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Activity-Targeted SIP-Metaproteomics of a Full-Scale Biogas Facility

ABSTRACT: In this work, two BONCAT enrichment techniques were evaluated against a conventional untargeted metaproteomics approach for their ability to enrich the SIP signal of SAOB in metaproteomics data from an anaerobic microbiome. The first BONCAT enrichment was based on sorting active cells using fluorescent activated cell sorting (FACS) prior to protein extraction for mass spectrometry. The second BONCAT technique involved protein extraction from bulk biomass followed by purification of newly-translated proteins using click chemistry and a biotin-streptavidin bead pulldown assay prior to preparation for mass spectrometry. We found that both of these techniques improved detection of a highly-active and low-abundance syntrophic bacterium that was responsible for significant acetate turnover within the study full-scale biogas facility. The BONCAT-SIP approaches described here therefore represent a powerful toolbox for unraveling the contributions of active microorganisms to microbial communities--a facet of microbial ecology that holds great potential for improving our comprehension of the untapped metabolic potentials contained within microbiomes. Mass spectra data were processed using quantms v1.3.1 of the nf-core collection of workflows, utilizing reproducible software environments from the Bioconda and Biocontainers projects and tools from OpenMS v3.2.0. The workflow was executed with Nextflow v24.04.4. Both 12C-acetate (non-SIP) and 13C-acetate (SIP) samples from all metaproteomic methods were treated identically for peptide identification. Mass spectra were searched against the metagenome-derived protein database using both MSGF+ v2021.03.22 and Comet v2023.01. A target-decoy competition strategy was employed to estimate the rate of false positive peptide-spectrum matches (PSMs). Mass tolerances were set to 5 ppm for precursor (peptide) ions and 0.03 Da for fragment ions. Up to two missed cleavages were allowed and peptide termini were required to match the tryptic cleavage conditions fully. The precursor charge was limited to 2-4 and the peptide length was limited to 6-40. The PSMs were then rescored using Percolator v3.05.0. The two search engine results were combined by retaining only the peptide sequence with the highest E-value for each spectrum and the consensus list of PSMs was filtered with a false discovery rate (FDR) threshold of 10% to reduce decoy and false positive matches. Quantification of peptides was performed on the 12C-acetate (non-SIP) samples using the OpenMS tool, proteomicsLFQ. Peptide intensities were scaled such that the median peptide intensity in every sample was equal. The peptides are then evaluated for uniqueness and used for protein inference. The quantification results were filtered with a protein FDR of 1% and a PSM FDR of 1%. Detection of isotopically labeled peptides in the labeled condition (13C-acetate) was performed using MetaProSIP v2.3.0 as implemented in OpenMS v2.8.0 in a KNIME Analytics Platform v4.6.3 workflow. The unlabeled condition (12C-acetate) paired samples were used to identify coeluting isotopologues in the labeled condition (13C-acetate).

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

ORGANISM(S): Anaerobic Digester Metagenome (ncbitaxon:1263854)

