Proteomics in cardiovascular disease: recent progress and clinical implication and implementation

Introduction: Although multiple efforts have been initiated to shed light into the molecular mechanisms underlying cardiovascular disease, it still remains one of the major causes of death worldwide. Proteomic approaches are unequivocally powerful tools that may provide deeper understanding into the molecular mechanisms associated with cardiovascular disease and improve its management. Areas covered: Cardiovascular proteomics is an emerging field and significant progress has been made during the past few years with the aim of defining novel candidate biomarkers and obtaining insight into molecular pathophysiology. To summarize the recent progress in the field, a literature search was conducted in PubMed and Web of Science. As a result, 704 studies from PubMed and 320 studies from Web of Science were retrieved. Findings from original research articles using proteomics technologies for the discovery of biomarkers for cardiovascular disease in human are summarized in this review. Expert commentary: Proteins associated with cardiovascular disease represent pathways in inflammation, wound healing and coagulation, proteolysis and extracellular matrix organization, handling of cholesterol and LDL. Future research in the field should target to increase proteome coverage as well as integrate proteomics with other omics data to facilitate both drug development as well as clinical implementation of findings

Ανακάλυψη πρωτεϊνών σχετικών με την παθολογία του καρκίνου του τραχήλου της μήτρας

Ο καρκίνος του τραχήλου της μήτρας αποτελεί την τέταρτη συχνότερη και πιο θανατηφόρα μορφή καρκίνου στις γυναίκες, παγκοσμίως. Ο εμβολιασμός και ο προληπτικός έλεγχος έχουν μειώσει τα περιστατικά όμως η ασθένεια ευθύνεται για σημαντικό αριθμό θανάτων ενώ η διαθέσιμες θεραπευτικές μέθοδοι επηρεάζουν τη γονιμότητα των ασθενών. Σκοπός της διδακτορικής αυτής διατριβής αποτελεί η χρήση τεχνικών πρωτεωμικής σε συνδυασμό με τη βιοπληροφορική ανάλυση και την ανασκόπηση της βιβλιογραφίας για την ανακάλυψη νέων πρωτεϊνών που σχετίζονται με την παθολογία του καρκίνου του τραχήλου της μήτρας με στόχο την καλύτερη κατανόηση των μοριακών μηχανισμών που διέπουν το σχηματισμό των δυσπλασιών. Για την επίτευξη του στόχου, χρησιμοποιήθηκαν τέσσερις κυτταρικές σειρές, αντιπροσωπευτικές των συχνότερων μολύνσεων από HPV (HeLa - HPV 18, SiHa - HPV 16), του καρκίνου του τραχήλου της μήτρας χωρίς μόλυνση από HPV (C33A), καθώς και φυσιολογικών κερατινοκυττάρων του τραχήλου της μήτρας (HCK1T). Η ανάλυση των πρωτεϊνικών εκχυλισμάτων πραγματοποιήθηκε με τη μέθοδο GeLC-MS/MS και προσέφερε ικανοποιητικό αριθμό αναγνωρίσεων (2.500-3.500 πρωτεΐνες ανά κυτταρική σειρά) με πολύ καλή αναπαραγωγιμότητα. Από τη σύγκριση της κάθε κυτταρικής σειράς καρκίνου του τραχήλου της μήτρας με τη τα φυσιολογικά κερατινοκύτταρα προέκυψαν ~800-1.400 διαφορικά εκφραζόμενες πρωτεΐνες, ανά σύγκριση, οι οποίες παρέχουν χρήσιμη πληροφορία για τις μεταβολές στη ρύθμιση των σηματοδοτικών μονοπατιών και των σημαντικών μορίων στον καρκίνο του τραχήλου της μήτρας. Η βιοπληροφορική ανάλυση των διαφορικά εκφραζόμενων πρωτεϊνών επιβεβαίωσε την ειδικότητα των αναγνωρίσεων και την ακρίβεια των μετρήσεων καθώς τα μοριακά μονοπάτια που εμφάνισαν αλλαγή στη ρύθμισή τους σχετίζονται με τις υψηλές ενεργειακές ανάγκες και τον αυξημένο κυτταρικό κύκλο που είναι γνωστό ότι υφίστανται στα καρκινικά κύτταρα. Για τη διαλογή πρωτεϊνών ενδιαφέροντος για περαιτέρω διερεύνηση επιλέχθηκαν οι 105 διαφορικά εκφραζόμενες πρωτεΐνες που εμφάνισαν κοινή τάση έκφρασης στις τρεις συγκρίσεις (HeLa/HCK1T, SiHa/CK1T και C33A/HCK1T) και μελετήθηκαν ενδελεχώς στη βιβλιογραφία. Οι πρωτεΐνες που δεν είχαν μελετηθεί προηγουμένως στον καρκίνο του τραχήλου της μήτρας αλλά υπάρχουν δεδομένα για αυτές σε άλλους τύπους καρκίνων με παρόμοια τάση έκφρασης όπως και στην παρούσα μελέτη, είναι συνολικά 21 και αποτελούν τη βάση για περαιτέρω μελέτη και πειράματα. Μάλιστα, μετά από βαθύτερη διερεύνηση, επιλέχθηκαν 7 από αυτές (LIM domain and actin-binding protein 1, Importin subunit alpha-1, Serum paraoxonase/arylesterase 2, Elongation factor 