Co-fractionation mass spectrometry couples native-like separations of protein-protein complexes with mass spectrometric proteome analysis for global characterization of protein networks. The technique allows for both
We developed mini-Complexome Profiling (mCP), a streamlined workflow with reduced requirements for fractionation and, thus, biological material and laboratory and instrument time. Soluble and membrane-associated protein complexes are extracted from biological material under mild conditions, and fractionated by Blue Native electrophoresis using commercial equipment. Each fraction is analysed by data-independent acquisition mass spectrometry, and known protein complexes are detected based on the coelution of known components using a novel R package with a controlled false discovery rate approach. The tool is available to the community on a GitHub repository.
mCP was benchmarked using HEK293 cell lysate and exhibited performance similar to established workflows, but from a significantly reduced number of fractions. We then challenged mCP by performing comparative complexome analysis of cardiomyocytes isolated from different chambers from a single mouse heart, where we identified subtle chamber-specific changes in mitochondrial OxPhos complexes.
The reduced sample and instrument time requirements open up new applications of co-fractionation mass spectrometry, specifically for the analysis of sparse samples such as human patient biopsies. The ability to identify subtle changes between similar tissue types (left/right ventricular and atrial cardiomyocytes) serves as a proof of principle for comparative analysis of mild/asymptomatic disease states.