Proteomic Analysis of Human Brown Adipose Tissue Reveals Utilization of Coupled and Uncoupled Energy Expenditure Pathways
Human brown adipose tissue (BAT) has become an attractive target to combat the current epidemical spread of obesity and its associated co-morbidities. Currently, information on its functional role is primarily derived from rodent studies. Here, we present the first comparative proteotype analysis of primary human brown adipose tissue versus adjacent white adipose tissue, which reveals significant quantitative differences in protein abundances and in turn differential functional capabilities. The majority of the 318 proteins with increased abundance in BAT are associated with mitochondrial metabolism and confirm the increased oxidative capacity. In addition to uncoupling protein 1 (UCP1), the main functional effector for uncoupled respiration, we also detected the mitochondrial creatine kinases (CKMT1A/B, CKMT2), as effective modulators of ATP synthase coupled respiration, to be exclusively expressed in BAT. The abundant expression and utilization of both energy expenditure pathways in parallel highlights the complex functional involvement of BAT in human physiology.
Sample Processing Protocol
Samples were prepared with the TMT10plex Isobaric Mass Tag Labeling Kit (Thermo Scientific, Lot number QD212963) and the Pierce High pH Reversed-Phase Peptide Fractionation Kit (Thermo Scientific) according to the manufacturer’s instructions.
The labeling efficiency of this method was tested with the TMTzero label reagent (Thermo Scientific) followed by mass spectrometric analysis and determined to be higher than 98% (data not shown).
Data Processing Protocol
The acquired raw data was analyzed with Proteome Discoverer 2.1 (Thermo Scientific) en bloc. For the identification of the MS2 spectra, Sequest HT was utilized, with a 10 ppm precursor mass tolerance, 0.8 Da as fragment mass tolerance and as database, the Swissprot part of Uniprot for the species human was used (version 2016_01; 20145 entries). As dynamic modifications, oxidation (DeltaMass="15.99492") for all methionine residues was set and acetylation (DeltaMass="42.01057") for all protein N-termini. As static modifications were treated, the TMT-label (DeltaMass="229.16293") at the peptide N-terminus and all lysine residius and carbamidomethylation (DeltaMass="57.02146") at all cysteine residues. The fasle- dicovery-rate (FDR) for the peptide and consecutive protein assignments were performed by the included Percolator software package, based on a decoy database search and a strict cut -off of a 0.01 % FDR was applied on the protein level. Reporter ion quantification was performed in the MS3 channel with a 20 ppm mass tolerance and corrected for the isotopic impurities provided for TMT lot QD212963. For the area calculations, the top 3 peptides were used and maximum fold change was set to 1000. No minimum vales were imputed. For the individual proteins, only unique and razor peptides were used for quantification. Overall the quantification was normalized by the total peptide amount in each channel. The data was enriched by multiple annotations, including gene ontology (GO) terms directly by Proteome Discoverer 2.1. The data was filtered to contain only proteins with a FDR < 0.01 and being the “Master” or “Master - candidate” proteins in their respective protein group. The dataset was exported to Spotfire 3.2.2 (Tipco) for further processing. For the quantificatio n, the normalized intensities on the protein level were averaged between the technical replicates. A protein was only deemed as consistently and reproducibly quantified, if a quantification value was obtained in both technical replicates in more than half of the individual samples.
Müller S, Balaz M, Stefanicka P, Varga L, Amri EZ, Ukropec J, Wollscheid B, Wolfrum C. Proteomic Analysis of Human Brown Adipose Tissue Reveals Utilization of Coupled and Uncoupled Energy Expenditure Pathways. Sci Rep. 2016 Jul 15;6:30030 PubMed: 27418403