skip to main content
Explore Scholarly Publications and Datasets in the NSF-PAR

  • NSF Public Access
  • Search Results
  • Accepted Manuscript: Using high-abundance proteins as guides for fast and effective peptide/protein identification from human gut metaproteomic data
Title: 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 more » 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. « less
Authors:
; ;
Award ID(s):
2025451
Publication Date:
NSF-PAR ID:
10297311
Journal Name:
Microbiome
Volume:
9
Issue:
1
ISSN:
2049-2618
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; ; ; ; ; ( , Scientific Reports)
    Abstract

    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 andCandida. 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.

  2. ; ; ; ; ; ( , mSystems)
    Hird, Sarah M. (Ed.)
    The gut microbiome provides vital functions for mammalian hosts, yet research on its variability and function across adult life spans and multiple generations is limited in large mammalian carnivores. Here, we used 16S rRNA gene and metagenomic high-throughput sequencing to profile the bacterial taxonomic composition, genomic diversity, and metabolic function of fecal samples collected from 12 wild spotted hyenas ( Crocuta crocuta ) residing in the Masai Mara National Reserve, Kenya, over a 23-year period spanning three generations. The metagenomic data came from four of these hyenas and spanned two 2-year periods. With these data, we determined the extent to which host factors predicted variation in the gut microbiome and identified the core microbes present in the guts of hyenas. We also investigated novel genomic diversity in the mammalian gut by reporting the first metagenome-assembled genomes (MAGs) for hyenas. We found that gut microbiome taxonomic composition varied temporally, but despite this, a core set of 14 bacterial genera were identified. The strongest predictors of the microbiome were host identity and age, suggesting that hyenas possess individualized microbiomes and that these may change with age during adulthood. The gut microbiome functional profiles of the four adult hyenas were also individual specificmore »and were associated with prey abundance, indicating that the functions of the gut microbiome vary with host diet. We recovered 149 high-quality MAGs from the hyenas’ guts; some MAGs were classified as taxa previously reported for other carnivores, but many were novel and lacked species-level matches to genomes in existing reference databases. IMPORTANCE There is a gap in knowledge regarding the genomic diversity and variation of the gut microbiome across a host’s life span and across multiple generations of hosts in wild mammals. Using two types of sequencing approaches, we found that although gut microbiomes were individualized and temporally variable among hyenas, they correlated similarly to large-scale changes in the ecological conditions experienced by their hosts. We also recovered 149 high-quality MAGs from the hyena gut, greatly expanding the microbial genome repertoire known for hyenas, carnivores, and wild mammals in general. Some MAGs came from genera abundant in the gastrointestinal tracts of canid species and other carnivores, but over 80% of MAGs were novel and from species not previously represented in genome databases. Collectively, our novel body of work illustrates the importance of surveying the gut microbiome of nonmodel wild hosts, using multiple sequencing methods and computational approaches and at distinct scales of analysis.« less
  3. ; ; ( , PLOS Computational Biology)
    Coelho, Luis Pedro (Ed.)
    Host-microbiome interactions and the microbial community have broad impact in human health and diseases. Most microbiome based studies are performed at the genome level based on next-generation sequencing techniques, but metaproteomics is emerging as a powerful technique to study microbiome functional activity by characterizing the complex and dynamic composition of microbial proteins. We conducted a large-scale survey of human gut microbiome metaproteomic data to identify generalist species that are ubiquitously expressed across all samples and specialists that are highly expressed in a small subset of samples associated with a certain phenotype. We were able to utilize the metaproteomic mass spectrometry data to reveal the protein landscapes of these species, which enables the characterization of the expression levels of proteins of different functions and underlying regulatory mechanisms, such as operons. Finally, we were able to recover a large number of open reading frames (ORFs) with spectral support, which were missed by de novo protein-coding gene predictors. We showed that a majority of the rescued ORFs overlapped with de novo predicted protein-coding genes, but on opposite strands or in different frames. Together, these demonstrate applications of metaproteomics for the characterization of important gut bacterial species.
  4. ; ; ; ; ( , Applied and Environmental Microbiology)
    Ercolini, Danilo (Ed.)
