{"doi":"10.1101/2024.04.01.587549","title":"IS-PRM-based peptide targeting informed by long-read sequencing for alternative proteome detection","abstract":"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 pre-defined 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 (LR RNAseq) data to predict potential protein isoforms with prior transcript evidence. Predicted tryptic isoform-specific peptides, which are specific to individual gene product isoforms, serve as \"triggers\" and \"targets\" in the IS-PRM method, Tomahto. Using the model human stem cell line WTC11, LR RNAseq data were generated and used to inform the generation of synthetic standards for 192 isoform-specific peptides (114 isoforms from 55 genes). These synthetic \"trigger\" peptides were labeled with super heavy tandem mass tags (TMT) and spiked into TMT-labeled WTC11 tryptic digest, predicted to contain corresponding endogenous \"target\" peptides. Compared to DDA mode, Tomahto increased detectability of isoforms by 3.6-fold, resulting in the identification of five previously unannotated isoforms. Our method detected protein isoform expression for 43 out of 55 genes corresponding to 54 resolved isoforms. This LR RNA seq-informed Tomahto targeted approach, called LRP-IS-PRM, is a new modality for generating protein-level evidence of alternative isoforms - a critical first step in designing functional studies and eventually clinical assays.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2024,"id":496175,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9528,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2024-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":851675,"name":"Erin D. Jeffery","orcid":"0000-0001-9511-7719","position":1,"is_corresponding":false},{"id":1343068,"name":"Saikat Bandyopadhyay","orcid":null,"position":2,"is_corresponding":false},{"id":571095,"name":"Ben T. Jordan","orcid":"0000-0003-2268-5226","position":3,"is_corresponding":false},{"id":1161432,"name":"Micah Lehe","orcid":"0000-0002-3219-6066","position":4,"is_corresponding":false},{"id":1342779,"name":"Emily F. Watts","orcid":"0000-0001-6018-4428","position":5,"is_corresponding":false},{"id":651017,"name":"Aidan M. Fenix","orcid":"0000-0002-1564-7347","position":6,"is_corresponding":false},{"id":237007,"name":"Mathias Wilhelm","orcid":"0000-0002-9224-3258","position":7,"is_corresponding":false},{"id":106260,"name":"Gloria Sheynkman","orcid":"0000-0002-4223-9947","position":8,"is_corresponding":false},{"id":1342778,"name":"Jennifer A. Korchak","orcid":"0000-0002-2679-721X","position":0,"is_corresponding":true}],"reference_count":72,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T02:09:23.333150Z","pmid":"38617311","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}