1-alpha 2, Caprin-1, Mitochondrial import receptor subunit TOM34, Ras GTPase-activating protein-binding protein 1) για πειράματα ανοσοϊστοχημείας σε δείγματα τραχήλου από υγιείς και ασθενείς με προκαρκινική δυσπλασία χαμηλής και υψηλής διαφοροποίησης καθώς και με και καρκίνο του τραχήλου της μήτρας, ως συνέχεια της παρούσας διδακτορικής διατριβής. Ακόμη, για τις πρωτεΐνες LIM domain and actin-binding protein 1, Importin subunit alpha-1 και Ras GTPase-activating protein-binding protein 1 έχουν σχεδιαστεί και πειράματα λειτουργικής διερεύνησης. Eν κατακλείδι, αποτελέσματα της παρούσας διδακτορικής διατριβής παρέχουν νέες γνώσεις για τους μοριακούς μηχανισμούς και τους βασικούς ρυθμιστές της καρκινογένεσης του τραχήλου της μήτρας και μπορούν να αποτελέσουν τη βάση για περαιτέρω έρευνες και προσεγγίσεις της βιολογίας συστημάτων και να συμβάλουν στην ανακάλυψη βιοδεικτών και στην αναγνώριση νέων φαρμακευτικών στόχων.Cervical cancer is the fourth most common and most lethal type of cancer in women, worldwide. Profylactic vaccination and regular screening have marked a decline on incidents, however the disease still accounts for a significant number of deaths and the available therapeutic approaches affect patients’ fertility. The purpose of this doctoral dissertation is the use of proteomic techniques in combination with bioinformatics and literature mining for the discovery of novel proteins involved in cervical cancer pathology with the aim of better understanding of the molecular mechanisms underlying the malignancies formation. To approach the goal, four cell lines, representative of the most common HPV infections (HeLa - HPV 18, SiHa - HPV 16), HPV-free cervical cancer (C33A) as well as normal cervical keratinocytes (HCK1T) were used. Analysis of the protein extracts was performed based on the GeLC-MS/MS protocol and provided a satisfactory number of protein identifications (2,500-3,500 proteins per cell line) with a very good reproducibility. The differentially expressed proteins that resulted from the comparison of each cervical cancer cell line with the normal keratinocytes were ~800-1,400 per comparison, and they provide useful information on the deregulation of signaling pathways and important molecules in cervical cancer. Bioinformatics analysis of the differentially expressed proteins validated the specificity of the identifications and the accuracy of the measurements as the deregulated molecular pathways are associated with the high metabolic demands and increased cell cycle that are known to occur in cancer cells. For the shortlisting of interesting proteins for further investigation, 105 differentially expressed proteins that followed the same expression trend in the three comparisons (HeLa/HCK1T, SiHa/CK1T και C33A/HCK1T) were chosen and extensively investigated in the literature. The proteins that were not studied in the context of cervical cancer before but have data on other types of cancers with similar expression trend as in the present study, are in total 21 and constitute the basis for further investigation and experiments. In addition, after a deeper investigation, 7 of these proteins were chosen (LIM domain and actin-binding protein 1, Importin subunit alpha-1, Serum paraoxonase/arylesterase 2, Elongation factor 1-alpha 2, Caprin-1, Mitochondrial import receptor subunit TOM34, Ras GTPase-activating protein-binding protein 1) for immunohistochemistry experiments on cervical specimens from healthy participants and patients with low and high grade squamous intraepithelial lesions and cervical cancer, as a next step to the present doctoral dissertation. Furthermore, functional investigation experiments for the proteins LIM domain and actin-binding protein 1, Importin subunit alpha-1 και Ras GTPase-activating protein-binding protein have been designed. In conclusion, the results of this doctoral dissertationprovide novel knowledge on the molecular mechanisms and the key regulators of cervical carcinogenesis and can be the basis for further investigations through systems biology approaches and contribute in the discovery of biomarkers and the identification of novel therapeutic targets

