.

Makridakis Manousos M Kontostathi Georgia G Petra Eleni E Stroggilos Rafael R Lygirou Vasiliki V Filip Szymon S Duranton Flore F Mischak Harald H Argiles Angel A Zoidakis Jerome J Vlahou Antonia A

Scientific reports 20200316 1

Current diagnostic measures for Chronic Kidney Disease (CKD) include detection of reduced estimated glomerular filtration rate (eGFR) and albuminuria, which have suboptimal accuracies in predicting disease progression. The disease complexity and heterogeneity underscore the need for multiplex quantification of different markers. The goal of this study was to determine the association of six previously reported CKD-associated plasma proteins [B2M (Beta-2-microglobulin), SERPINF1 (Pigment epitheli ...[more]

PMID: 32179759

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Project description:BackgroundType 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.MethodsThe 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.ResultsA 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%.ConclusionsTargeted 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.

Project description:Exercise is extremely beneficial to whole body health reducing the risk of a number of chronic human diseases. Some of these physiological benefits appear to be mediated via the secretion of peptide/protein hormones into the blood stream. The plasma peptidome contains the entire complement of low molecular weight endogenous peptides derived from secretion, protease activity and PTMs, and is a rich source of hormones. In the current study we have quantified the effects of intense exercise on the plasma peptidome to identify novel exercise regulated secretory factors in humans. We developed an optimized 2D-LC-MS/MS method and used multiple fragmentation methods including HCD and EThcD to analyze endogenous peptides. This resulted in quantification of 5,548 unique peptides during a time course of exercise and recovery. The plasma peptidome underwent dynamic and large changes during exercise on a time-scale of minutes with many rapidly reversible following exercise cessation. Among acutely regulated peptides, many were known hormones including insulin, glucagon, ghrelin, bradykinin, cholecystokinin and secretogranins validating the method. Prediction of bioactive peptides regulated with exercise identified C-terminal peptides from Transgelins, which were increased in plasma during exercise. In vitro experiments using synthetic peptides identified a role for transgelin peptides on the regulation of cell-cycle, extracellular matrix remodeling and cell migration. We investigated the effects of exercise on the regulation of PTMs and proteolytic processing by building a site-specific network of protease/substrate activity. Collectively, our deep peptidomic analysis of plasma revealed that exercise rapidly modulates the circulation of hundreds of bioactive peptides through a network of proteases and PTMs. These findings illustrate that peptidomics is an ideal method for quantifying changes in circulating factors on a global scale in response to physiological perturbations such as exercise. This will likely be a key method for pinpointing exercise regulated factors that generate health benefits.

Dataset Information

Multiplexed MRM-based protein quantification of putative prognostic biomarkers for chronic kidney disease progression in plasma.

Publications

Multiplexed MRM-based protein quantification of putative prognostic biomarkers for chronic kidney disease progression in plasma.

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