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

Jiaxi Cai1,2, Quan Yun1, Cindy Yuxuan Zhang1

  • 1Department of Cellular and Molecular Medicine, University of California, San Diego, California.

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|August 2, 2024
PubMed
Summary
This summary is machine-generated.

A new method, concatemer-assisted stoichiometry analysis (CASA), precisely quantifies protein complex subunits. CASA revealed cell-cycle controlled Cse4CENP-A as a limiting factor in yeast kinetochore assembly.

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Area of Science:

  • Molecular and Cellular Biology
  • Biochemistry
  • Proteomics

Background:

  • Multi-protein machines are crucial for biological processes.
  • Accurate stoichiometric determination of protein complexes in native states is challenging.
  • Existing methods lack precision and accuracy.

Purpose of the Study:

  • To introduce concatemer-assisted stoichiometry analysis (CASA) for robust protein complex quantification.
  • To address limitations in accuracy and precision of current stoichiometric determination tools.
  • To apply CASA for analyzing protein stoichiometries in budding yeast kinetochores.

Main Methods:

  • Development of concatemer-assisted stoichiometry analysis (CASA).
  • Utilizing stable isotope-labeled concatemers.
  • Employing liquid chromatography parallel reaction monitoring mass spectrometry (LC-PRM-MS) for sub-femtomole sensitivity quantification.

Main Results:

  • CASA achieved robust quantification of protein complex subunits with high sensitivity.
  • Stoichiometries were determined for various budding yeast kinetochore components.
  • Absolute quantification revealed Cse4CENP-A as a cell-cycle controlled limiting factor for kinetochore assembly.

Conclusions:

  • CASA provides accurate and precise stoichiometric determination of protein complexes.
  • The method is applicable to complex biological assemblies like kinetochores.
  • Cse4CENP-A regulation impacts kinetochore assembly dynamics.