Naturally occurring fragments of collagen type I alpha 1 chain (COL1A1) have been previously associated with chronic kidney disease (CKD), with some fragments showing positive and others negative associations. Using urinary peptidome data from healthy individuals (n = 1131) and CKD patients (n = 5585) this aspect was investigated in detail. Based on the hypothesis that many collagen peptides are derived not from the full, mature collagen molecule, but from (larger) collagen degradation products, relationships between COL1A1 peptides containing identical sequences were investigated, with the smaller (offspring) peptide being a possible degradation product of the larger (parent) one. The strongest correlations were found for relationships where the parent differed by a maximum of three amino acids from the offspring, indicating an exopeptidase‐regulated stepwise degradation process. Regression analysis indicated that CKD affects this degradation process. A comparison of matched CKD patients and control individuals (n = 612 each) showed that peptides at the start of the degradation process were consistently downregulated in CKD, indicating an attenuation of COL1A1 endopeptidase‐mediated degradation. However, as these peptides undergo further degradation, likely mediated by exopeptidases, this downregulation can become less significant or even reverse, leading to an upregulation of later‐stage fragments and potentially explaining the inconsistencies observed in previous studies.

Previous studies have established the association of sex with gene and protein expression. This study investigated the association of sex with the abundance of endogenous urinary peptides, using capillary electrophoresis‐coupled to mass spectrometry (CE‐MS) datasets from 2008 healthy individuals and patients with type II diabetes, divided in one discovery and two validation cohorts. Statistical analysis using the Mann–Whitney test, adjusted for multiple testing, revealed 143 sex‐associated peptides in the discovery cohort. Of these, 90 peptides were associated with sex in at least one of the validation cohorts and showed agreement in their regulation trends across all cohorts. The 90 sex‐associated peptides were fragments of 29 parental proteins. Comparison with previously published transcriptomics data demonstrated that the genes encoding 16 of these parental proteins had sex‐biased expression. The 143 sex‐associated peptides were combined into a support vector machine‐based classifier that could discriminate males from females in two independent sets of healthy individuals and patients with type II diabetes, with an AUC of 89% and 81%, respectively. Collectively, the urinary peptidome contains multiple sex‐associated differences, which may enable a better understanding of sex‐biased molecular mechanisms and the development of more accurate diagnostic, prognostic, or predictive classifiers for each individual sex.

Collagen type I (COL(I)) is a key component of the extracellular matrix (ECM) and is involved in cell signaling and migration through cell receptors. Collagen degradation produces bioactive peptides (matrikines), which influence cellular processes. In this study, we investigated the biological effects of nine most abundant, naturally occurring urinary COL(I)-derived peptides on human endothelial cells at physiological concentrations, using cell migration assays, mass spectrometry-based proteomics, flow cytometry, and AlphaFold 3. While none of the peptides significantly altered endothelial migration by themselves at physiological concentrations, full-length COL(I) increased cell migration, which was inhibited by Peptide 1 (²²⁹NGDDGEAGKPGRPGERGPpGp²⁴⁹). This peptide uniquely contains the DGEA and GRPGER motifs, interacting with integrin α2β1. Flow cytometry confirmed the presence of integrin α2β1 on human endothelial cells, and AlphaFold 3 modeling predicted an interaction between Peptide 1 and integrin α2. Mass spectrometry-based proteomics investigating signaling pathways revealed that COL(I) triggered phosphorylation events linked to integrin α2β1 activation and cell migration, which were absent in COL(I) plus peptide 1-treated cells. These findings identify Peptide 1 as a biologically active COL(I)-derived peptide at a physiological concentration capable of modulating collagen-induced cell migration, and provide a foundation for further investigation into its mechanisms of action and role in urine excretion.

