biostudies-literatureUnknownCai JNIH HHSNIGMS NIH HHSLarge multi-protein machines are central to multiple 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 <i>ex vivo</i> reconstituted kinetochore components, including the canonical H3 nucleosomes, centromeric (Cse4^CENP-A) nubioRxiv : the preprint server for biology2024.07.26.605382https://www.ebi.ac.uk/biostudies/studies/S-EPMC11291133biostudies-literatureConcatemer Assisted Stoichiometry Analysis (CASA): targeted mass spectrometry for protein quantification.PMC11291133S10 OD023498R01 GM116897R01 GM151191Suhandynata RTCai JWang ZZhou HHinshaw SMYun QZhang CYfalseConcatemer Assisted Stoichiometry Analysis (CASA): targeted mass spectrometry for protein quantification.Large multi-protein machines are central to multiple 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 <i>ex vivo</i> reconstituted kinetochore components, including the canonical H3 nucleosomes, centromeric (Cse4^CENP-A) nu2024-01-01T00:00:00Z2024 Jul2025-04-04T10:13:21.003Z2025-04-04T10:13:21.003ZS-EPMC112911333909176910.1101/2024.07.26.605382