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Research Group Presentation at the 2022Association of Biomolecular Resource Facilities (ABRF) Annual Meeting. One of the primary missions of the Proteomics Research Group (PRG) is education and outreach through multi-laboratory studies. This year, the focus of our planned study is glycoproteomics using egg whites to provide an easily accessible material for an multi-lab study. Here, we discuss the planned study motivation, preliminary design, and goals as part of announcing the study to the community.&nbsp

Introduction Beer is one of the oldest and most widely consumed beverages in the world. It contains a complex mixture of proteins from several organisms including plants (barley, hops, rice, wheat) and yeast, depending on the beer. The Proteomics Research Group (PRG) in the Association of Biomolecular Resource Facilities brought together an international consortium of proteomics laboratories (69 laboratories from 33 countries) to perform beer proteomics. An aliquot of specially brewed beer from the UC Davis Brewing Program was sent to 52 labs, and participants were encouraged to also use a widely available commercial beer (Heineken) and any other beer. In addition to helping connect scientists in trying times, we have also demonstrated the utility of multi-center studies with accessible materials. Methods Control beer brewed at UC Davis was shipped to participating laboratories at room temperature. Where shipping proved impractical, a widely available commercial beer was suggested as an alternative (Heineken). The PRG suggested a general digestion method, approximately 1 h (± 15 min) LC gradient, and using data-dependent acquisition. The suggested method consisted of precipitation, reduction, alkylation, and digestion with trypsin or LysC plus trypsin, but participants could use other methods. Raw mass spectrometry data (357 injections) and methods were deposited to MassIVE (MassIVE MSV000088080). Database searching was performed with MetaMorpheus against five species databases (barley, hops, rice, wheat, yeast) and contaminants. Mass tolerances were set automatically, carbamidomethylation was fixed, oxidized methionine was variable, and a 1 % FDR identification cutoff. Preliminary Data Of the 69 laboratories that signed up for the study, 35 returned data. Of these, 32 analyzed the PRG Beer and 17 analyzed Heineken (14 analyzed both). When shipping of the PRG Beer was prohibitive, participants were encouraged to use Heineken due to its global availability and perceived quality control. Participants were also encouraged to analyze other beers of their choosing, and 79 other beers were analyzed. In total there were 357 beer injections on 13 different types of mass spectrometers around the world. On average, 753.1 proteins were identified using MetaMorpheus, with the most being 2907 identified in the PRG Beer with a Thermo QE classic. Though database searching used non-UniProtKB non-RefSeq databases for barley, hops, and wheat, a downstream orthology conversion with BLAST found that despite their small size, the UniProtKB databases of these species adequately describe the mass specomtery data. Next, the 50 most abundant proteins identified in the PRG Beer and Heineken will be used for unsupervised clustering to determine how well beer proteomics performs across the world using the same or very similar beer. Finally, we will use the compareMS2 software tool to cluster data between beers and labs in an ID-free method. Though these studies focused only on the main grain additives to yeast, future work will expand the search space to include microorganisms that are part of the brewing process in some beers (i.e., bacteria in sours). Novel Aspect Multi-center proteomic studies using accessible materials, such as beer, can be successful, generate valuable results, and facilitate community building.

Research Group Presentation at the 2021 Virtual Association of Biomolecular Resource Facilities (ABRF) Annual Meeting. One of the primary missions of the Proteomics Research Group (PRG) is education and outreach through multi-laboratory studies. Here we discuss the status of past, present and future studies. The data from 45 participants in the 2019 DIA study is available on MassIVE, described on bioRxiv, and is currently in review. Last fall we engaged the community in a "bridge" study, resulting in 32 labs conducting beer proteomics and returning data, including that of a control beer. Finally, we are planning our next study that will focus on glycoproteomic methods and analysis. These studies highlight the engagement of the ABRF community, and future opportunities.

Additional file 6: Figure S6. Accumulation of intracellular defects in WE-68 cells. WE-68 cells were treated with 45 nM AUY922 (B), 2 µM VE821 (C) and their combination (D). DMSO was used for control (A). Intracellular structures were analyzed by transmission electron microscopy (TEM) and are labeled in red: lysosome (lyso), mitochondria (mito), endoplasmic reticulum (ER), nucleus (nuc), autophagosomes (auto), β-glycagon granule (glycagon), lipid droplets, vesicles and lipid-filled vesicles. Red arrows indicate sites of cell rupture. TEM pictures show one representative experiment.

Additional file 4: Figure S4. Analysis of molecular alterations. (A) Otherwise isogenic p53 wild-type (wt) and p53 null (p53-/-) HCT116 cells were treated with 45 nM AUY922 ± 2 µM VE821 for 24 h. Analysis of indicated proteins was done by Western blot. ⍺-tubulin and vinculin were used to control protein loading. Immunoblots are representative for at least two independent experiments. (B) Schematic workflow of the BALB/c cell transformation assay (BALB-CTA). WE-68 cells were treated with 30 nM of AUY922, 1 µM of VE821, 7.5 µM of KU55933 and their combinations; A673 cells were treated with 15 nM of AUY922, 1 µM of VE821, 5 µM of KU55933 and their combinations. DMSO was used for control. (C-D) The mRNA expression of indicated gene was analyzed after 24 h by qPCR. All graphs show the mean ± SEM of three independent experiments (* p

Additional file 3: Figure S3: Analysis of cell cycle distribution. (A) WE-68 and (B) A673 cells were treated with 15–45 nM of AUY922, 2 µM of VE821, 5 µM of KU55933 and their combinations for 24 h. (C-E) WE-68 and A673 cells were treated with indicated concentrations of AUY922 (C), VE821 (D) or KU55933 (E) for 48 h. Cell cycle distributions were determined by flow-cytometric analysis of PI-stained ethanol-fixed cells. All graphs show the mean ± SEM of at least two independent experiments.

Additional file 7: Figure S7. Accumulation of intracellular defects in A673 cells. A673 cells were treated with 45 nM AUY922 (B), 2 µM VE821 (C) and their combination (D). DMSO was used for control (A). Intracellular structures were analyzed by transmission electron microscopy (TEM) and are labeled in red: lysosome (lyso), mitochondria (mito), endoplasmic reticulum (ER), nucleus (nuc), autophagosomes (auto), β-glycagon granule (glycagon), lipid droplets, vesicles and lipid-filled vesicles. A673 cells were treated with 0.4–5 µg/ml of tunicamycin for 24 h. (E) Analysis of indicated proteins was done by Western blot. α-tubulin was used to control protein loading. TEM pictures show one representative experiment; immunoblots are representative for at least two independent experiments.

Additional file 10: Figure S10. Ingenuity pathway analysis of the proteome data set. A673 cells were treated with 45 nM AUY922 ± 2 µM VE821 for 24 h, and a quantitative whole proteome analysis was done by mass spectrometry from three individual experiments. The proteome data set was further processed by ingenuity pathway analysis (IPA; cutoff: q