    ABSTRACT Dietary polyphenols can significantly benefit human health, but their bioavailability is metabolically controlled by human gut microbiota. To facilitate the study of polyphenol metabolism for human gut health, we have manually curated experimentally characterized polyphenol utilization proteins (PUPs) from published literature. This resulted in 60 experimentally characterized PUPs (named seeds) with various metadata, such as species and substrate. Further database search found 107,851 homologs of the seeds from UniProt and UHGP (unified human gastrointestinal protein) databases. All PUP seeds and homologs were classified into protein classes, families, and subfamilies based on Enzyme Commission (EC) numbers, Pfam (protein family) domains, and sequence similarity networks. By locating PUP homologs in the genomes of UHGP, we have identified 1,074 physically linked PUP gene clusters (PGCs), which are potentially involved in polyphenol metabolism in the human gut. The gut microbiome of Africans was consistently ranked the top in terms of the abundance and prevalence of PUP homologs and PGCs among all geographical continents. This reflects the fact that dietary polyphenols are consumed by the African population more commonly than by other populations, such as Europeans and North Americans. A case study of the Hadza hunter-gatherer microbiome verified the feasibility of using dbPUP tomore »profile metagenomic data for biologically meaningful discovery, suggesting an association between diet and PUP abundance. A Pfam domain enrichment analysis of PGCs identified a number of putatively novel PUP families. Lastly, a user-friendly web interface ( https://bcb.unl.edu/dbpup/ ) provides all the data online to facilitate the research of polyphenol metabolism for improved human health. IMPORTANCE Long-term consumption of polyphenol-rich foods has been shown to lower the risk of various human diseases, such as cardiovascular diseases, cancers, and metabolic diseases. Raw polyphenols are often enzymatically processed by gut microbiome, which contains various polyphenol utilization proteins (PUPs) to produce metabolites with much higher bioaccessibility to gastrointestinal cells. This study delivered dbPUP as an online database for experimentally characterized PUPs and their homologs in human gut microbiome. This work also performed a systematic classification of PUPs into enzyme classes, families, and subfamilies. The signature Pfam domains were identified for PUP families, enabling conserved domain-based PUP annotation. This standardized sequence similarity-based PUP classification system offered a guideline for the future inclusion of new experimentally characterized PUPs and the creation of new PUP families. An in-depth data analysis was further conducted on PUP homologs and physically linked PUP gene clusters (PGCs) in gut microbiomes of different human populations.« less
  5. ; ; ( , Biodiversity Information Science and Standards)
    Large systematic revisionary projects incorporating data for hundreds or thousands of taxa require an integrative approach, with a strong biodiversity-informatics core for efficient data management to facilitate research on the group. Our original biodiversity informatics platform, 3i (Internet-accessible Interactive Identification) combined a customized MS Access database backend with ASP-based web interfaces to support revisionary syntheses of several large genera of leafhopers (Hemiptera: Auchenorrhyncha: Cicadellidae). More recently, for our National Science Foundation sponsored project, “GoLife: Collaborative Research: Integrative genealogy, ecology and phenomics of deltocephaline leafhoppers (Hemiptera: Cicadellidae), and their microbial associates”, we selected the new open-source platform TaxonWorks as the cyberinfrastructure. In the scope of the project, the original “3i World Auchenorrhyncha Database” was imported into TaxonWorks. At the present time, TaxonWorks has many tools to automatically import nomenclature, citations, and specimen based collection data. At the time of the initial migration of the 3i database, many of those tools were still under development, and complexity of the data in the database required a custom migration script, which is still probably the most efficient solution for importing datasets with long development history. At the moment, the World Auchenorrhyncha Database comprehensively covers nomenclature of the group and includes data on 70 validmore »families, 6,816 valid genera, 47,064 valid species as well as synonymy and subsequent combinations (Fig. 1). In addition, many taxon records include the original citation, bibliography, type information, etymology, etc. The bibliography of the group includes 37,579 sources, about 1/3 of which are associated with PDF files. Species have distribution records, either derived from individual specimens or as country and state level asserted distribution, as well as biological associations indicating host plants, predators, and parasitoids. Observation matrices in TaxonWorks are designed to handle morphological data associated with taxa or specimens. The matrices may be used to automatically generate interactive identification keys and taxon descriptions. They can also be downloaded to be imported, for example, into Lucid builder, or to perform phylogenetic analysis using an external application. At the moment there are 36 matrices associated with the project. The observation matrix from GoLife project covers 798 taxa by 210 descriptors (most of which are qualitative multi-state morphological descriptors) (Fig. 2). Illustrations are provided for 9,886 taxa and organized in the specialized image matrix and could be used as a pictorial key for determination of species and taxa of a higher rank. For the phylogenetic analysis, a dataset was constructed for 730 terminal taxa and >160,000 nucleotide positions obtained using anchored hybrid enrichment of genomic DNA for a sample of leafhoppers from the subfamily Deltocephalinae and outgroups. The probe kit targets leafhopper genes, as well as some bacterial genes (endosymbionts and plant pathogens transmitted by leafhoppers). The maximum likelihood analyses of concatenated nucleotide and amino acid sequences as well as coalescent gene tree analysis yielded well-resolved phylogenetic trees (Cao et al. 2022). Raw sequence data have been uploaded to the Sequence Read Archive on GenBank. Occurrence and morphological data, as well as diagnostic images, for voucher specimens have been incorporated into TaxonWorks. Data in TaxonWorks could be exported in raw format, get accessed via Application Programming Interface (API), or be shared with external data aggregators like Catalogue of Life, GBIF, iDigBio.« less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email