A Robust Workflow for Native Mass Spectrometric Analysis of Affinity-Isolated Endogenous Protein Assemblies

Area of Science:

• Biochemistry
• Structural Biology
• Cellular Biology

Background:

• Macromolecular assemblies are central to cellular functions.
• Understanding subunit stoichiometry and connectivity is crucial for structural and functional characterization.
• Native mass spectrometry (MS) is a direct method for determining the mass of molecular assemblies.

Purpose of the Study:

• To develop a robust workflow for analyzing endogenous protein complexes.
• To overcome limitations in obtaining sufficient concentrations of cell-derived assemblies for native MS.
• To enable detailed characterization of stoichiometry and intersubunit connectivity in native protein complexes.

Main Methods:

• Coupling rapid affinity isolation of endogenous protein complexes with sensitive native MS.
• Development of a workflow for analyzing intact macromolecular assemblies.
• Utilizing native MS for accurate mass determination of cellular components.

Main Results:

• A robust workflow for analyzing endogenous protein complexes has been established.
• The workflow facilitates the study of protein assemblies expressed at endogenous levels.
• Overcomes challenges related to low concentrations and artifacts from overexpression.

Conclusions:

• The presented workflow significantly advances the study of endogenous protein complexes.
• Provides crucial data for integrative structural elucidation of biological systems.
• Enables detailed insights into the stoichiometry and connectivity of native cellular machinery.