One More Bottleneck towards Biomarker Validation and Clinical Implementation

ELISA is the main approach for the sensitive quantification of protein biomarkers in body fluids and is currently employed in clinical laboratories for the measurement of clinical markers. As such, it also constitutes the main methodological approach for biomarker validation and further qualification. For the latter, specific assay performance requirements have to be met, as described in respective guidelines of regulatory agencies. Even though many clinical ELISA assays in serum are regularly used, ELISA clinical applications in urine are significantly less. The scope of our study was to evaluate ELISA assay analytical performance in urine for a series of potential biomarkers for bladder cancer, as a first step towards their large scale clinical validation. Seven biomarkers (Secreted protein acidic and rich in cysteine, Survivin, Slit homolog 2 protein, NRC-Interacting Factor 1, Histone 2B, Proteinase-3 and Profilin-1) previously described in the literature as having differential expression in bladder cancer were included in the study. A total of 11 commercially available ELISA tests for these markers were tested by standard curve analysis, assay reproducibility, linearity and spiking experiments. The results show disappointing performance with coefficients of variation>20% for the vast majority of the tests performed. Only 3 assays (for Secreted protein acidic and rich in cysteine, Survivin and Slit homolog 2 protein) passed the accuracy thresholds and were found suitable for further application in marker quantification. These results collectively reflect the difficulties in developing urine-based ELISA assays of sufficient analytical performance for clinical application, presumably attributed to the urine matrix itself and/or presence of markers in various isoforms

Plasma proteomic analysis reveals altered protein abundances in cardiovascular disease

Background: Cardiovascular disease (CVD) describes the pathological conditions of the heart and blood vessels. Despite the large number of studies on CVD and its etiology, its key modulators remain largely unknown. To this end, we performed a comprehensive proteomic analysis of blood plasma, with the scope to identify disease-associated changes after placing them in the context of existing knowledge, and generate a well characterized dataset for further use in CVD multi-omics integrative analysis. Methods: LC–MS/MS was employed to analyze plasma from 32 subjects (19 cases of various CVD phenotypes and 13 controls) in two steps: discovery (13 cases and 8 controls) and test (6 cases and 5 controls) set analysis. Following label-free quantification, the detected proteins were correlated to existing plasma proteomics datasets (plasma proteome database; PPD) and functionally annotated (Cytoscape, Ingenuity Pathway Analysis). Differential expression was defined based on identification confidence (≥ 2 peptides per protein), statistical significance (Mann–Whitney p value ≤ 0.05) and a minimum of twofold change. Results: Peptides detected in at least 50% of samples per group were considered, resulting in a total of 3796 identified proteins (838 proteins based on ≥ 2 peptides). Pathway annotation confirmed the functional relevance of the findings (representation of complement cascade, fibrin clot formation, platelet degranulation, etc.). Correlation of the relative abundance of the proteins identified in the discovery set with their reported concentrations in the PPD was significant, confirming the validity of the quantification method. The discovery set analysis revealed 100 differentially expressed proteins between cases and controls, 39 of which were verified (≥ twofold change) in the test set. These included proteins already studied in the context of CVD (such as apolipoprotein B, alpha-2-macroglobulin), as well as novel findings (such as low density lipoprotein receptor related protein 2 [LRP2], protein SZT2) for which a mechanism of action is suggested. Conclusions: This proteomic study provides a comprehensive dataset to be used for integrative and functional studies in the field. The observed protein changes reflect known CVD-related processes (e.g. lipid uptake, inflammation) but also novel hypotheses for further investigation including a potential pleiotropic role of LPR2 but also links of SZT2 to CVD

