Data independent acquisition (DIA) mass spectrometry is a powerful technique that is improving the reproducibility and throughput of proteomics studies. We introduce a new experimental workflow that uses this technique to construct chromatogram libraries that capture fragment ion chromatographic peak shape and retention time for every detectable peptide in an experiment. These coordinates calibrate information in spectrum libraries or protein databases to a specific mass spectrometer and chromatography setup, and enable sensitive peptide detection in quantitative experiments. We also present EncyclopeDIA, a software tool for generating and searching chromatogram libraries, and demonstrate the performance of our workflow by quantifying proteins in human and yeast cells. We find that by exploiting calibrated retention time and fragmentation specificity in chromatogram libraries, EncyclopeDIA can detect and quantify >50% more peptides from DIA experiments than with DDAbased spectrum libraries alone.

Stimulating brown adipose tissue (BAT) energy expenditure could be a therapy for obesity and related metabolic diseases. Achieving this requires a systems-level understanding of the biochemical underpinnings of thermogenesis. To identify novel metabolic features of active BAT, we measured protein abundance, protein acetylation, and metabolite levels in BAT isolated from mice living in their thermoneutral zone or in colder environments. We find that the enzymes which synthesize lipids from cytosolic acetyl-coA are among the most robustly increased proteins after cold acclimation, consistent with recent studies highlighting the importance of anabolic de novo lipogenesis in BAT. In addition, many mitochondrial proteins are hyperacetylated by cold acclimation, including several sites on UCP1, which may have functional relevance. Metabolomics analysis further reveals cold-dependent increases to acetylated carnitine and several amino acids. This BAT multi-omics resource highlights widespread proteomic and metabolic changes linked to acetyl-CoA synthesis and utilization that may be useful in unraveling the remarkable metabolic properties of active BAT. RESULTS Cold adaptation upregulates lipid synthesis, glucose metabolism, and electron transport chain complex I-II proteinsFirst, we explored proteomic signatures of chronic cold-induced BAT activation by acclimating mice to three different temperature conditions: thermoneutrality (30°C, which is preferred by mice 14 ), room temperature (which is mildly cold for mice, 22°C) and severe cold (6°C) (Fig 1A). Following the temperature acclimation period, BAT was harvested and subjected to proteome analysis. The number of confidently detected proteins is consistent across all samples, ranging from 1857 to 2432 ( Fig 1B). Differential protein expression was determined by comparing each cold condition to thermoneutrality. We identified 60 proteins altered (~3%, 26 increasing, 34 decreasing) in response to mild cold and 232 proteins altered (~11%, 119 increasing, 113 decreasing) in response to severe cold ( Fig 1C) indicating a thermogenic proteome response that scales with cold severity.We would like to thank all members of the Villen lab for critical discussions and feedback on the project. This work was supported by NIH grants R01 CA196986 (to D.A.G.); R35 GM119536, R01 AG056359, and R01 NS098329 (to J.V.); R37 DK030898 and R01 DK103047 (to M.P.C.). S.W.E. and R.T.L were both supported by Samuel and Althea Stroum Endowed Graduate Fellowships.