Cryo-slicing blue native-mass spectrometry (csBN-MS), a novel technology for high resolution complexome profiling
23.11.2015 Müller CS, Bildl W, Haupt A, Ellenrieder L, Becker T, Hunte C, Fakler B, Schulte U. Mol Cell Proteomics. 2016;15(2):669-81 Mol Cell Proteomics. online article Blue-native gel electrophoresis (BN) is a powerful method for protein separation. Combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) it enables large-scale identification of protein complexes and their subunits. Current BN-MS approaches, however, are limited in size resolution, comprehensiveness and quantification. Here we present a new methodology combining defined sub-millimeter slicing of BN gels by a cryo-microtome with high-performance LC-MS/MS and label-free quantification of protein amounts. Application of this cryo-slicing BN-MS approach (csBN- MS) to mitochondria from rat brain demonstrated a high degree of comprehensiveness, accuracy and size resolution. The technique provided abundance-mass profiles for 774 mitochondrial proteins including all canonical subunits of the oxidative respiratory chain assembled into 13 distinct (super-)complexes. Moreover, the data revealed COX7R as a constitutive subunit of distinct super-complexes and identified novel assemblies of VDACs/porins and TOM proteins. Together, csBN-MS enables quantitative profiling of complexomes with resolution close to the limits of native gel electrophoresis. 