A novel pipeline for drug repurposing for bladder cancer based on patients’ omics signatures

Multi-omics signatures of patients with bladder cancer (BC) can guide the identification of known de-risked therapeutic compounds through drug repurposing, an approach not extensively explored yet. In this study, we target drug repurposing in the context of BC, driven by tissue omics signatures. To identify compounds that can reverse aggressive high-risk Non-Muscle Invasive BC (NMIBC) to less aggressive low-risk molecular subtypes, the next generation Connectivity Map (CMap) was employed using as input previously published proteomics and transcriptomics respective signatures. Among the identified compounds, the ATP-competitive inhibitor of mTOR, WYE-354, showed a consistently very high score for reversing the aggressive BC molecular signatures. WYE-354 impact was assessed in a panel of eight multi-origin BC cell lines and included impaired colony growth and proliferation rate without any impact on apoptosis. Overall, with this study we introduce a promising pipeline for the repurposing of drugs for BC treatment, based on patients’ omics signatures

Proteomics analysis of bladder cancer invasion: targeting EIF3D for therapeutic intervention

Patients with advanced bladder cancer have poor outcomes, indicating a need for more efficient therapeutic approaches. This study characterizes proteomic changes underlying bladder cancer invasion aiming for the better understanding of disease pathophysiology and identification of drug targets. High resolution liquid chromatography coupled to tandem mass spectrometry analysis of tissue specimens from patients with non-muscle invasive (NMIBC, stage pTa) and muscle invasive bladder cancer (MIBC, stages pT2+) was conducted. Comparative analysis identified 144 differentially expressed proteins between analyzed groups. These included proteins previously associated with bladder cancer and also additional novel such as PGRMC1, FUCA1, BROX and PSMD12, which were further confirmed by immunohistochemistry. Pathway and interactome analysis predicted strong activation in muscle invasive bladder cancer of pathways associated with protein synthesis e.g. eIF2 and mTOR signaling. Knock-down of eukaryotic translation initiation factor 3 subunit D (EIF3D) (overexpressed in muscle invasive disease) in metastatic T24M bladder cancer cells inhibited cell proliferation, migration, and colony formation in vitro and decreased tumor growth in xenograft models. By contrast, knocking down GTP-binding protein Rheb (which is upstream of EIF3D) recapitulated the effects of EIF3D knockdown in vitro, but not in vivo. Collectively, this study represents a comprehensive analysis of NMIBC and MIBC providing a resource for future studies. The results highlight EIF3D as a potential therapeutic target

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Discovery of proteins associated with cervical cancer pathology

