Concatemer 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 reconstituted kinetochore components, including the canonical H3 nucleosomes, centromeric (Cse4) nucleosomes, centromere proximal factors (Cbf1 and CBF3 complex), inner kinetochore proteins (Mif2, Ctf19 complex), and outer kinetochore proteins (KMN network). Absolute quantification by CASA revealed Cse4 as a cell-cycle controlled limiting factor for kinetochore assembly. These findings demonstrate that CASA is applicable for stoichiometry analysis of multi-protein assemblies.