Cryo-slicing Blue Native-Mass Spectrometry (csBN-MS), a Novel Technology for High Resolution Complexome Profiling.
Mol Cell Proteomics
; 15(2): 669-81, 2016 Feb.
Article
em En
| MEDLINE
| ID: mdl-26598645
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.
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Base de dados:
MEDLINE
Assunto principal:
Espectrometria de Massas
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Biossíntese de Proteínas
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Cromatografia Líquida
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Proteínas Mitocondriais
Idioma:
En
Ano de publicação:
2016
Tipo de documento:
Article