Format

Send to

Choose Destination
Mol Cell Proteomics. 2016 Feb;15(2):669-81. doi: 10.1074/mcp.M115.054080. Epub 2015 Nov 23.

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

Author information

1
From the ‡Institute of Physiology, University of Freiburg, Hermann-Herder-Strasse 7, 79104 Freiburg;
2
§Institute for Biochemistry and Molecular Biology, Stefan-Meier-Strasse 17, 79104 Freiburg;
3
§Institute for Biochemistry and Molecular Biology, Stefan-Meier-Strasse 17, 79104 Freiburg; ¶Center for Biological Signaling Studies (BIOSS), Schänzlestrasse 18, 79104 Freiburg;
4
From the ‡Institute of Physiology, University of Freiburg, Hermann-Herder-Strasse 7, 79104 Freiburg; ¶Center for Biological Signaling Studies (BIOSS), Schänzlestrasse 18, 79104 Freiburg;
5
From the ‡Institute of Physiology, University of Freiburg, Hermann-Herder-Strasse 7, 79104 Freiburg; ¶Center for Biological Signaling Studies (BIOSS), Schänzlestrasse 18, 79104 Freiburg; ‖Logopharm GmbH, Schlossstrasse 14, 79232 March-Buchheim, Germany uwe.schulte@physiologie.uni-freiburg.de.

Abstract

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.

PMID:
26598645
PMCID:
PMC4739680
DOI:
10.1074/mcp.M115.054080
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central
Loading ...
Support Center