Cervical cancer is the fourth most common and most lethal type of cancer in women, worldwide. Profylactic vaccination and regular screening have marked a decline on incidents, however the disease still accounts for a significant number of deaths and the available therapeutic approaches affect patients’ fertility. The purpose of this doctoral dissertation is the use of proteomic techniques in combination with bioinformatics and literature mining for the discovery of novel proteins involved in cervical cancer pathology with the aim of better understanding of the molecular mechanisms underlying the malignancies formation. To approach the goal, four cell lines, representative of the most common HPV infections (HeLa - HPV 18, SiHa - HPV 16), HPV-free cervical cancer (C33A) as well as normal cervical keratinocytes (HCK1T) were used. Analysis of the protein extracts was performed based on the GeLC-MS/MS protocol and provided a satisfactory number of protein identifications (2,500-3,500 proteins per cell line) with a very good reproducibility. The differentially expressed proteins that resulted from the comparison of each cervical cancer cell line with the normal keratinocytes were ~800-1,400 per comparison, and they provide useful information on the deregulation of signaling pathways and important molecules in cervical cancer. Bioinformatics analysis of the differentially expressed proteins validated the specificity of the identifications and the accuracy of the measurements as the deregulated molecular pathways are associated with the high metabolic demands and increased cell cycle that are known to occur in cancer cells. For the shortlisting of interesting proteins for further investigation, 105 differentially expressed proteins that followed the same expression trend in the three comparisons (HeLa/HCK1T, SiHa/CK1T και C33A/HCK1T) were chosen and extensively investigated in the literature. The proteins that were not studied in the context of cervical cancer before but have data on other types of cancers with similar expression trend as in the present study, are in total 21 and constitute the basis for further investigation and experiments. In addition, after a deeper investigation, 7 of these proteins were chosen (LIM domain and actin-binding protein 1, Importin subunit alpha-1, Serum paraoxonase/arylesterase 2, Elongation factor 1-alpha 2, Caprin-1, Mitochondrial import receptor subunit TOM34, Ras GTPase-activating protein-binding protein 1) for immunohistochemistry experiments on cervical specimens from healthy participants and patients with low and high grade squamous intraepithelial lesions and cervical cancer, as a next step to the present doctoral dissertation. Furthermore, functional investigation experiments for the proteins LIM domain and actin-binding protein 1, Importin subunit alpha-1 και Ras GTPase-activating protein-binding protein have been designed. In conclusion, the results of this doctoral dissertationprovide novel knowledge on the molecular mechanisms and the key regulators of cervical carcinogenesis and can be the basis for further investigations through systems biology approaches and contribute in the discovery of biomarkers and the identification of novel therapeutic targets.Ο καρκίνος του τραχήλου της μήτρας αποτελεί την τέταρτη συχνότερη και πιο θανατηφόρα μορφή καρκίνου στις γυναίκες, παγκοσμίως. Ο εμβολιασμός και ο προληπτικός έλεγχος έχουν μειώσει τα περιστατικά όμως η ασθένεια ευθύνεται για σημαντικό αριθμό θανάτων ενώ η διαθέσιμες θεραπευτικές μέθοδοι επηρεάζουν τη γονιμότητα των ασθενών. Σκοπός της διδακτορικής αυτής διατριβής αποτελεί η χρήση τεχνικών πρωτεωμικής σε συνδυασμό με τη βιοπληροφορική ανάλυση και την ανασκόπηση της βιβλιογραφίας για την ανακάλυψη νέων πρωτεϊνών που σχετίζονται με την παθολογία του καρκίνου του τραχήλου της μήτρας με στόχο την καλύτερη κατανόηση των μοριακών μηχανισμών που διέπουν το σχηματισμό των δυσπλασιών. Για την επίτευξη του στόχου, χρησιμοποιήθηκαν τέσσερις κυτταρικές σειρές, αντιπροσωπευτικές των συχνότερων μολύνσεων από HPV (HeLa - HPV 18, SiHa - HPV 16), του καρκίνου του τραχήλου της μήτρας χωρίς μόλυνση από HPV (C33A), καθώς και φυσιολογικών κερατινοκυττάρων του τραχήλου της μήτρας (HCK1T). Η ανάλυση των πρωτεϊνικών εκχυλισμάτων πραγματοποιήθηκε με τη μέθοδο GeLC-MS/MS και προσέφερε ικανοποιητικό αριθμό αναγνωρίσεων (2.500-3.500 πρωτεΐνες ανά κυτταρική σειρά) με πολύ καλή αναπαραγωγιμότητα. Από τη σύγκριση της κάθε κυτταρικής σειράς καρκίνου του τραχήλου της μήτρας με τη τα φυσιολογικά κερατινοκύτταρα προέκυψαν ~800-1.400 διαφορικά εκφραζόμενες πρωτεΐνες, ανά σύγκριση, οι οποίες παρέχουν χρήσιμη πληροφορία για τις μεταβολές στη ρύθμιση των σηματοδοτικών μονοπατιών και των σημαντικών μορίων στον καρκίνο του τραχήλου της μήτρας. Η βιοπληροφορική ανάλυση των διαφορικά εκφραζόμενων πρωτεϊνών επιβεβαίωσε την ειδικότητα των αναγνωρίσεων και την ακρίβεια των μετρήσεων καθώς τα μοριακά μονοπάτια που εμφάνισαν αλλαγή στη ρύθμισή τους σχετίζονται με τις υψηλές ενεργειακές ανάγκες και τον αυξημένο κυτταρικό κύκλο που είναι γνωστό ότι υφίστανται στα καρκινικά κύτταρα. Για τη διαλογή πρωτεϊνών ενδιαφέροντος για περαιτέρω διερεύνηση επιλέχθηκαν οι 105 διαφορικά εκφραζόμενες πρωτεΐνες που εμφάνισαν κοινή τάση έκφρασης στις τρεις συγκρίσεις (HeLa/HCK1T, SiHa/CK1T και C33A/HCK1T) και μελετήθηκαν ενδελεχώς στη βιβλιογραφία. Οι πρωτεΐνες που δεν είχαν μελετηθεί προηγουμένως στον καρκίνο του τραχήλου της μήτρας αλλά υπάρχουν δεδομένα για αυτές σε άλλους τύπους καρκίνων με παρόμοια τάση έκφρασης όπως και στην παρούσα μελέτη, είναι συνολικά 21 και αποτελούν τη βάση για περαιτέρω μελέτη και πειράματα. Μάλιστα, μετά από βαθύτερη διερεύνηση, επιλέχθηκαν 7 από αυτές (LIM domain and actin-binding protein 1, Importin subunit alpha-1, Serum paraoxonase/arylesterase 2, Elongation factor 1-alpha 2, Caprin-1, Mitochondrial import receptor subunit TOM34, Ras GTPase-activating protein-binding protein 1) για πειράματα ανοσοϊστοχημείας σε δείγματα τραχήλου από υγιείς και ασθενείς με προκαρκινική δυσπλασία χαμηλής και υψηλής διαφοροποίησης καθώς και με και καρκίνο του τραχήλου της μήτρας, ως συνέχεια της παρούσας διδακτορικής διατριβής. Ακόμη, για τις πρωτεΐνες LIM domain and actin-binding protein 1, Importin subunit alpha-1 και Ras GTPase-activating protein-binding protein 1 έχουν σχεδιαστεί και πειράματα λειτουργικής διερεύνησης. Eν κατακλείδι, αποτελέσματα της παρούσας διδακτορικής διατριβής παρέχουν νέες γνώσεις για τους μοριακούς μηχανισμούς και τους βασικούς ρυθμιστές της καρκινογένεσης του τραχήλου της μήτρας και μπορούν να αποτελέσουν τη βάση για περαιτέρω έρευνες και προσεγγίσεις της βιολογίας συστημάτων και να συμβάλουν στην ανακάλυψη βιοδεικτών και στην αναγνώριση νέων φαρμακευτικών στόχων

