期刊
MOLECULAR & CELLULAR PROTEOMICS
卷 15, 期 2, 页码 669-681出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/mcp.M115.054080
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资金
- Deutsche Forschungsgemeinschaft [BE4679/2-1, SFB 746/TP22, SFB 746/TP16, SFB TRR 152/TP02, SFB 746/TP20, SFB TRR 152/TP05]
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 cryoslicing 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.
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