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23rd Human Proteome Organization World Congress

Metaproteomics from bench to clinic

Keynote speaker

Keynote lecture

Advancing medical diagnosis with fully untargeted proteomics-based virus identification

Microbiology and Microbiome Analysis

Introduction:The fast and accurate identification of microorganisms is essential for medical diagnosis. Traditional microbiological methods rely on cultivation, which is time-consuming and fails to detect all microorganisms. DNA amplification-based methods, such as PCR, require prior knowledge of the microorganism's genome and allow only the detection of the targeted species. MALDI-TOF only permits the detection of species included in a dedicated spectral library and performs poorly on unknown pathogens or in the presence of host material. In this context, a universal method that does not require prior knowledge of the sample components and is not hindered by host proteins would greatly advance the detection and identification of pathogens. Here, we present a groundbreaking tandem mass spectrometry proteotyping method for rapid and universal virus identification, eliminating the need for predefined targets, and permitting detection from multiple matrices of clinical interest.Methods:We evaluated our proteotyping method on a range of viruses including SARS-CoV-2, Vaccinia virus, and Monkeypox virus, among others. To demonstrate its clinical relevance, we tested the method using various clinically significant matrices, such as positive saliva and feces samples. Proteins were extracted and digested via microplate-adapted SP3 protocol. The resulting peptides were subjected to a 30 min LC-MS/MS analysis using a Vanquish Neo UHPLC coupled to an Exploris 480 mass spectrometer (ThermoFisher). The resulting spectra were analyzed using an in-house tool, based on a cascade search permitting the taxonomical identification of the sample components using a generalist database.Results:Based on the combination of shared and taxa-specific peptides, our method successfully identified all viruses. Additionally, the other Eukaryotic and Prokaryotic organisms present in the sample were identified. Notably, SARS-CoV-2 and Vaccinia virus were detected from saliva, a complex biological matrix, from 106 pfu/ml, a clinically relevant concentration. In addition, rotavirus from gastroenteritis fecal samples was successfully identified, along with the different microorganisms, depicting the full sample microbial pictures in a single query. The entire experimental workflow, including digestion in 96-well plates and short LC-MS/MS analyses is straightforwardly amenable to high throughput. Such detection procedures pave the way for a rapid, accurate, and non-invasive diagnosis.

Oral presentation

Mapping the Crosstalk Between Cellular or Viral-mediated Proteolysis and Ubiquitination and Phosphorylation During Norovirus Infection

Human noroviruses are the leading cause of viral gastroenteritis accounting for&gt;200,000 deaths per annum and major disruption to healthcare systems worldwide. Despite its medical relevance, our knowledge of human norovirus biology is limited given difficulties in propagating the virus life cycle in vitro. Alternatively, Murine Norovirus (MNV) represents a useful cell culture model for studying infection and replication of noroviruses. It&quot;s known that during MNV replication viral proteins can be post-translationally modified (PTM), with regulated proteolysis being one essential PTM for viral replication. MNV encodes a chymotrypsin-like protease (NS6 or 3CL protease) that mediates the proteolytic processing of the ORF1 polyprotein encoded in the viral genome, thus releasing functional non-structural proteins required for viral replication. Although polyprotein processing is essential and conserved in many other positive-sense single-stranded RNA (+ssRNA) viruses, mounting evidence suggests that other PTMs such as ubiquitination and phosphorylation a regulated during MNV replication playing an important role in the outcome of the infection. Therefore, this work aims to characterise quantitatively proteolytic processing of viral and cellular proteins via N-terminomics and investigate the interplay of proteolysis with protein ubiquitination and phosphorylation. For this, we performed a time course infection of murine BV2 cells with Murine Norovirus, collecting cells from 0-12 hours post-infection (psi) following LC-MS analysis. Our newly developed TMT-HUNTER methodology using multiple proteases was applied for the characterisation of neo-N-termini generated in response to MNV infection. PTM profiling, including ubiquitination and phosphorylation, was performed using both open search workflow of Fragpipe v22 software. Over 3,700 neo-N-termini were detected across all infection time points revealing a temporal regulation of proteolysis during MNV infection. Motif analysis showed that a fraction of neo-n-termini identified in murine proteins matched the conserved 3CL proteases cleavage motif, suggesting that cellular proteins are also substrates for viral proteases. On the other hand, neo-n-termini matching caspase cleavage motif has been detected in viral non-structural proteins, indicating a convoluted network in which proteases and their substrates can have either viral or cellular origin. Ubiquitinome and phosphoproteome profiling are being carried out to assess the interplay of these PTMs and proteolysis. Our N-terminomics results provided an expanded repertoire of cellular substrates potentially targeted by MNV protease. The role of the proteolysis substrates and other PTMs in subverting host cells and causing disease will be further investigated.

Population-based metaproteomics reveals functional associations between gut microbiota and phenotypes

The association of the gut microbial community with various physiological disorders underscores the significance of understanding its biological functions, which have yet to be fully explored. In this study, we characterized 86,876 microbial proteins and 1,277 human proteins in 1,967 Chinese stool specimens of GNHS cohort. Our findings demonstrate that, compared to metagenomic data, metaproteomic data can effectively capture the microbial biodiversity of an entire population using about 500 individuals. Additionally, it reveals greater inter-individual variability in microbial functions and serves as a more reliable indicator for assessing the temporal stability of the microbiota. We delineated the highly expressed functions of microbes among population and identified the core microbes across individuals that are likely to contribute to SCFA and vitamin B production. Moreover, our analysis discovered 10,714 associations between metaproteomic taxon, microbial function, or human proteins with 39 phenotypes, uncovering 1,381 microbial taxa, functions, and host proteins that related to Type 2 Diabetes (T2D). Among them, five features exhibited consistent and significant associations with T2D in both the GNHS cohort and an independent cohort. Finally, we demonstrated that Megasphaera elsdenii could mitigate elevated blood glucose levels in the host by metabolizing lactate to produce butyric acid, suggesting a protective mechanism against T2D. Our study underscores the power of population-based metaproteomics to offer unique functional insights into the gut microbiota.

Conference room 5-6

Session

Microbiome analysis in health and environment

Session
Microbiome analysis in health and environment

Appointment

Chairs

Program

SessionMicrobiome analysis in health and environment

Appointment

Chairs

Program

Session
Microbiome analysis in health and environment