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

Area of Science:

• Molecular Biology
• Biochemistry
• Proteomics

Background:

• Large multiprotein machines are essential for biological processes.
• Accurate stoichiometric determination of protein complex subunits in native states is difficult.
• Existing methods lack precision and accuracy.

Purpose of the Study:

• To introduce a novel method, concatemer-assisted stoichiometry analysis (CASA), for accurate protein complex stoichiometry determination.
• To overcome the limitations of current stoichiometric analysis tools.
• To apply CASA for quantifying components of the budding yeast kinetochore.

Main Methods:

• Development and application 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 protein quantification.

Main Results:

• CASA achieved robust quantification of proteins with high sensitivity.
• Stoichiometries were determined for various ex vivo reconstituted kinetochore components.
• Cse4^CENP-A was identified as a cell cycle-controlled limiting factor in kinetochore assembly.

Conclusions:

• CASA provides a powerful and sensitive approach for stoichiometry analysis of multiprotein assemblies.
• The method enables accurate quantification of protein complex subunits.
• Findings highlight the cell cycle regulation of kinetochore assembly through Cse4^CENP-A levels.