3 research outputs found

    mini-Complexome Profiling (mCP), an FDR-controlled workflow for global targeted detection of protein complexes

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    IntroductionCo-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 de novo detection of complexes and for the detection of subtle changes in their protein composition. The typical requirement for fine-grained fractionation of >80 fractions, however, translates into significant demands on sample quantity and mass spectrometric instrument time, and represents a significant barrier to experimental replication and the use of scarce sample material (ex. patient biopsies).MethodsWe 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.ResultsmCP 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.DiscussionThe 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
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