Cryo-slicing Blue Native-Mass Spectrometry (csBN-MS), a Novel Technology for High Resolution Complexome Profiling

Blue native (BN) gel electrophoresis 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,...

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Bibliographic Details
Published in:Molecular & cellular proteomics 2016-02, Vol.15 (2), p.669-681
Main Authors: Müller, Catrin S., Bildl, Wolfgang, Haupt, Alexander, Ellenrieder, Lars, Becker, Thomas, Hunte, Carola, Fakler, Bernd, Schulte, Uwe
Format: Article
Language:English
Subjects:
Animals
Brain - metabolism
Chromatography, Liquid - methods
Electron Transport - genetics
Electrophoresis, Gel, Two-Dimensional
Mass Spectrometry - methods
Mitochondria - metabolism
Mitochondrial Proteins - biosynthesis
Mitochondrial Proteins - genetics
Protein Biosynthesis - genetics
Rats
Tandem Mass Spectrometry - methods
Technological Innovation and Resources
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Summary:Blue native (BN) gel electrophoresis 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 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 voltage-dependent anion channels/porins and TOM proteins. Together, cryo-slicing BN-MS enables quantitative profiling of complexomes with resolution close to the limits of native gel electrophoresis.
ISSN:1535-9476
1535-9484
DOI:10.1074/mcp.M115.054080