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bioRxiv · 10.1101/2024.07.26.605382

Concatemer Assisted Stoichiometry Analysis (CASA): a targeted mass spectrometry method for protein quantification

Cai, J.·Quan, Y.·Zhang, C. Y.·Wang, Z.·Hinshaw, S. M.·Zhou, H.·Suhandynata, R. T.
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Abstract

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 ex vivo reconstituted kinetochore components, including the canonical H3 nucleosomes, centromeric (Cse4CENP-A) nucleosomes, centromere proximal factors (Cbf1 and CBF3 complex), inner kinetochore proteins (Mif2CENP-C, Ctf19CCAN complex), and outer kinetochore proteins (KMN network). Absolute quantification by CASA revealed Cse4CENP-A as a cell-cycle controlled limiting factor for kinetochore assembly. These findings demonstrate that CASA is applicable for stoichiometry analysis of multi-protein assemblies. SummaryThis study presents Concatemer-Assisted Stoichiometry Analysis (CASA) to address a common challenge in cell biological research: quantifying the number of each protein subunit in a native protein complex.

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Cai, J., Quan, Y., Zhang, C. Y., Wang, Z., Hinshaw, S. M., Zhou, H., Suhandynata, R. T.. 2024-07-27. Concatemer Assisted Stoichiometry Analysis (CASA): a targeted mass spectrometry method for protein quantification. https://doi.org/10.1101/2024.07.26.605382

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