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Development of a capillary electrophoresis coupled to mass spectrometry pipeline for comparable assessment of the plasma peptidome

ABSTRACT: Although capillary electrophoresis coupled to mass spectrometry (CE-MS) holds promise for urinary peptide profiling, only a limited number of studies have used CE-MS to study plasma peptides. Here we describe the establishment of a workflow, including sample preparation, CE-MS and MS/MS analyses, data processing and normalisation optimized for the analysis of plasma peptides. Using 293 plasma samples from 136 patients with end stage kidney failure (including pre- and post-dialysis samples) and 22 patients with chronic kidney disease, we identified and quantified the abundance of 3920 unique plasma peptides. The repeatability and intermediate precision of the analysis were high (with a coefficient of variation of 15% on average for all peptides). 644 out of 3920 peptides were sequenced by CE-MS/MS. These peptide fragments belonged to 129 parent proteins that were also found in Human Plasma PeptideAtlas. Using the pipeline, we identified 188 sequenced plasma peptides with different plasma abundance before and after dialysis. These peptides combined in a support vector machine (SVM) classifier successfully discriminated between pre- and post- dialysis samples in a blinded validation cohort of 45 dialysis patients. Enriched peptides after dialyses were for the major part associated to inflammation. In conclusion, this high-throughput strategy could contribute to address the need for reliable plasma biomarkers of disease.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Plasma

SUBMITTER: Carine Froment

LAB HEAD: Joost-Peter Schanstra

PROVIDER: PXD056224 | Pride | 2025-05-16

REPOSITORIES: Pride

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Development and Validation of a Capillary Electrophoresis Coupled to Mass Spectrometry Pipeline for Comparable Assessment of the Plasma Peptidome.

Fernandez Lucie L Breuil Benjamin B Froment Carine C Seye Mouhamed M Sylla Babacar B Estanco Marina M Chaubet Adeline A Delecroix Eléonore E Chaoui Karima K Vu Jeanne Pierrette JP Ardeleanu Serban S Faguer Stanislas S Burlet-Schiltz Odile O Buffin-Meyer Bénédicte B Schanstra Joost P JP Klein Julie J

Proteomics 20250316 11-12

Although capillary electrophoresis coupled to mass spectrometry (CE-MS) holds promise for urinary peptide profiling, only a limited number of studies have used CE-MS to study plasma peptides. Here we describe the establishment of a workflow, including sample preparation, CE-MS analysis, data processing and normalization optimized for the analysis of plasma peptides. Using 291 plasma samples from 136 patients with end stage kidney failure (including pre- and post-dialysis samples) and 20 patients ...[more]

PMID: 40091299

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Project description:Background. Chronic renal failure is characterized by progressive renal scarring and accelerated cardiovascular disease. In animal models, this is thought to be due to non-dialyzable uremic toxins M-bM-^@M-^S small, protein-bound molecules normally secreted via Organic Anion Transporters (OATs) in the proximal renal tubule,rather than filtered at the glomerulus. The best studied of these is indoxyl sulfate (IS). Methods. We examined global gene expression responses in cultured normal human renal tubular cells incubated with control plasma (n=5) or pre- and post-dialysis uremic plasma (n=10). Results. GeneSpring analysis of microarrays revealed significantly altered expression of 2016 genes. The expression of 537 genes M-bM-^@M-^\normalizedM-bM-^@M-^] in post-dialysis plasma, suggesting removal of M-bM-^@M-^\low molecular weight uremic toxinsM-bM-^@M-^] by dialysis. The expression of the majority of dysregulated genes (1479) was not normalized in post-dialysis plasma. These likely represent the effects of substances not effectively removed by dialysis (M-bM-^@M-^\protein-bound uremic toxinsM-bM-^@M-^]). Addition of indoxyl sulfateto control plasma simulated most effects (81.1%) of uremic plasma, while the addition of probenecid, an OAT inhibitor, to uremic plasma reversed most changes in gene expression. Analysis of molecular programs with the Gene Set Enrichment Analysis and the DAVID database revealed patterns common to indoxyl sulfate-treated control plasma, pre-dialysis, and post-dialysis uremic plasma. These included increased cell cycle, pro-inflammatory, and pro-fibrotic molecular programs, particularly genes in the TGF-M-NM-2 pathway. Conclusion: These findings provide insight into the role of non-dialyzable, protein-bound uremic toxins in the pathogenesis of renal scarring and uremic vasculopathy. The GSEA patterns confirm that inflammatory and fibrotic programs are active. Transcriptomic remodeling in response to external stimulus RNA from non-immortalized cultured renal cortical cells incubated with plasma samples from 5 controls, 10 uremics pre-dialysis, 10 uremics post-dialysis, 5 controls with indoxyl sulfate added, 5 uremics pre-dialysis with probenecid added, 5 uremics post-dialysis with indoxyl sulfate added

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	EXP_DESIGN.xlsx	Xlsx
	OFBGB230207_10.raw	Raw
	OFBGB230207_10_C96-4-12.msf	Msf
	OFBGB230207_12.raw	Raw
	OFBGB230207_12_C94-1-1.msf	Msf