The human cervical-vaginal area contains proteins derived from microorganisms that may prevent or predispose women to gynecological conditions. The liquid Pap test fixative is an unexplored resource for analysis of microbial communities and the microbe-host interaction. Previously, we showed that the residual cell-free fixative from discarded Pap tests of healthy women could be used for mass spectrometry (MS) based proteomic identification of cervical-vaginal proteins. In this study, we reprocessed these MS raw data files for metaproteomic analysis to characterize the microbial community composition and function of microbial proteins in the cervical-vaginal region. This was accomplished by developing a customized protein sequence database encompassing microbes likely present in the vagina. High-mass accuracy data were searched against the protein FASTA database using a two-step search method within the Galaxy for proteomics platform. Data was analyzed by MEGAN6 (MetaGenomeAnalyzer) for phylogenetic and functional characterization. We identified over 300 unique peptides from a variety of bacterial phyla and
Using high-abundance proteins as guides for fast and effective peptide/protein identification from human gut metaproteomic data
Abstract Background A few recent large efforts significantly expanded the collection of human-associated bacterial genomes, which now contains thousands of entities including reference complete/draft genomes and metagenome assembled genomes (MAGs). These genomes provide useful resource for studying the functionality of the human-associated microbiome and their relationship with human health and diseases. One application of these genomes is to provide a universal reference for database search in metaproteomic studies, when matched metagenomic/metatranscriptomic data are unavailable. However, a greater collection of reference genomes may not necessarily result in better peptide/protein identification because the increase of search space often leads to fewer spectrum-peptide matches, not to mention the drastic increase of computation time. Methods Here, we present a new approach that uses two steps to optimize the use of the reference genomes and MAGs as the universal reference for human gut metaproteomic MS/MS data analysis. The first step is to use only the high-abundance proteins (HAPs) (i.e., ribosomal proteins and elongation factors) for metaproteomic MS/MS database search and, based on the identification results, to derive the taxonomic composition of the underlying microbial community. The second step is to expand the search database by including all proteins from identified abundant species. We call our approach HAPiID (HAPs guided metaproteomics IDentification). Results We tested our approach using human gut metaproteomic datasets from a previous study and compared it to the state-of-the-art reference database search method MetaPro-IQ for metaproteomic identification in studying human gut microbiota. Our results show that our two-steps method not only performed significantly faster but also was able to identify more peptides. We further demonstrated the application of HAPiID to revealing protein profiles of individual human-associated bacterial species, one or a few species at a time, using metaproteomic data. Conclusions The HAP guided profiling approach presents a novel effective way for constructing target database for metaproteomic data analysis. The HAPiID pipeline built upon this approach provides a universal tool for analyzing human gut-associated metaproteomic data.
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- Award ID(s):
- 2025451
- NSF-PAR ID:
- 10297311
- Date Published:
- Journal Name:
- Microbiome
- Volume:
- 9
- Issue:
- 1
- ISSN:
- 2049-2618
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Candida . Peptides corresponding to proteins involved in carbohydrate metabolism, oxidation-reduction, and transport were identified. By identifying microbial peptides in Pap test supernatants it may be possible to acquire a functional signature of these microbes, as well as detect specific proteins associated with cervical health and disease. -
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1186/s40168-021-01035-8
