Detection of PTMs in Arginine-and Lysine-Rich Peptides via Sequential IP and Proteinase K Digestion of Tagged Protein

Detection of PTMs in Arginine-and Lysine-Rich Peptides via Sequential IP and Proteinase K Digestion of Tagged Protein | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 6 August 2025 V1 Latest version Share on Detection of PTMs in Arginine-and Lysine-Rich Peptides via Sequential IP and Proteinase K Digestion of Tagged Protein Author : Shobith Suresh 0000-0001-6336-1418 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.175449370.07645799/v1 156 views 144 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Post-translational modifications (PTMs) are critical regulators of protein function, especially within intrinsically disordered and arginine/lysine-rich regions such as nuclear localization signals and RNA-binding motifs. However, the dense clustering of arginine (R) and lysine (K) residues often complicates protease-based peptide recovery for downstream mass spectrometry (MS). Here, we describe a method optimized for detecting PTMs in such regions, particularly focusing on the peptide sequence RKKRRQRR, which resists cleavage by Proteinase K. The method combines FLAG-based immunoprecipitation, in-bead Proteinase K digestion, and a secondary antibody pull-down against the target peptide. The protocol includes specific strategies to eliminate co-eluted antibody contaminants and prepares samples for high-resolution LC-MS/MS analysis. This workflow facilitates reliable identification of PTMs in peptides refractory to traditional enzymatic digestion. Supplementary Material File (ptm of peptides via ip and proteinase k digestion (1).pdf) Download 485.35 KB Information & Authors Information Version history V1 Version 1 06 August 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords antibody pull-down arginine-rich peptides flag tag immunoprecipitation lc-ms/ms post-translational modifications proteinase k proteomics rkkrrqrr Authors Affiliations Shobith Suresh 0000-0001-6336-1418 [email protected] Jawaharlal Nehru Centre for Advanced Scientific Research View all articles by this author Metrics & Citations Metrics Article Usage 156 views 144 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Shobith Suresh. Detection of PTMs in Arginine-and Lysine-Rich Peptides via Sequential IP and Proteinase K Digestion of Tagged Protein. Authorea . 06 August 2025. DOI: https://doi.org/10.22541/au.175449370.07645799/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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