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Cryo-slicing BN-MS – a novel technology for high-resolution complexome profiling

Catrin Swantje Müller, Wolfgang Bildl, Alexander Haupt, Lars Ellenrieder, Thomas Becker, Carola Hunte, Bernd Fakler, and Uwe Schulte

Dec 22, 2015

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 (csBNMS)
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.

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