Native Multidimensional Protein Complex Identification and Topology Characterization (Native MudPIT)

The initiation and progression of diseases are usually due to biological processes going awry, driven by proteins in complexes that often assemble via noncovalent interactions of monomeric or multimeric subunits. A broader view of protein complex dynamics in cells is vital for advancing our understanding of the molecular mechanisms of diseases. Here, we present a novel mass spectrometry (MS)-based native proteomics technology, native multi-dimensional protein complex identification and topology characterization (Native MudPIT), to achieve proteome-scale protein complex characterization. Native MudPIT integrates native size exclusion chromatography (SEC), capillary zone electrophoresis-MS (nCZE-MS), surface-induced dissociation (SID), and multilevel proteomics. The technique enabled the detection of 124 endogenous proteins/protein complexes with masses up to 1.5 MDa from an Escherichia coli whole cell lysate, identified 46 protein complexes in a discovery mode, and provided topological information on 39 protein complexes. This work represents a pivotal advance in native proteomics for global and comprehensive characterization of protein complexes in complex biological systems (i.e., cells, tissues, and biological fluids).