Unified workflow for the rapid and in-depth characterization of bacterial proteomes

ABSTRACT: Among the kingdoms of life, bacteria are by far the most abundant, but also the most diverse organisms. Due to this excessive diversity, finding a unified, comprehensive and safe workflow for quantitative bacterial proteomics is highly challenging. In this study, we have systematically evaluated and optimized sample preparation, mass spectrometric data acquisition and data analysis strategies in the context of bacterial proteomics. In order to mimic bacterial diversity, we investigated workflow performances on six representative species with highly different physiologic properties. The best sample preparation strategy was a cell lysis protocol in 100% trifluoroacetic acid followed by an in solution digest. Subsequently, proteomes were acquired in a 30-minute microflow data-independent acquisition mass spectrometric measurement and analyzed with DIA-NN using a predicted spectral library ('library-free'). Performance was evaluated according to the number of identified proteins, quantitative precision, throughput, costs and biological safety. With this rapid workflow, on average over 40% of all encoded genes could be detected per bacterial species. Finally, we demonstrate the general applicability of our workflow on a set of 23 taxonomically and physiologically bacterial species. In this dataset we could confidently identify over 30,000 bacterial proteins which have not been described before. Our work thereby provides a valuable resource for the microbial scientific community. The fast and biological safe proteomic workflow we present in this manuscript does not require any specialized equipment or commercial software and can be easily applied by other laboratories to support and accelerate the proteomic exploration of the bacterial kingdom

INSTRUMENT(S): Orbitrap Fusion Lumos, Q Exactive HF-X, Orbitrap Exploris 480

ORGANISM(S): Bacillus Subtilis (ncbitaxon:1423) Enterococcus Faecalis (ncbitaxon:1351) Vibrio Proteolyticus (ncbitaxon:671) Lactobacillus Delbrueckii (ncbitaxon:1584) Burkholderia Cepacia (ncbitaxon:292) Streptococcus Mitis (ncbitaxon:28037) Staphylococcus Aureus (ncbitaxon:1280) Escherichia Coli (ncbitaxon:562) Bifidobacterium Bifidum (ncbitaxon:1681) Sphingobacterium Spiritivorum (ncbitaxon:258) Deinococcus Radiodurans (ncbitaxon:1299) Prevotella Melaninogenica (ncbitaxon:28132) Micrococcus Luteus (ncbitaxon:1270) Cutibacterium Acnes (ncbitaxon:1747) Brevundimonas Diminuta (ncbitaxon:293) Pseudomonas Aeruginosa (ncbitaxon:287) Anoxybacillus Flavithermus (ncbitaxon:33934) Acetobacter Aceti (ncbitaxon:435) Mycolicibacterium Smegmatis (ncbitaxon:1772) Listeria Monocytogenes (ncbitaxon:1639) Clostridium Sporogenes (ncbitaxon:1509) Bacillus Cereus (ncbitaxon:1396) Corynebacterium Glutamicum (ncbitaxon:1718)

SUBMITTER: Christina Ludwig  

PROVIDER: MSV000091088 | MassIVE | Wed Jan 18 09:18:00 GMT 2023

SECONDARY ACCESSION(S): PXD039543

REPOSITORIES: MassIVE