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Data repository for commentary on Zuckerkandl et al. 1960

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.

Introduction Bats can harbor many pathogens without showing disease. However, the mechanisms by which bats resolve these infections or limit pathology remain unclear. To illuminate the bat immune response to coronaviruses, viruses with high public health significance, we will use serum proteomics to assess broad differences in immune proteins of uninfected and infected vampire bats (Desmodus rotundus). In contrast to global profiling techniques of blood such as transcriptomics, proteomics provides a unique perspective into immunology, as the serum proteome includes proteins from not only blood but also those secreted from proximal tissues. We will use enrichment analyses alongside generalized linear models of targeted serum proteins involved in important immune pathways to identify up- and downregulated bat immune responses to coronavirus shedding. Methods We sampled vampire bats in April 2019 in the Lamanai Archeological Reserve of northern Belize. For the 19 individuals included in this study, we used a harp trap and mist nets to capture bats upon leaving a single roost site. All individuals were identified by sex, age, and reproductive status. For serum proteomics, we collected blood by lancing the propatagial vein followed by collection into serum separator tubes prior to centrifugation and storage at −80 °C. Saliva and rectal swab samples were also collected and stored in DNA/RNA Shield for coronavirus detection. We used a semi-nested pan-coronavirus PCR targeting the RNA-dependent RNA polymerase gene (RdRp), followed by amplicon sequencing and NCBI BLAST to identify virus genera. Preliminary data Because CDC regulatory guidelines require any vampire bat samples to undergo heat inactivation (56 °C for 1 h) prior to importation into the U.S., we used four serum samples previously analyzed to evaluate the effects of this treatment on proteomic analysis. Since these prior samples had not been heat inactivated, a paired analysis of heat inactivation was possible (results not discussed in poster, will be in forthcoming paper). Next, using the 19 samples in the current coronavirus study, we used the S-trap method for tryptic digestion, followed by data-independent acquisition bottom-up proteomics at 300 nL/min with an Ultimate 3000 interfaced to a Fusion Lumos Orbitrap mass spectrometer. Data will be processed in a library-free manner with DIA-NN using the RefSeq annotated vampire bat genome. We identified 586 proteins over five orders of magnitude. Similar to our previous study, we converted the vampire bat protein identifications to human orthologs to aid in downstream analysis. Of our included 19 vampire bats, four were infected with coronaviruses, and phylogenetic analyses identified all as alphacoronaviruses specifically. We used receiver operator characteristic curves to identify candidate markers of alphacoronaviruses shedding status. Though this is a small sample set, it will provide invaluable information of the proteome changes, specifically immune related, in a natural host in response to alphacoronavirus. Novel aspect These results will provide much needed insight into changes in the bat serum proteome in response to coronavirus infection.

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.

The field of proteomics is on the cusp of realizing the promise of Data-Independent Acquisition (DIA), but there is still a need to develop best practices and improve opportunity for adoption. The Proteomics Research Group (PRG) within the Association of Biomolecular Resource Facilities (ABRF) conducted a study to promote adoption of DIA. We developed a study sample set consisting of a HeLa digest spiked with four non-human proteins at different concentrations. These samples were sent to 63 labs from 20 countries around the globe. We provided participants recommended LC gradients and DIA methods for specific instrument platforms, as well as guidelines for method generation on other platforms. Samples were injection-ready, and data was analyzed by PRG members. Of the 63 labs, 40 labs uploaded data as of January 2019, 53 labs filled out survey, and 35 data sets were used for preliminary analysis. Based on survey information, most participants were experienced LCMS users, but few had previously used DIA. Data from most participants reflects expected fold-change of spike-in proteins. Interestingly, accuracy of quantitation was not correlated to previous DIA experience. Overall, by providing a standard sample set and recommended methods for different instrument platforms, and extended trial software licenses for users, the PRG provided an opportunity for labs to evaluate their readiness to carry out DIA analysis. Ongoing work will analyze the complete data set as more labs completed the study after this pilot analysis, providing additional opportunities to evaluate LC and instrument parameters.