Biology subjects

Miotello, G.

Publications and source records attributed to Miotello, G..

5 recordsLinked to original sources

bioRxiv2025

Critical Assessment of MetaProteome Investigation 2 (CAMPI-2): Multi-laboratory assessment of sample processing methods to stabilize fecal microbiome for functional analysis

BackgroundFecal samples are widely used as a proxy for studying gut microbiome composition in both human and animal research. Fecal metaproteomics provides valuable insights by tracking changes in the relative abundance of microbial taxa and their protein functions. To ensure reliable results, it is crucial to minimize alterations in the metaproteome occurring from sample collection to protein extraction. Therefore, employing effective stabilization methods is essential to preserve the integrity of the fecal metaproteome from sample collection to laboratory analysis, particularly over long distances or when rapid freezing options are not readily available. In line with these needs, the second edition of the Critical Assessment of MetaProteome Investigation (CAMPI-2) was specifically focused on testing sample stabilization protocols to be applied before metaproteomic analysis. ResultsThis collaborative multicenter study assessed the ability of five different stabilization methods, based on two commercial devices and three specific reagents (acetone, lithium dodecyl sulfate, and an RNAlater-like buffer), respectively, to stabilize the fecal metaproteome during room-temperature storage (14 days) and shipment to mass spectrometry facilities. The five methods were tested simultaneously by eight different laboratories across Europe, using aliquots from the same fecal sample. After protein extraction and digestion, duplicate aliquots of the resulting peptides were analyzed independently by two mass spectrometry facilities at distinct international locations. Analysis of the mass spectrometric data using two different search engines revealed that the fecal metaproteome profile differed considerably depending on the stabilization method used in terms of richness, alpha and beta diversity, reproducibility, and quantitative distribution of main taxa and functions. Although each method showed unique strengths and weaknesses, a commercial swab-based device stood out for its remarkable reproducibility and ranked highest for most of the metrics measured. ConclusionsCAMPI-2 allowed a robust evaluation of five different methods for preserving fecal metaproteome samples. The present investigation provides useful data for the design of metaproteomics and multi-omics studies where fecal sampling cannot be immediately followed by long-term storage at -80{degrees}C. Further optimization of the tested protocols is necessary to improve stabilization efficiency and control bias in the taxonomic and functional profile of the gut microbiome.

microbiology↗

bioRxiv2021

Critical Assessment of Metaproteome Investigation (CAMPI): a Multi-Lab Comparison of Established Workflows

Metaproteomics has matured into a powerful tool to assess functional interactions in microbial communities. While many metaproteomic workflows are available, the impact of method choice on results remains unclear. Here, we carried out the first community-driven, multi-laboratory comparison in metaproteomics: the critical assessment of metaproteome investigation study (CAMPI). Based on well-established workflows, we evaluated the effect of sample preparation, mass spectrometry, and bioinformatic analysis using two samples: a simplified, laboratory-assembled human intestinal model and a human fecal sample. We observed that variability at the peptide level was predominantly due to sample processing workflows, with a smaller contribution of bioinformatic pipelines. These peptide-level differences largely disappeared at the protein group level. While differences were observed for predicted community composition, similar functional profiles were obtained across workflows. CAMPI demonstrates the robustness of present-day metaproteomics research, serves as a template for multi-laboratory studies in metaproteomics, and provides publicly available data sets for benchmarking future developments.

microbiology↗

bioRxiv2020

Proteotyping SARS-CoV-2 virus from nasopharyngeal swabs: a proof-of-concept focused on a 3 min mass spectrometry window

Rapid but yet sensitive, specific and high-throughput detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in clinical samples is key to diagnose infected people and to better control the spread of the virus. Alternative methodologies to PCR and immunodiagnostic that would not require specific reagents are worth to investigate not only for fighting the COVID-19 pandemic, but also to detect other emergent pathogenic threats. Here, we propose the use of tandem mass spectrometry to detect SARS-CoV-2 marker peptides in nasopharyngeal swabs. We documented that the signal from the microbiota present in such samples is low and can be overlooked when interpreting shotgun proteomic data acquired on a restricted window of the peptidome landscape. Simili nasopharyngeal swabs spiked with different quantities of purified SARS-CoV-2 viral material were used to develop a nanoLC-MS/MS acquisition method, which was then successfully applied on COVID-19 clinical samples. We argue that peptides ADETQALPQR and GFYAQGSR from the nucleocapsid protein are of utmost interest as their signal is intense and their elution can be obtained within a 3 min window in the tested conditions. These results pave the way for the development of time-efficient viral diagnostic tests based on mass spectrometry.

microbiology↗

bioRxiv2020

Shotgun proteomics of SARS-CoV-2 infected cells and its application to the optimisation of whole viral particle antigen production for vaccines

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has resulted in a pandemic and continues to spread quickly around the globe. Currently, no effective vaccine is available to prevent COVID-19 and an intense global development activity is in progress. In this context, the different technology platforms face several challenges resulting from the involvement of a new virus still not fully characterised. Finding of the right conditions for virus amplification for the development of vaccines based on inactivated or attenuated whole viral particles is among them. Here, we describe the establishment of a workflow based on shotgun tandem mass spectrometry data to guide the optimisation of the conditions for viral amplification. In parallel, we analysed the dynamic of the host cell proteome following SARS-CoV-2 infection providing a global overview of biological processes modulated by the virus and that could be further explored to identify drug targets to address the pandemic.

microbiology↗

bioRxiv2018

The Rhodobacter sphaeroides methionine sulfoxide reductase MsrP can reduce R- and S- diastereomers of methionine sulfoxide from a broad-spectrum of protein substrates.

Methionine (Met) is prone to oxidation and can be converted to Met sulfoxide (MetO), which exists as R- and S-diastereomers. MetO can be reduced back to Met by the ubiquitous methionine sulfoxide reductase (Msr) enzymes. Canonical MsrA and MsrB were shown as absolutely stereospecific for the reduction of S- and R-diastereomer, respectively. Recently, the molybdenum-containing protein MsrP, conserved in all gram-negative bacteria, was shown to be able to reduce MetO of periplasmic proteins without apparent stereospecificity in Escherichia coli. Here, we describe the substrate specificity of the Rhodobacter sphaeroides MsrP. Proteomics analysis coupled to enzymology approaches indicate that it reduces a broad spectrum of periplasmic oxidized proteins. Moreover, using model proteins, we demonstrated that RsMsrP preferentially reduces unfolded oxidized proteins and we confirmed that this enzyme, like its E. coli homolog, can reduce both R- and S-diastereomers of MetO with similar efficiency.

biochemistry↗