biostudies-literatureKorchak JANHLBI NIH HHSNational Heart, Lung, and Blood InstituteNational Institute of General Medical SciencesNIGMS NIH HHS2614-2630https://www.ebi.ac.uk/biostudies/studies/S-EPMC11544703biostudies-literatureUnknown35(11)Alternative splicing is a major contributor of transcriptomic complexity, but the extent to which transcript isoforms are translated into stable, functional protein isoforms is unclear. Furthermore, detection of relatively scarce isoform-specific peptides is challenging, with many protein isoforms remaining uncharted due to technical limitations. Recently, a family of advanced targeted MS strategies, termed internal standard parallel reaction monitoring (IS-PRM), have demonstrated multiplexed, sensitive detection of predefined peptides of interest. Such approaches have not yet been used to confirm existence of novel peptides. Here, we present a targeted proteogenomic approach that leverages sample-matched long-read RNA sequencing (lrRNA-seq) data to predict potential protein isoforms with Journal of the American Society for Mass SpectrometryIS-PRM-Based Peptide Targeting Informed by Long-Read Sequencing for Alternative Proteome Detection.PMC11544703T32HL007284R35 GM142647R35GM142647T32 HL007284Jordan BTFenix AWatts EFKorchak JABandyopadhyay SLehe MDJeffery EDSheynkman GMWilhelm MfalseIS-PRM-Based Peptide Targeting Informed by Long-Read Sequencing for Alternative Proteome Detection.Alternative splicing is a major contributor of transcriptomic complexity, but the extent to which transcript isoforms are translated into stable, functional protein isoforms is unclear. Furthermore, detection of relatively scarce isoform-specific peptides is challenging, with many protein isoforms remaining uncharted due to technical limitations. Recently, a family of advanced targeted MS strategies, termed internal standard parallel reaction monitoring (IS-PRM), have demonstrated multiplexed, sensitive detection of predefined peptides of interest. Such approaches have not yet been used to confirm existence of novel peptides. Here, we present a targeted proteogenomic approach that leverages sample-matched long-read RNA sequencing (lrRNA-seq) data to predict potential protein isoforms with 2024-01-01T00:00:00Z2024 Nov2026-06-03T00:35:09.584Z2025-04-19T14:38:55.662ZS-EPMC115447033901205410.1021/jasms.4c00119