SUMMARY Protein interactions form a network whose structure drives cellular function and whose organization informs biological inquiry. Using high-throughput affinity-purification mass spectrometry, we identify interacting partners for 2,594 human proteins in HEK293T cells. The resulting network (BioPlex) contains 23,744 interactions among 7,668 proteins with 86% previously undocumented. BioPlex accurately depicts known complexes, attaining 80-100% coverage for most CORUM complexes. The network readily subdivides into communities that correspond to complexes or clusters of functionally related proteins. More generally, network architecture reflects cellular localization, biological process, and molecular function, enabling functional characterization of thousands of proteins. Network structure also reveals associations among thousands of protein domains, suggesting a basis for examining structurally-related proteins. Finally, BioPlex, in combination with other approaches can be used to reveal interactions of biological or clinical significance. For example, mutations in the membrane protein VAPB implicated in familial Amyotrophic Lateral Sclerosis perturb a defined community of interactors.

Multiplexed quantitation via isobaric chemical tags (e.g., tandem mass tags (TMT) and isobaric tags for relative and absolute quantitation (iTRAQ)) has the potential to revolutionize quantitative proteomics. However, until recently the utility of these tags was questionable due to reporter ion ratio distortion resulting from fragmentation of coisolated interfering species. These interfering signals can be negated through additional gas-phase manipulations (e.g., MS/MS/MS (MS3) and proton-transfer reactions (PTR)). These methods, however, have a significant sensitivity penalty. Using isolation waveforms with multiple frequency notches (i.e., synchronous precursor selection, SPS), we coisolated and cofragmented multiple MS2 fragment ions, thereby increasing the number of reporter ions in the MS3 spectrum 10-fold over the standard MS3 method (i.e., MultiNotch MS3). By increasing the reporter ion signals, this method improves the dynamic range of reporter ion quantitation, reduces reporter ion signal variance, and ultimately produces more high-quality quantitative measurements. To demonstrate utility, we analyzed biological triplicates of eight colon cancer cell lines using the MultiNotch MS3 method. Across all the replicates we quantified 8 378 proteins in union and 6 168 proteins in common. Taking into account that each of these quantified proteins contains eight distinct cell-line measurements, this data set encompasses 174 704 quantitative ratios each measured in triplicate across the biological replicates. Herein, we demonstrate that the MultiNotch MS3 method uniquely combines multiplexing capacity with quantitative sensitivity and accuracy, drastically increasing the informational value obtainable from proteomic experiments.

The physiology of a cell can be viewed as the product of thousands of proteins acting in concert to shape the cellular response. Coordination is achieved in part through networks of protein-protein interactions that assemble functionally related proteins into complexes, organelles, and signal transduction pathways. Understanding the architecture of the human proteome has the potential to inform cellular, structural, and evolutionary mechanisms and is critical to elucidation of how genome variation contributes to disease1–3. Here, we present BioPlex 2.0 (Biophysical Interactions of ORFEOME-derived complexes), which employs robust affinity purification-mass spectrometry (AP-MS) methodology4 to elucidate protein interaction networks and co-complexes nucleated by more than 25% of protein coding genes from the human genome, and constitutes the largest such network to date. With >56,000 candidate interactions, BioPlex 2.0 contains >29,000 previously unknown co-associations and provides functional insights into hundreds of poorly characterized proteins while enhancing network-based analyses of domain associations, subcellular localization, and co-complex formation. Unsupervised Markov clustering (MCL)5 of interacting proteins identified more than 1300 protein communities representing diverse cellular activities. Genes essential for cell fitness6,7 are enriched within 53 communities representing central cellular functions. Moreover, we identified 442 communities associated with more than 2000 disease annotations, placing numerous candidate disease genes into a cellular framework. BioPlex 2.0 exceeds previous experimentally derived interaction networks in depth and breadth, and will be a valuable resource for exploring the biology of incompletely characterized proteins and for elucidating larger-scale patterns of proteome organization.

