Isabel R Uribe, Emily Zahn, Richard Searfoss, Han-Byeol Kim, Morgan Dasovich, Jim Voorneveld, Sabrina R Hunt, Ugochi C Onuoha, Catherine Valadez, Dmitri V Filippov, Chan Hyun Na, Benjamin A Garcia, Benjamin C Orsburn, Anthony K L Leung
{"title":"dELTA-MS: A Mass Spectrometry-Based Proteomics Approach for Identifying ADP-Ribosylation Sites and Forms.","authors":"Isabel R Uribe, Emily Zahn, Richard Searfoss, Han-Byeol Kim, Morgan Dasovich, Jim Voorneveld, Sabrina R Hunt, Ugochi C Onuoha, Catherine Valadez, Dmitri V Filippov, Chan Hyun Na, Benjamin A Garcia, Benjamin C Orsburn, Anthony K L Leung","doi":"10.1021/acs.jproteome.4c00890","DOIUrl":null,"url":null,"abstract":"<p><p>ADP-ribosylation, characterized by the addition of adenosine diphosphate ribose, can occur in both monomeric (MARylation) and polymeric (PARylation) forms. Little is known about the specific contributions of MARylation and PARylation to cellular processes due to a lack of tools for jointly investigating these individual forms. We present a novel mass spectrometry (MS)-based proteomics approach that preserves information about the native ADP-ribosylation form associated with the modification site within a single proteomics experiment. Our workflow enables the simplified, binary identification of ADP-ribosylation forms, avoiding some challenges typically presented by PARylated peptides during MS analysis. Our method uses the coronaviral enzyme NS2 to reverse our previous labeling approach, ELTA, which enzymatically labels the terminal ADP-ribose. NS2 deconjugates ELTA-labeled free or peptide-conjugated ADP-ribose monomers and polymers (thereby termed dELTA), leaving behind a signature phosphate. Our dELTA-MS workflow involves ELTA labeling, dELTA deconjugation, and further processing using <i>Deinococcus radiodurans</i> poly(ADP-ribose) glycohydrolase (DrPARG), resulting in two distinct mass shifts for MARylation and PARylation sites. We demonstrate the feasibility of this workflow for proteomics analyses using proof-of-principle peptide standards. dELTA-MS thus creates possibilities to reveal the fundamental biology of ADP-ribosylation and explore its dysregulation, in terms of both sites and forms, associated with disease progression.</p>","PeriodicalId":48,"journal":{"name":"Journal of Proteome Research","volume":" ","pages":""},"PeriodicalIF":3.8000,"publicationDate":"2025-03-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Proteome Research","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1021/acs.jproteome.4c00890","RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
dELTA-MS: A Mass Spectrometry-Based Proteomics Approach for Identifying ADP-Ribosylation Sites and Forms.
ADP-ribosylation, characterized by the addition of adenosine diphosphate ribose, can occur in both monomeric (MARylation) and polymeric (PARylation) forms. Little is known about the specific contributions of MARylation and PARylation to cellular processes due to a lack of tools for jointly investigating these individual forms. We present a novel mass spectrometry (MS)-based proteomics approach that preserves information about the native ADP-ribosylation form associated with the modification site within a single proteomics experiment. Our workflow enables the simplified, binary identification of ADP-ribosylation forms, avoiding some challenges typically presented by PARylated peptides during MS analysis. Our method uses the coronaviral enzyme NS2 to reverse our previous labeling approach, ELTA, which enzymatically labels the terminal ADP-ribose. NS2 deconjugates ELTA-labeled free or peptide-conjugated ADP-ribose monomers and polymers (thereby termed dELTA), leaving behind a signature phosphate. Our dELTA-MS workflow involves ELTA labeling, dELTA deconjugation, and further processing using Deinococcus radiodurans poly(ADP-ribose) glycohydrolase (DrPARG), resulting in two distinct mass shifts for MARylation and PARylation sites. We demonstrate the feasibility of this workflow for proteomics analyses using proof-of-principle peptide standards. dELTA-MS thus creates possibilities to reveal the fundamental biology of ADP-ribosylation and explore its dysregulation, in terms of both sites and forms, associated with disease progression.
期刊介绍:
Journal of Proteome Research publishes content encompassing all aspects of global protein analysis and function, including the dynamic aspects of genomics, spatio-temporal proteomics, metabonomics and metabolomics, clinical and agricultural proteomics, as well as advances in methodology including bioinformatics. The theme and emphasis is on a multidisciplinary approach to the life sciences through the synergy between the different types of "omics".