Concatemer-assisted stoichiometry analysis: targeted mass spectrometry for protein quantification

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.