Biology subjects

Peters, S. L.

Publications and source records attributed to Peters, S. L..

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↗

bioRxiv2024

Sporadic distribution of a new archaeal genetic code with all TAG codons as pyrrolysine

Numerous genetic codes developed during the evolution of Eukaryotes and three are known in Bacteria, yet no alternative genetic code has been established for Archaea. Some bacterial and archaeal proteins include selenocysteine or pyrrolysine, the 21st and 22nd amino acids, but no evidence establishes the adoption of a genetic code in which a stop codon universally encodes either amino acid. Here, we used proteomics to confirm the prediction that certain Archaea consistently incorporate pyrrolysine at TAG codons, supporting a new archaeal genetic code which we designate Genetic Code 34. This genetic code has 62 sense codons encoding 21 amino acids, and only two stop codons. In contrast with monophyletic genetic code distributions in bacteria, Code 34 occurs sporadically. This, combined with evidence for lateral gene transfer of the code change machinery and anticipated barriers to code reversal, suggests Code 34 arose independently in multiple lineages. TAG codon distribution patterns in Code 34 genomes imply a wide range in time since code switch. We identified many new enzymes containing Pyl residues, raising questions about potential roles of this amino acid in protein structure and function. We used five new PylRS/tRNAPyl pairs from Code 34 archaea to introduce new-to-nature pyrrolysine analogs into proteins in E. coli, demonstrating their utility for genetic code expansion. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=157 SRC="FIGDIR/small/615893v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@f363a5org.highwire.dtl.DTLVardef@112f061org.highwire.dtl.DTLVardef@63ad59org.highwire.dtl.DTLVardef@9eddb_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

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

Microbiota functional activity biosensors for characterizing nutrient utilization in vivo

Methods for measuring gut microbiota biochemical activities in vivo are needed to characterize its functional states in health and disease. To illustrate one approach, an arabinan-containing polysaccharide was purified from pea fiber, its structure defined, and forward genetic and proteomic analyses used to compare its effects, versus unfractionated pea fiber and sugar beet arabinan, on a human gut bacterial strain consortium in gnotobiotic mice. We produced Microbiota Functional Activity Biosensors (MFABs) consisting of glycans covalently-linked to the surface of fluorescent paramagnetic microscopic glass beads. Three MFABs, each containing a unique glycan/fluorophore combination, were simultaneously orally gavaged into gnotobiotic mice, recovered from their intestines, and analyzed to directly quantify bacterial metabolism of structurally distinct arabinans in different human diet contexts. Colocalizing pea-fiber arabinan and another polysaccharide (glucomannan) on the bead surface enhanced in vivo metabolism of glucomannan. MFABs represent a potentially versatile platform for developing new prebiotics and more nutritious foods.

microbiology↗

bioRxiv2020

Genetic and behavioral adaptation of Candida parapsilosis to the microbiome of hospitalized infants revealed by in situ genomics, transcriptomics and proteomics

Candida parapsilosis is a common cause of invasive candidiasis, especially in newborn infants, and infections have been increasing over the past two decades. C. parapsilosis has been primarily studied in pure culture, leaving gaps in understanding of its function in microbiome context. Here, we reconstructed five unique C. parapsilosis genomes from premature infant fecal samples and analyzed their genome structure, population diversity and in situ activity relative to reference strains in pure culture. All five genomes contain hotspots of single nucleotide variants, some of which are shared by strains from multiple hospitals. A subset of environmental and hospital-derived genomes share variants within these hotspots suggesting derivation of that region from a common ancestor. Four of the newly reconstructed C. parapsilosis genomes have four to sixteen copies of the gene RTA3, which encodes a lipid translocase and is implicated in antifungal resistance, potentially indicating adaptation to hospital antifungal use. Time course metatranscriptomics and metaproteomics on fecal samples from a premature infant with a C. parapsilosis blood infection revealed highly variable in situ expression patterns that are distinct from those of similar strains in pure cultures. For example, biofilm formation genes were relatively less expressed in situ, whereas genes linked to oxygen utilization were more highly expressed, indicative of growth in a relatively aerobic environment. In gut microbiome samples, C. parapsilosis coexisted with Enterococcus faecalis that shifted in relative abundance over time, accompanied by changes in bacterial and fungal gene expression and proteome composition. The results reveal potentially medically relevant differences in Candida function in gut vs. laboratory environments, and constrain evolutionary processes that could contribute to hospital strain persistence and transfer into premature infant microbiomes.

microbiology↗