{"database":"biostudies-literature","file_versions":[],"scores":null,"additional":{"submitter":["Smith IR"],"funding":["European Molecular Biology Organization","Danmarks Frie Forskningsfond","NIA NIH HHS","University of Washington","NHGRI NIH HHS","NLM NIH HHS","NINDS NIH HHS","National Institute of General Medical Sciences","NIGMS NIH HHS"],"pagination":["15198-15206"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/S-EPMC9851627"],"repository":["biostudies-literature"],"omics_type":["Unknown"],"volume":["94(44)"],"pubmed_abstract":["Stable-isotope labeling with amino acids in cell culture (SILAC)-based metabolic labeling is a widely adopted proteomics approach that enables quantitative comparisons among a variety of experimental conditions. Despite its quantitative capacity, SILAC experiments analyzed with data-dependent acquisition (DDA) do not fully leverage peptide pair information for identification and suffer from undersampling compared to label-free proteomic experiments. Herein, we developed a DDA strategy that coisolates and fragments SILAC peptide pairs and uses y-ions for their relative quantification. To facilitate the analysis of this type of data, we adapted the Comet sequence database search engine to make use of SILAC peptide paired fragments and developed a tool to annotate and quantify MS/MS spectra of coisolated SILAC pairs. This peptide pair coisolation approach generally improved expectation scores compared to the traditional DDA approach. Fragment ion quantification performed similarly well to precursor quantification in the MS1 and achieved more quantifications. Lastly, our method enables reliable MS/MS quantification of SILAC proteome mixtures with overlapping isotopic distributions. This study shows the feasibility of the coisolation approach. Coupling this approach with intelligent acquisition strategies has the potential to improve SILAC peptide sampling and quantification."],"journal":["Analytical chemistry"],"pubmed_title":["Coisolation of Peptide Pairs for Peptide Identification and MS/MS-Based Quantification."],"pmcid":["PMC9851627"],"funding_grant_id":["R01 AG056359","T32 LM012419","R35GM119536","0131-00031B","R01 NS098329","ALTF 481-2020","RM1 HG010461","R35 GM119536","UWPR95794","T32 HG000035"],"pubmed_authors":["Llovet A","Villen J","Rodriguez-Mias RA","Eng JK","Barente AS","Smith IR","Hogrebe A"],"additional_accession":[]},"is_claimable":false,"name":"Coisolation of Peptide Pairs for Peptide Identification and MS/MS-Based Quantification.","description":"Stable-isotope labeling with amino acids in cell culture (SILAC)-based metabolic labeling is a widely adopted proteomics approach that enables quantitative comparisons among a variety of experimental conditions. Despite its quantitative capacity, SILAC experiments analyzed with data-dependent acquisition (DDA) do not fully leverage peptide pair information for identification and suffer from undersampling compared to label-free proteomic experiments. Herein, we developed a DDA strategy that coisolates and fragments SILAC peptide pairs and uses y-ions for their relative quantification. To facilitate the analysis of this type of data, we adapted the Comet sequence database search engine to make use of SILAC peptide paired fragments and developed a tool to annotate and quantify MS/MS spectra of coisolated SILAC pairs. This peptide pair coisolation approach generally improved expectation scores compared to the traditional DDA approach. Fragment ion quantification performed similarly well to precursor quantification in the MS1 and achieved more quantifications. Lastly, our method enables reliable MS/MS quantification of SILAC proteome mixtures with overlapping isotopic distributions. This study shows the feasibility of the coisolation approach. Coupling this approach with intelligent acquisition strategies has the potential to improve SILAC peptide sampling and quantification.","dates":{"release":"2022-01-01T00:00:00Z","publication":"2022 Nov","modification":"2026-05-28T15:20:30.779Z","creation":"2025-04-04T14:16:04.98Z"},"accession":"S-EPMC9851627","cross_references":{"pubmed":["36306373"],"doi":["10.1021/acs.analchem.2c01711"]}}