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

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 Cse4^CENP-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.
• Cse4^CENP-A regulation impacts kinetochore assembly dynamics.