Analysis of 953 human proteins from a mitochondrial HEK293 fraction by complexome profiling.
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.
Department of Laboratory Medicine, Laboratory of Genetic Endocrine and Metabolic Disorders, Radboud University Medical Centre, Nijmegen, The Netherlands. j.wessels@labgk.umcn.nl
Hans J C T
HJ
Wessels
Rutger O
RO
Vogel
Robert N
RN
Lightowlers
Johannes N
JN
Spelbrink
Richard J
RJ
Rodenburg
Lambert P
LP
van den Heuvel
Alain J
AJ
van Gool
Jolein
J
Gloerich
Jan A M
JA
Smeitink
Leo G
LG
Nijtmans
D002853:Chromatography, Liquid; D016000:Cluster Analysis; D030562:Databases, Protein; D057809:HEK293 Cells; D006801:Humans; D013058:Mass Spectrometry; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D012269:Ribosomal Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism
D024101:Mitochondrial Proteins; D046912:Multiprotein Complexes; D020543:Proteome; D012269:Ribosomal Proteins