A Clinical Metaproteomics Workflow Implemented within Galaxy Bioinformatics Platform to Analyze Host-Microbiome Interactions Underlying Human Disease

Summary

Clinical metaproteomics offers insights into the human microbiome and its contributions to disease. We harnessed the computational power of the Galaxy platform to develop a modular bioinformatics workflow that facilitates complex mass spectrometry-based metaproteomic analysis and characterization of diverse clinical sample types relevant to studies of disease.

Abstract

Clinical metaproteomics reveals host-microbiome interactions underlying diseases. However, challenges to this approach exist. In particular, the characterization of microbial proteins present in low abundance relative to host proteins is difficult. Other significant challenges are attributed to using very large protein sequence databases, which impedes sensitivity and accuracy during peptide and protein identification from mass spectrometry data in addition to retrieving taxonomy and functional annotations and performing statistical analysis. To address these problems, we present an integrated bioinformatics workflow for mass spectrometry-based metaproteomics that combines custom protein sequence database generation, peptide-spectrum match generation and verification, quantification, taxonomic and functional annotations, and statistical analysis. This workflow also offers characterization of human proteins (while prioritizing microbial proteins), thus offering insights into host-microbe dynamics in disease. The tools and workflow are deployed in the Galaxy ecosystem, enabling the development, optimization, and dissemination of these computational resources. We have applied this workflow for metaproteomic analysis of numerous clinical sample types, such as nasopharyngeal swabs and bronchoalveolar lavage fluid. Here, we demonstrate its utility via the analysis of residual fluid from cervical swabs. The complete workflow and accompanying training resources are accessible on the Galaxy Training Network to equip non-experts and experienced researchers with the necessary knowledge and tools to analyze their data.

Introduction

Mass spectrometry (MS)-based metaproteomics identifies and quantifies microbial and human proteins from clinical samples. This approach provides a new understanding of microbiome responses to disease and uncovers potential mediators of host-microbiome interactions^1,2. Although metaproteomic analysis of clinical samples can uncover the microbiome's interactions with its host environment, the field still faces many challenges. One main challenge is the relatively high abundance of host (human) proteins, which hampers the identification of lower abundant microbial proteins. Moreover, MS-based metaproteomics d....

Protocol

MS/MS spectral data were obtained from de-identified residual PTF samples that were collected using procedures that followed institutional board-approved guidelines and regulations, as previously described^21,29,30.

NOTE: Figure 1 provides an overview of the complete workflow, which consists of five modules. All inputs, outputs, and software tools are summarized in

Discussion

Clinical metaproteomics research offers potential breakthroughs for clinical studies, but challenges in its implementation persist. The lower abundance of microbial proteins relative to the host proteins in most samples hinders the detection and characterization of non-host proteins^6,10. Dependence on large protein sequence databases for accurate peptide and protein identification and quantification, along with complexities of taxonomically and functionally annot.......

Materials

Name	Company	Catalog Number	Comments
Collapse Collection	GalaxyP	Galaxy Version 5.1.1	Combines a dataset list collection into a single file (in the order of the list)
Concatenate datasets	GalaxyP	Galaxy Version 0.1.1	Concatenate files tail-to-head
Cut	GalaxyP	Galaxy Version 1.0.2	Cut (select) specified columns from a file
FASTA Merge Files and Filter Unique Sequences	GalaxyP	Galaxy Version 1.2.0	Concatenate FASTA database files together
FastaCLI	GalaxyP	Galaxy Version 4.0.41+galaxy1	Appends decoy sequences to FASTA files
FASTA-to-Tablular	GalaxyP	Galaxy Version 1.1.0	Convert FASTA-formatted sequences to TAB-delimited format
Filter	GalaxyP	Galaxy Version 1.1.1	Filter columns using simple expressions
Filter Tabular	GalaxyP	Galaxy Version 3.3.0	Filter a tabular file via line filters
Galaxy Europe (EU) server	GalaxyP		https://usegalaxy.eu/
Group	GalaxyP	Galaxy Version 2.1.4	Group a file by a particular column and perform aggregate functions
Identification Parameters	GalaxyP	Galaxy Version 4.0.41+galaxy1	Set identification parameters for SearchGUI/PeptideShaker
Learning Pathway: Clinical metaproteomics workflows within Galaxy	GalaxyP		https://training.galaxyproject.org/training-material/learning-pathways/clinical-metaproteomics.html
MaxQuant	GalaxyP	Galaxy Version 2.0.3.0+galaxy0 (Discovery module); Galaxy Version 1.6.17.0+galaxy4 (Quantification module)	Quantitative proteomics software package for analysis of large mass spectrometric data files
MetaNovo	GalaxyP	Galaxy Version 1.9.4+galaxy4	Search MS/MS data against a FASTA database (of known proteins) to produce a targeted database (of matched proteins) for mass spectrometry analysis
msconvert	GalaxyP	Galaxy Version 3.0.20287.2	Convert and/or filter mass spectrometry files
MSstatsTMT	GalaxyP	Galaxy Version 2.0.0+galaxy1	R-based package for detection of differentially abundant proteins in shotgun mass spectrometry-based proteomic experiments using tandem mass tag (TMT) labeling
PepQuery2	GalaxyP	Galaxy Version 2.0.2+galaxy0	Peptide-centric search engine for identification and/or validating known and novel peptides of interest
PeptideShaker	GalaxyP	Galaxy Version 2.0.33+galaxy1	Interpret results from SearchGUI for protein identification
Protein Database Downloader	GalaxyP	Galaxy Version 0.3.4	Download specified protein sequences as a FASTA file
Query Tabular	GalaxyP	Galaxy Version 3.3.0	Load tabular files intoa  SQLite database
Remove beginning	GalaxyP	Galaxy Version 1.0.0	Remove the specified number of (header) lines from a file
SearchGUI	GalaxyP	Galaxy Version 4.0.41+galaxy1	Run search engines on MGF peak lists and prepare results for input to Peptide Shaker
Select	GalaxyP	Galaxy Version 1.0.4	Select lines that match an expression
Unipept	GalaxyP	Galaxy Version 4.5.1	Retrieve UniProt entries and taxonomic information for tryptic peptides
UniProt	GalaxyP	Galaxy Version 2.3.0	Download proteome as a XML (UniProtXML) or FASTA file from UniProtKB