Tandem mass spectrometry-based proteotyping allows characterizing microorganisms in terms of taxonomy and is becoming an important tool for investigating microbial diversity from several ecosystems. Fast and automatable sample preparation for obtaining peptide pools amenable to tandem mass spectrometry is necessary for enabling proteotyping as a high-throughput method. First, the protocol to increase the yield of lysis of several representative bacterial and eukaryotic microorganisms was optimized by using a long and drastic bead-beating setting with 0.1 mm silica beads, 0.1 and 0.5 mm glass beads, in presence of detergents. Then, three different methods to obtain greater digestion yield from these extracts were tested and optimized for improve efficiency and reduce application time: denaturing electrophoresis of proteins and in-gel proteolysis, suspension-trapping filter-based approach (S-Trap) and, solid-phase-enhanced sample preparation named SP3. The latter method outperforms the other two in terms of speed and delivers also more peptides and proteins than with the in-gel proteolysis (2.2 fold for both) and S-trap approaches (1.3 and 1.2 fold, respectively). Thus, SP3 directly improves tandem mass spectrometry proteotyping.

Metaproteomics is an emerging approach for studying microbiomes, offering the ability to characterize proteins that underpin microbial functionality within diverse ecosystems. As the primary catalytic and structural components of microbiomes, proteins provide unique insights into the active processes and ecological roles of microbial communities. By integrating metaproteomics with other omics disciplines, researchers can gain a comprehensive understanding of microbial ecology, interactions, and functional dynamics. This review, developed by the Metaproteomics Initiative (www.metaproteomics.org), serves as a practical guide for both microbiome and proteomics researchers, presenting key principles, state‐of‐the‐art methodologies, and analytical workflows essential to metaproteomics. Topics covered include experimental design, sample preparation, mass spectrometry techniques, data analysis strategies, and statistical approaches.

Post-transcriptional control of protein abundance is a highly important, underexplored regulatory process by which organisms respond to their environments. Here we describe an important and previously unidentified regulatory pathway involving the ribosomal modification protein RimK, its regulator proteins RimA and RimB, and the widespread bacterial second messenger cyclic-di-GMP (cdG). Disruption of rimK affects motility and surface attachment in pathogenic and commensal Pseudomonas species, with rimK deletion significantly compromising rhizosphere colonisation by the commensal soil bacterium P. fluorescens, and plant infection by the pathogens P. syringae and P. aeruginosa. RimK functions as an ATP-dependent glutamyl ligase, adding glutamate residues to the C-terminus of ribosomal protein RpsF and inducing specific effects on both ribosome protein complement and function. Deletion of rimK in P. fluorescens leads to markedly reduced levels of multiple ribosomal proteins, and also of the key translational regulator Hfq. In turn, reduced Hfq levels induce specific downstream proteomic changes, with significant increases in multiple ABC transporters, stress response proteins and non-ribosomal peptide synthetases seen for both ΔrimK and Δhfq mutants. The activity of RimK is itself controlled by interactions with RimA, RimB and cdG. We propose that control of RimK activity represents a novel regulatory mechanism that dynamically influences interactions between bacteria and their hosts; translating environmental pressures into dynamic ribosomal changes, and consequently to an adaptive remodeling of the bacterial proteome. Post-transcriptional control of protein abundance is a significant and underexplored regulatory process by which organisms respond to environmental change. We have discovered an important new mechanism for this control in bacteria, based on the covalent modification of a small ribosomal protein by the widespread enzyme RimK. Here we show that the activity of RimK has specific effects on the levels of ribosomal proteins in the cell, which in turn affects the abundance of the important translational regulator Hfq. RimK is itself controlled by binding to the small regulatory proteins RimA and RimB and the widespread signalling molecule cyclic-di-GMP. Deletion of rimK compromises motility, virulence and plant colonisation/infection in several different Pseudomonas species. We propose that changes in intracellular RimK activity enable Pseudomonas to respond to environmental pressures by changing the nature of their ribosomes, leading in turn to an adaptive phenotypic response to their surroundings. This promotes motility and virulence during the initial stages of plant contact, and phenotypes including attachment, metabolite transport and stress control during long-term environmental adaptation.

Detection of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is a crucial tool for fighting the COVID‐19 pandemic. This dataset brief presents the exploration of a shotgun proteomics dataset acquired on SARS‐CoV‐2 infected Vero cells. Proteins from inactivated virus samples were extracted, digested with trypsin, and the resulting peptides were identified by data‐dependent acquisition tandem mass spectrometry. The 101 peptides reporting for six viral proteins were specifically analyzed in terms of their analytical characteristics, species specificity and conservation, and their proneness to structural modifications. Based on these results, a shortlist of 14 peptides from the N, S, and M main structural proteins that could be used for targeted mass‐spectrometry method development and diagnostic of the new SARS‐CoV‐2 is proposed and the best candidates are commented.

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has resulted in a pandemic and is continuing to spread rapidly around the globe. No effective vaccine is currently available to prevent COVID-19, and intense efforts are being invested worldwide into vaccine development. In this context, all technology platforms must overcome several challenges resulting from the use of an incompletely characterized new virus. These include finding the right conditions for virus amplification for the development of vaccines based on inactivated or attenuated whole viral particles. Here, we describe a shotgun tandem mass spectrometry workflow, the data produced can be used to guide optimization of the conditions for viral amplification. In parallel, we analysed the changes occurring in the host cell proteome following SARS-CoV-2 infection to glean information on the biological processes modulated by the virus that could be further explored as potential drug targets to deal with the pandemic.