Background and Aims Chronic kidney disease (CKD) significantly contributes to global morbidity and mortality. Early, targeted intervention offers an ideal strategy for mitigating this burden. Peptidomic changes inform on CKD onset and progression and hold insights for treatment strategies. We investigated the molecular effects of six different therapeutic interventions in silico in all possible combinations on the urine peptidome, aiming to identify the most beneficial treatment for individual patients. Method This study predicted major adverse kidney events (MAKE), defined as a ≥40% decline in estimated glomerular filtration rate (eGFR) or kidney failure (median follow up time of 1.50 (95%CI 0.35, 5.0)), using the urinary peptidomic classifier CKD273 in a retrospective cohort of 935 participants. The impact of various treatments on urinary peptidomic profiles, assessed from previous studies of four different drug-based interventions (MRA, SGLT2i, GLP1-RA and ARB), one dietary intervention (olive oil) or from exercise, was applied. Treatment effects were quantified through fold changes in peptide abundance after treatment, recalibrated to align with outcomes observed in randomized controlled trials and applied to patient-specific urinary profiles, simulating intervention effects and recalculation of CKD273 scores for each individual. For combination treatments, the effects of multiple interventions were combined to model their cumulative impact. Results Simulated interventions demonstrated a significant reduction in median CKD273 scores, from 0.57 (IQR: 0.19–0.81) before to 0.039 (IQR: -0.192–0.363) after intervention (paired Wilcoxon test, P < 0.0001), when the most beneficial treatment or combination of treatments was applied individually. The combination of all available treatments was found optimal only for 17.6% of the patients and not the most frequently predicted optimal intervention. Patients with higher baseline CKD273 scores required more complex intervention combinations to achieve the greatest reduction in scores. The findings present potential individualized treatment strategies in CKD management. Conclusion This study supports the feasibility of in silico predicting effects of therapeutic interventions on CKD progression. By identifying the most beneficial treatment combinations for individual patients, this approach paves the way for precision medicine in CKD. Urinary peptidomics data from different types of intervention were applied in silico to predict the impact of treatment, with the extent of the impact being calibrated to fit the observed effect in large randomized trials. The personalized optimal combination of interventions was determined in 935 patients, based on the impact on CKD273. The results indicate that guiding personalized treatment based on urine peptide data is possible and may substantially improve patient management.

Proteins and peptides indicate physiological or pathological states and are investigated to identify markers to scrutinize health and disease surveillance. Saliva contains many proteins and peptides that could serve as biomarkers, offering a potential noninvasive approach for disease detection. To enable the assessment of the saliva proteome and peptidome as sources of biomarkers, protocols for sampling, sample preparation, and measurements have to be developed. We present the results of peptidome analysis from saliva samples collected at different time points before and after breakfast from 14 healthy adults (50% male, mean age 42.7 ± 10.3 years). While similar methods have been previously applied to urine, our aim was to adapt and demonstrate the effectiveness of these protocols for saliva. Specifically, we aimed to establish a salivary peptide dataspace, including peptide amino acid sequences, and to evaluate the impact of food intake and time of sampling. Capillary electrophoresis‐mass spectrometry (CE‐MS) and CE‐MS/MS were used for peptidome analysis. Per sample, 3147 ± 559 peptides were detectable, without significant differences in the number of detected peptides between sample collection times. However, some peptides differed significantly in their abundance between samples collected before breakfast and 1, 2 and 4 h after breakfast. Samples collected after breakfast were more consistent in their peptide content. Sequencing identified 630 peptides, fragments of 82 proteins, with the majority derived from proline‐rich proteins. The data indicate that saliva for peptidomics is best collected 1 to 4 h after breakfast.

Collagen type I (COL(I)) is a key component of the extracellular matrix (ECM) and is involved in cell signalling and migration through cell receptors. Collagen degradation produces bioactive peptides (matrikines), which influence cellular processes. In this study, we investigated the biological effects of nine most abundant, naturally occurring urinary COL(I)-derived peptides on human endothelial cells at physiological concentrations, using cell migration assays, mass spectrometry-based proteomics, flow cytometry, and AlphaFold3. While none of the peptides significantly altered endothelial migration by themselves at physiological concentrations, full-length COL(I) increased cell migration, which was inhibited by Peptide 1 ( ²²⁹ NGDDGEAGKPGRPGERGPpGp ²⁴⁹ ). This peptide uniquely contains the DGEA and GRPGER motifs, interacting with integrin α2β1. Flow cytometry confirmed the presence of integrin α2β1 on human endothelial cells, and AlphaFold3 modelling predicted an interaction between Peptide 1 and integrin α2. Mass spectrometry-based proteomics investigating signalling pathways revealed that COL(I) triggered phosphorylation events linked to integrin α2β1 activation and cell migration, which were absent in COL(I) plus peptide 1-treated cells. These findings identify Peptide 1 as a biologically active COL(I)-derived peptide at a physiological concentration capable of modulating collagen-induced cell migration, and provide a foundation for further investigation into its mechanisms of action and role in urine excretion.