MassIVE MSV000096205

Complete Public PXD057167

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

Subscribe Comment Reanalyze Spectra Compare Results Add Reanalysis

Description

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). [doi:10.25345/C5Z02ZM6H] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: anaerobic digestion ; bioorthogonal noncanonical amino acid tagging ; boncat ; proteinSIP ; stable isotope probing ; metaproteomics ; DatasetType:Proteomics

Contact

Principal Investigators:
(in alphabetical order) 		Ryan Ziels, University of British Columbia, Canada
Submitting User: skylerbio
Number of Files:
Total Size:
Spectra:
Subscribers:
 
Owner Reanalyses
Experimental Design
    Conditions:
    Biological Replicates:
    Technical Replicates:
 
Identification Results
    Proteins (Human, Remapped):
    Proteins (Reported):
    Peptides:
    Variant Peptides:
    PSMs:
 
Quantification Results
    Differential Proteins:
    Quantified Proteins:
 
Browse Dataset Files Browse Results
Browse Quantification Results
 
FTP Download Link (click to copy):

Species

Instrument

Modifications

- Dataset Reanalyses

+ Dataset History

Click here to queue conversion of this dataset's submitted spectrum files to open formats (e.g. mzML). This process may take some time.

When complete, the converted files will be available in the "ccms_peak" subdirectory of the dataset's FTP space (accessible via the "FTP Download" link to the right).
Number of distinct conditions across all analyses (original submission and reanalyses) associated with this dataset.

Distinct condition labels are counted across all files submitted in the "Metadata" category having a "Condition" column in this dataset.

"N/A" means no results of this type were submitted.
Number of distinct biological replicates across all analyses (original submission and reanalyses) associated with this dataset.

Distinct replicate labels are counted across all files submitted in the "Metadata" category having a "BioReplicate" or "Replicate" column in this dataset.

"N/A" means no results of this type were submitted.
Number of distinct technical replicates across all analyses (original submission and reanalyses) associated with this dataset.

The technical replicate count is defined as the maximum number of times any one distinct combination of condition and biological replicate was analyzed across all files submitted in the "Metadata" category. In the case of fractionated experiments, only the first fraction is considered.

"N/A" means no results of this type were submitted.
Originally identified proteins that were automatically remapped by MassIVE to proteins in the SwissProt human reference database.

"N/A" means no results of this type were submitted.
Number of distinct protein accessions reported across all analyses (original submission and reanalyses) associated with this dataset.

"N/A" means no results of this type were submitted.
Number of distinct unmodified peptide sequences reported across all analyses (original submission and reanalyses) associated with this dataset.

"N/A" means no results of this type were submitted.
Number of distinct peptide sequences (including modified variants or peptidoforms) reported across all analyses (original submission and reanalyses) associated with this dataset.

"N/A" means no results of this type were submitted.
Total number of peptide-spectrum matches (i.e. spectrum identifications) reported across all analyses (original submission and reanalyses) associated with this dataset.

"N/A" means no results of this type were submitted.
Number of distinct proteins quantified across all analyses (original submission and reanalyses) associated with this dataset.

Distinct protein accessions are counted across all files submitted in the "Statistical Analysis of Quantified Analytes" category having a "Protein" column in this dataset.

"N/A" means no results of this type were submitted.
Number of distinct proteins found to be differentially abundant in at least one comparison across all analyses (original submission and reanalyses) associated with this dataset.

A protein is differentially abundant if its change in abundance across conditions is found to be statistically significant with an adjusted p-value <= 0.05 and lists no issues associated with statistical tests for differential abundance.

Distinct protein accessions are counted across all files submitted in the "Statistical Analysis of Quantified Analytes" category having a "Protein" column in this dataset.

"N/A" means no results of this type were submitted.
This dataset may not contain all raw spectra data as originally deposited in PRIDE. It has been imported to MassIVE for reanalysis purposes, so its spectra data here may consist solely of processed peak lists suitable for reanalysis with most software.