Highlights d Quantified the proteomes of 375 cell lines from diverse lineages in the CCLE d Correlated expression of proteins across many pathways d Downregulation of multiple protein complexes in microsatellite instability d Protein complexes associated with sensitivity to gene knockdown and mutation

SUMMARY Thermogenic brown and beige adipose tissues dissipate chemical energy as heat, and their thermogenic activities can combat obesity and diabetes. Herein the functional adaptations to cold of brown and beige adipose depots are examined using quantitative mitochondrial proteomics. We identify arginine/creatine metabolism as a beige adipose signature and demonstrate that creatine enhances respiration in beige fat mitochondria when ADP is limiting. In murine beige fat, cold exposure stimulates mitochondrial Creatine Kinase activity and induces coordinated expression of genes associated with creatine metabolism. Pharmacological reduction of creatine levels decreases whole body energy expenditure after administration of a β3-agonist and reduces the adipose metabolic rate. Genes of creatine metabolism are compensatorily induced when UCP1-dependent thermogenesis is ablated, and creatine reduction in Ucp1-deficient mice reduces core body temperature. These findings link a futile cycle of creatine metabolism to adipose tissue energy expenditure and thermal homeostasis.

Brown adipose tissue (BAT) can dissipate chemical energy as heat through thermogenic respiration, which requires uncoupling protein 1 (UCP1)1,2. Thermogenesis from BAT and beige adipose can combat obesity and diabetes3, encouraging investigation of factors that control UCP1-dependent respiration in vivo. Herein we show that acutely activated BAT thermogenesis is defined by a substantial increase in mitochondrial reactive oxygen species (ROS) levels. Remarkably, this process supports in vivo BAT thermogenesis, as pharmacological depletion of mitochondrial ROS results in hypothermia upon cold exposure, and inhibits UCP1-dependent increases in whole body energy expenditure. We further establish that thermogenic ROS alter BAT cysteine thiol redox status to drive increased respiration, and Cys253 of UCP1 is a key target. UCP1 Cys253 is sulfenylated during thermogenesis, while mutation of this site desensitizes the purine nucleotide inhibited state of the carrier to adrenergic activation and uncoupling. These studies identify BAT mitochondrial ROS induction as a mechanism that drives UCP1-dependent thermogenesis and whole body energy expenditure, which opens the way to develop improved therapeutic strategies for combating metabolic disorders.

Multiplexed quantitative analyses of complex proteomes enable deep biological insight. While a multitude of workflows have been developed for multiplexed analyses, the most quantitatively accurate method (SPS-MS3) suffers from long acquisition duty cycles. We built a new, real-time database search (RTS) platform, Orbiter, to combat the SPS-MS3 method's longer duty cycles. RTS with Orbiter eliminates SPS-MS3 scans if no peptide matches to a given spectrum. With Orbiter's online proteomic analytical pipeline, which includes RTS and false discovery rate analysis, it was possible to process a single spectrum database search in less than 10 ms. The result is a fast, functional means to identify peptide spectral matches using Comet, filter these matches, and more efficiently quantify proteins of interest. Importantly, the use of Comet for peptide spectral matching allowed for a fully featured search, including analysis of post-translational modifications, with well-known and extensively validated scoring. These data could then be used to trigger subsequent scans in an adaptive and flexible manner. In this work we tested the utility of this adaptive data acquisition platform to improve the efficiency and accuracy of multiplexed quantitative experiments. We found that RTS enabled a 2-fold increase in mass spectrometric data acquisition efficiency. Orbiter's RTS quantified more than 8000 proteins across 10 proteomes in half the time of an SPS-MS3 analysis (18 h for RTS, 36 h for SPS-MS3).

Artificial human gut microbial communities implanted into germ-free mice provide insights into how species-level responses to changes in diet give rise to community-level structural and functional reconfiguration and how types of bacteria prioritize use of available nutrients in vivo.