Research of endogenous S-cyanylation in clinical human samples requires the development of dedicated experimental methods

Research of endogenous S-cyanylation in clinical human samples requires the development of dedicated experimental methods | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Research of endogenous S-cyanylation in clinical human samples requires the development of dedicated experimental methods Gonçalo da Silva, Konstantin Barylyuk, Tasso Miliotis, Karin Jennbacken, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9472761/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Endogenous cyanide has recently been identified in mammals and implicated in regulating mitochondrial function and the response to hypoxia. It is hypothesized to act as a gasotransmitter with functional significance comparable to nitric oxide. Importantly, a fraction of endogenous cyanide exists as a covalently bound posttranslational modification, called S- cyanylation, and may be more feasible to study in clinical samples than cyanide in its gaseous form. We conducted a feasibility study to assess whether cyanylation can be detected and quantified in human Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD). We used anonymized, published clinical proteomics PXD051911 and standard computational tools. Cyanylation was detectable but very low abundance. We observed <1% cyanylated cysteines in every liver sample. In both control and MASLD livers, cyanylated proteins were observed in fatty acid oxidation and NRF2-mediated oxidative stress response pathways, as well as in pathways involved in degradation of alcohol, serine and adrenaline. The total amount of cyanylation was not associated with the presence of MASLD or fibrosis severity in the liver. However, granular quantitative analysis for cyanylated peptides was impossible because most of the unique peptide sequences were observed in only one or a few liver samples. We conclude that the quantitative studies of cyanylation in clinical samples are not feasible with standard methods. To enable meaningful analysis of this modification and optimize the use of experimental resources, future work will require targeted mass spectrometry assays with labelled cyanylated peptide standards or the development of other dedicated methodologies. Analytical Biochemistry Computational Biology Proteomics Cyanylation MASLD Full Text Additional Declarations The authors declare potential competing interests as follows: The authors are employees of AstraZeneca and may own company stocks/shares. Supplementary Files TableS1.xlsx Table S1. Full list of cyanylated peptides detected with Spectronaut in the MASLD liver data. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9472761","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":626339559,"identity":"87ba8308-31b7-414c-82b1-b582ac96a0dd","order_by":0,"name":"Gonçalo da Silva","email":"","orcid":"https://orcid.org/0000-0002-1252-2709","institution":"AstraZeneca","correspondingAuthor":false,"prefix":"","firstName":"Gonçalo","middleName":"da","lastName":"Silva","suffix":""},{"id":626339560,"identity":"18e306af-d3cc-4364-8d63-2b10d9ef30de","order_by":1,"name":"Konstantin Barylyuk","email":"","orcid":"https://orcid.org/0000-0002-3580-0345","institution":"AstraZeneca","correspondingAuthor":false,"prefix":"","firstName":"Konstantin","middleName":"","lastName":"Barylyuk","suffix":""},{"id":626362487,"identity":"b8d766e6-9184-4f39-b0d1-b2313a8f8de8","order_by":2,"name":"Tasso Miliotis","email":"","orcid":"https://orcid.org/0000-0003-4917-0724","institution":"AstraZeneca","correspondingAuthor":false,"prefix":"","firstName":"Tasso","middleName":"","lastName":"Miliotis","suffix":""},{"id":626363554,"identity":"d9edb78a-f3ce-4839-8323-1455ad9b4e25","order_by":3,"name":"Karin Jennbacken","email":"","orcid":"https://orcid.org/0000-0003-1254-5158","institution":"AstraZeneca","correspondingAuthor":false,"prefix":"","firstName":"Karin","middleName":"","lastName":"Jennbacken","suffix":""},{"id":626363555,"identity":"a4f6b028-b500-4d3c-8efa-cd54a1140a6f","order_by":4,"name":"Maria Ryaboshapkina","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0003-1385-8869","institution":"AstraZeneca","correspondingAuthor":true,"prefix":"","firstName":"Maria","middleName":"","lastName":"Ryaboshapkina","suffix":""}],"badges":[],"createdAt":"2026-04-20 13:17:29","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":true,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-9472761/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9472761/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107490060,"identity":"dd75712c-374d-4a1e-9c06-07f61ef5038a","added_by":"auto","created_at":"2026-04-22 02:49:51","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":799150,"visible":true,"origin":"","legend":"","description":"","filename":"CyanylationProjectPSO.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9472761/v1_covered_219833e6-5bef-4857-abd6-2dc2979d0957.pdf"},{"id":107432993,"identity":"52ee936b-52b4-4b82-9a7f-9b0f5edacd1f","added_by":"auto","created_at":"2026-04-21 12:44:28","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":23583,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S1. 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It is hypothesized to act as a gasotransmitter with functional significance comparable to nitric oxide. Importantly, a fraction of endogenous cyanide exists as a covalently bound posttranslational modification, called S- cyanylation, and may be more feasible to study in clinical samples than cyanide in its gaseous form.\u003c/p\u003e\n\u003cp\u003eWe conducted a feasibility study to assess whether cyanylation can be detected and quantified in human Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD). We used anonymized, published clinical proteomics PXD051911 and standard computational tools. Cyanylation was detectable but very low abundance. We observed \u0026lt;1% cyanylated cysteines in every liver sample. In both control and MASLD livers, cyanylated proteins were observed in fatty acid oxidation and NRF2-mediated oxidative stress response pathways, as well as in pathways involved in degradation of alcohol, serine and adrenaline. 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