{"doi":"10.26508/lsa.202403007","title":"Concatemer-assisted stoichiometry analysis: targeted mass spectrometry for protein quantification","abstract":"Large multiprotein machines are central to many biological processes. However, stoichiometric determination of protein complex subunits in their native states presents a significant challenge. This study addresses the limitations of current tools in accuracy and precision by introducing concatemer-assisted stoichiometry analysis (CASA). CASA leverages stable isotope-labeled concatemers and liquid chromatography–parallel reaction monitoring–mass spectrometry (LC-PRM-MS) to achieve robust quantification of proteins with sub-femtomole sensitivity. As a proof of concept, CASA was applied to study budding yeast kinetochores. Stoichiometries were determined for ex vivo reconstituted kinetochore components, including the canonical H3 nucleosomes, centromeric (Cse4 CENP-A ) nucleosomes, centromere proximal factors (Cbf1 and CBF3 complex), inner kinetochore proteins (Mif2 CENP-C , Ctf19 CCAN complex), and outer kinetochore proteins (KMN network). Absolute quantification by CASA revealed Cse4 CENP-A as a cell cycle–controlled limiting factor for kinetochore assembly. These findings demonstrate that CASA is applicable for stoichiometry analysis of multiprotein assemblies.","journal":"Life Science Alliance","year":2024,"id":508178,"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":0.9629,"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":782454,"name":"Yun Quan","orcid":"0000-0003-3230-6189","position":1,"is_corresponding":false},{"id":1351230,"name":"Cindy Yuxuan Zhang","orcid":null,"position":2,"is_corresponding":false},{"id":1359827,"name":"Ziyi Wang","orcid":"0009-0008-2961-3173","position":3,"is_corresponding":false},{"id":782455,"name":"Stephen M. Hinshaw","orcid":"0000-0003-4215-5206","position":4,"is_corresponding":false},{"id":567173,"name":"Huilin Zhou","orcid":"0000-0002-1350-4430","position":5,"is_corresponding":false},{"id":314429,"name":"Raymond T. Suhandynata","orcid":"0000-0002-4767-7639","position":6,"is_corresponding":false},{"id":822682,"name":"Jiaxi Cai","orcid":"0000-0002-4487-5613","position":0,"is_corresponding":true}],"reference_count":78,"raw_metadata":null,"created_at":"2026-07-19T02:11:06.395600Z","pmid":"39741008","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":[]}