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

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.

SUBMITTER: Muller CS

PROVIDER: S-EPMC4739680 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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

Müller Catrin S CS Bildl Wolfgang W Haupt Alexander A Ellenrieder Lars L Becker Thomas T Hunte Carola C Fakler Bernd B Schulte Uwe U

Molecular & cellular proteomics : MCP 20151123 2

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 quantif ...[more]

PMID: 26598645

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Project description:Mass spectrometric sequencing of low abundance, integral membrane proteins, particularly the transmembrane domains, presents challenges that span the multiple phases of sample preparation including solubilization, purification, enzymatic digestion, peptide extraction, and chromatographic separation. We describe a method through which we have obtained high peptide coverage for 12 ?-aminobutyric acid type A receptor (GABAA receptor) subunits from 2 picomoles of affinity-purified GABAA receptors from rat brain neocortex. Focusing on the ?? subunit, we identified peptides covering 96% of the protein sequence from fragmentation spectra (MS2) using a database searching algorithm and deduced 80% of the amino acid residues in the protein from de novo sequencing of Orbitrap spectra. The workflow combined microscale membrane protein solubilization, protein delipidation, in-solution multi-enzyme digestion, multiple stationary phases for peptide extraction, and acquisition of high-resolution full scan and fragmentation spectra. For de novo sequencing of peptides containing the transmembrane domains, timed digestions with chymotrypsin were utilized to generate peptides with overlapping sequences that were then recovered by sequential solid phase extraction using a C4 followed by a porous graphitic carbon stationary phase. The specificity of peptide identifications and amino acid residue sequences was increased by high mass accuracy and charge state assignment to parent and fragment ions. Analysis of three separate brain samples demonstrated that 78% of the sequence of the ?? subunit was observed in all three replicates with an additional 13% covered in two of the three replicates, indicating a high degree of sequence coverage reproducibility. Label-free quantitative analysis was applied to the three replicates to determine the relative abundances of 11 ?-aminobutyric acid type A receptor subunits. The deep sequence MS data also revealed two N-glycosylation sites on the ?? subunit, confirmed two splice variants of the ?? subunit (??L and ??S) and resolved a database discrepancy in the sequence of the ?? subunit.

Dataset Information

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

Publications

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

Similar Datasets

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