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Analysis of 953 Human Proteins from a Mitochondrial HEK293 Fraction by Complexome Profiling

Lookup NU author(s): Professor Robert Lightowlers

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Abstract

Complexome profiling is a novel technique which uses shotgun proteomics to establish protein migration profiles from fractionated blue native electrophoresis gels. Here we present a dataset of blue native electrophoresis migration profiles for 953 proteins by complexome profiling. By analysis of mitochondrial ribosomal complexes we demonstrate its potential to verify putative protein-protein interactions identified by affinity purification - mass spectrometry studies. Protein complexes were extracted in their native state from a HEK293 mitochondrial fraction and separated by blue native gel electrophoresis. Gel lanes were cut into gel slices of even size and analyzed by shotgun proteomics. Subsequently, the acquired protein migration profiles were analyzed for co-migration via hierarchical cluster analysis. This dataset holds great promise as a comprehensive resource for de novo identification of protein-protein interactions or to underpin and prioritize candidate protein interactions from other studies. To demonstrate the potential use of our dataset we focussed on the mitochondrial translation machinery. Our results show that mitoribosomal complexes can be analyzed by blue native gel electrophoresis, as at least four distinct complexes. Analysis of these complexes confirmed that 24 proteins that had previously been reported to co-purify with mitoribosomes indeed co-migrated with subunits of the mitochondrial ribosome. Co-migration of several proteins involved in biogenesis of inner mitochondrial membrane complexes together with mitoribosomal complexes suggested the possibility of co-translational assembly in human cells. Our data also highlighted a putative ribonucleotide complex that potentially contains MRPL10, MRPL12 and MRPL53 together with LRPPRC and SLIRP.


Publication metadata

Author(s): Wessels HJCT, Vogel RO, Lightowlers RN, Spelbrink JN, Rodenburg RJ, van den Heuvel LP, van Gool AJ, Gloerich J, Smeitink JAM, Nijtmans LG

Publication type: Article

Publication status: Published

Journal: PLoS ONE

Year: 2013

Volume: 8

Issue: 7

Online publication date: 23/07/2013

Acceptance date: 28/05/2013

Date deposited: 19/01/2015

ISSN (electronic): 1932-6203

Publisher: Public Library of Science

URL: http://dx.doi.org/10.1371/journal.pone.0068340

DOI: 10.1371/journal.pone.0068340


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