Photo Cross‐Linking Probes Containing ϵ‐N‐Thioacyllysine and ϵ‐N‐Acyl‐(δ‐aza)lysine Residues

• Published in: Chemistry : a European journal Vol. 26; no. 17; pp. 3862 - 3869
• Main Authors: Bæk, Michael, Martín‐Gago, Pablo, Laursen, Jonas S., Madsen, Julie L. H., Chakladar, Saswati, Olsen, Christian A.
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 23-03-2020; John Wiley and Sons Inc
• Subjects: activity-based probes; Adducts; affinity-based probes; Alternative splicing; Amino Acid Sequence; Chemistry; Electrophoresis; Fluorescence; Gel electrophoresis; histone deacetylases; Humans; Hydrolysis; Localization; Lysine; Lysine - chemistry; Peptides; Peptides - chemistry; photo-affinity labeling; posttranslational modifications; Probes; Protein Processing, Post-Translational; Protein transport; Proteins; Proteome - metabolism; Residues; sirtuins; photo-affinity labeling; histone deacetylases; affinity-based probes; posttranslational modifications; activity-based probes; sirtuins
• ISSN: 0947-6539; 1521-3765
• DOI: 10.1002/chem.201905338

Posttranslational modifications (PTMs) are important in the regulation of protein function, trafficking, localization, and marking for degradation. This work describes the development of peptide activity/affinity‐based probes for the discovery of proteins that recognize novel acyl‐based PTMs on lysine residues in the proteome. The probes contain surrogates of ϵ‐N‐acyllysine by introduction of either hydrazide or thioamide functionalities to circumvent hydrolysis of the modification during the experiments. In addition to the modified PTMs, the developed chemotypes were analyzed with respect to the effect of peptide sequence. The photo cross‐linking conditions and subsequent functionalization of the covalent adducts were systematically optimized by applying fluorophore labeling and gel electrophoresis (in‐gel fluorescence measurements). Finally, selected probes, containing the ϵ‐N‐glutaryllysine and ϵ‐N‐myristoyllysine analogues, were successfully applied for the enrichment of native, endogenous proteins from cell lysate, recapitulating the expected interactions of SIRT5 and SIRT2, respectively. Interestingly, the latter mentioned was able to pull down two different splice variants of SIRT2, which has not been achieved with a covalent probe before. Based on this elaborate proof‐of‐concept study, we expect that the technology will have broad future applications for pairing of novel PTMs with the proteins that target them in the cell.
A new strategy for activity‐based probes is reported to address the members of the proteome that target ϵ‐N‐acyllysine posttranslational modifications, which are highly prevalent and constitute a continuously expanding collection of chemical groups. In addition, this work provides the first investigation of the importance of peptide sequence on performance of ϵ‐N‐acyllysine‐containing photo cross‐linking probes, screening the efficiency of a combined series of 36 probe constructs against all 7 human sirtuins.