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

Here, we demonstrate pilot development of native multi-dimensional protein complex identification and topology characterization (Native MudPIT) in complex biological samples via coupling native size exclusion chromatography (SEC), online native capillary electrophoresis-mass spectrometry (nCE-MS), surface-induced dissociation (SID), and multi-level proteomics. With semi-manual data interpretation because optimized algorithms do not yet exist for this approach, the Native MudPIT approach enabled the detection of 124 unique native endogenous protein complexes with masses up to 1.5 MDa from fractions of a whole cell lysate, identified 46 unique protein complexes in a discovery mode, and provided topological information for 39 protein complexes. The Native MudPIT approach is expected to be broadly applicable for characterizing protein complexes in cells, tissues, and biological fluids.