{"doi":"10.1101/2024.12.12.628274","title":"A click chemistry-based biorthogonal approach for the detection and identification of protein lysine malonylation for osteoarthritis research","abstract":"Abstract Lysine malonylation is a post-translational modification where a malonyl group, characterized by a negatively charged carboxylate, is covalently attached to the ε-amino side chain of lysine, influencing protein structure and function. Our laboratory identified Mak upregulation in cartilage under aging and obesity, contributing to osteoarthritis (OA). Current antibody-based detection methods face limitations in identifying Mak targets. Here, we introduce an alkyne-functionalized probe, MA-diyne, which metabolically incorporates into proteins, enabling copper(I) ion-catalyzed click reactions to conjugate labeled proteins with azide-based fluorescent dyes or affinity purification tags. In-gel fluorescence confirms MA-diyne incorporation into proteins across various cell types and species, including mouse chondrocytes, adipocytes, Hek293T cells, and C. elegans . Pull-down experiments identified known Mak proteins such as GAPDH and Aldolase. The extent of MA-diyne modification was higher in Sirtuin 5-deficient cells suggesting these modified proteins are Sirtuin 5 substrates. Pulse-chase experiments confirmed the dynamic nature of protein malonylation. Quantitative proteomics identified 1136 proteins corresponding to 8903 peptides with 429 proteins showing 1-fold increase in labeled group. Sirtuin 5 regulated 374 of these proteins. Pull down of newly identified proteins such as β-actin and Stat3 was also done. This study highlights MA-diyne as a powerful chemical tool to investigate the molecular targets and functions of lysine malonylation in OA conditions.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2024,"id":507472,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9559,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2024-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1359004,"name":"Pandurangan Nanjan","orcid":"0000-0001-5213-9264","position":1,"is_corresponding":false},{"id":1359326,"name":"Kavya Chellamuthu","orcid":null,"position":2,"is_corresponding":false},{"id":356580,"name":"Huanhuan Liu","orcid":"0000-0003-1790-3073","position":3,"is_corresponding":false},{"id":560740,"name":"Shouan Zhu","orcid":"0000-0002-4973-1504","position":4,"is_corresponding":false},{"id":1331200,"name":"Anupama Binoy","orcid":"0000-0001-6425-9570","position":0,"is_corresponding":true}],"reference_count":35,"raw_metadata":null,"created_at":"2026-07-19T02:11:02.460057Z","pmid":"39713453","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}