SUBMITTER: Ryan Ziels

PROVIDER: MSV000096205 | MassIVE | Thu Oct 24 18:06:00 BST 2024

SECONDARY ACCESSION(S): PXD057167

REPOSITORIES: MassIVE

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Project description:Project description In many anoxic environments, syntrophic acetate oxidation (SAO) is a key pathway mediating the conversion of acetate into methane, and is accomplished through obligate mutualistic cross-feeding between SAO bacteria and methanogenic archaea. This pathway is particularly important in anaerobic digestion (AD) systems operating at thermophilic temperatures and/or with high ammonia. Despite the widespread importance of the SAO functional guild to the stability and efficiency of the AD process, little is known about their in situ ecophysiologies due to their difficulty of isolation as well as low biomass abundances. Here, we performed a long-term (300 day) continuous enrichment of a thermophilic (55C) SAO community from a municipal AD system. Over 80% of the enriched bioreactor metagenome collectively belonged to a three-member consortium, including an acetate-oxidizing DTU068 bacterium encoding for CO2, H2, and formate production, along with two methanogenic Methanothermobacter_A archaeal species. Stable isotope probing was coupled with metaproteogenomics to quantify carbon flux into each community member during acetate conversion and inform metabolic reconstruction and genome-scale modeling. Sample processing After 300 days of chemostat operation, batch microcosms were established in 40 ml glass serum bottles flushed with 80:20 N2:CO2 by anoxically transferring 18 ml of digestate from a single bioreactor (R2) and sealing with butyl rubber septa. Four different incubation conditions were established in triplicate: (1) blank control (e.g. no amendment); (2) 50 mM [12C]-acetate; (3) 50 mM [2 13C]-acetate (e.g. methyl-labeled); (4) 50 mM [1,2 13C]-acetate (universally labeled). Acetate was added to the microcosms (2 ml) as anoxic sterilized basal medium containing [12C], [2 13C], or [1,2 13C] sodium acetate (isotope purity >98%, Cambridge Isotopes). Twelve replicate bottles were established for all universally-labeled acetate-amended microcosms, allowing for three triplicate sets to be sacrificed for protein extraction at 24h, 144 hr, and 408 hr, and a single triplicate set for liquid sampling throughout for VFA analysis. Biomass was pelleted from 10 ml liquid samples via centrifugation (10,000xg) and stored at minus 20C until protein extraction. Protein from cell pellet samples were extracted in an 8M urea solution, then reduced and alkylated. Proteins were then digested with trypsin and subsequently desalted using C18 solid phase extraction. MS analysis was performed using 0.1 ug/ul of peptide solution injected into a Q Exactive HF X mass spectrometer (Thermo Scientific). Data processing Mass spectrometry (MS) data for each biological replicate at all time points (n=18) were analyzed using an implementation of OpenMS implemented in KNIME. Briefly, MS/MS spectra were searched using the MS-GF+ tool against a protein database consisting of all ORFs from the de-replicated set of MAGs, concatenated with reversed (decoy) sequences of all protein entries. Peptide spectra matches (PSMs) were filtered at a 5% false discovery rate (FDR) with Percolator.

Project description:All large-scale LC-MS/MS post-translational methylation site discovery experiments require methylpeptide spectrum matches (methyl-PSMs) to be identified at acceptably low false discovery rates (FDRs). To meet estimated methyl-PSM FDRs, methyl-PSM filtering criteria are often determined using the target-decoy approach. The efficacy of this methyl-PSM filtering approach has, however, yet to be thoroughly evaluated. Here we conduct a systematic analysis of methyl-PSM FDRs across a range of sample preparation workflows (each differing in their exposure to the alcohols methanol and isopropanol) and mass spectrometric instrument platforms (each employing a different mode of MS/MS dissociation). Through 13CD3-methionine labeling (heavy-methyl SILAC) of S. cerevisiae cells and in-depth manual data inspection, accurate lists of true positive methyl-PSMs were determined, allowing methyl-PSM FDRs to be compared to target-decoy approach-derived methyl-PSM FDR estimates. These results show that global FDR estimates produce extremely unreliable methyl-PSM filtering criteria; we demonstrate that this is an unavoidable consequence of the high number of amino acid combinations capable of producing peptide sequences that are isobaric to methylated peptides of a different sequence. Separate methyl-PSM FDR estimates were also found to be unreliable due to prevalent sources of false positive methyl-PSMs that produce high peptide identity score distributions. Incorrect methylation site localizations, peptides containing cysteinyl-S-β-propionamide, and methylated glutamic or aspartic acid residues can partially, but not wholly, account for these false positive methyl-PSMs. Together these results indicate that the target-decoy approach is an unreliable means of estimating methyl-PSM FDRs and methyl-PSM filtering criteria. We suggest that orthogonal methylpeptide validation (e.g. heavy-methyl SILAC or its offshoots) should be considered a prerequisite for obtaining high confidence methyl-PSMs in large-scale LC-MS/MS methylation site discovery experiments, and make recommendations on how to reduce methyl-PSM FDRs in samples not amenable to heavy isotope labeling.

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Activity-Targeted SIP-Metaproteomics of a Full-Scale Biogas Facility

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