Novel structural approaches concerning HPV proteins: Insight into targeted therapies for cervical cancer (Review)

Cervical cancer incidence is tightly linked to HPV infection, and particularly virus types 16 and 18 cause the majority of cases presenting with pre-cancerous stages of cervical intraepithelial neoplasia (CIN). Structural and functional information concerning HPV proteins can offer novel insight into the mechanism(s) of cancer progression in the cervical epithelium. Recently, novel structural determinants of the interactions of viral proteins with their targets in keratinocytes have been elucidated. These exciting findings open the way for the development of targeted anti-oncogenic therapies, and may eventually allow the introduction of novel approaches for a rational cervical cancer treatment

Urine peptidome analysis in cardiorenal syndrome reflects molecular processes

Abstract The cardiorenal syndrome (CRS) is defined as the confluence of heart-kidney dysfunction. This study investigates the molecular differences at the level of the urinary peptidome between CRS patients and controls and their association to disease pathophysiology. The urinary peptidome of CRS patients (n = 353) was matched for age and sex with controls (n = 356) at a 1:1 ratio. Changes in the CRS peptidome versus controls were identified after applying the Mann–Whitney test, followed by correction for multiple testing. Proteasix tool was applied to investigate predicted proteases involved in CRS-associated peptide generation. Overall, 559 differentially excreted urinary peptides were associated with CRS patients. Of these, 193 peptides were specifically found in CRS when comparing with heart failure and chronic kidney disease urinary peptide profiles. Proteasix predicted 18 proteases involved in > 1% of proteolytic cleavage events including multiple forms of MMPs, proprotein convertases, cathepsins and kallikrein 4. Forty-four percent of the cleavage events were produced by 3 proteases including MMP13, MMP9 and MMP2. Pathway enrichment analysis supported that ECM-related pathways, fibrosis and inflammation were represented. Collectively, our study describes the changes in urinary peptides of CRS patients and potential proteases involved in their generation, laying the basis for further validation