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 and 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.
Highlights
The proteome of cervical-vaginal fluid (CVF) is extremely complex, containing proteins predominantly synthesized by the endocervix and vaginal cells, and from the endometrial and tubal secretions and from the peritoneal fluid[1]
We previously demonstrated the feasibility of using the residual fixative from discarded Pap tests for the identification of human cervical-vaginal proteins by mass spectrometry[21]
This study evaluates the use of residual Pap test samples for taxonomic and functional characterization of bacteria present in the female genital tract using a metaproteomic approach
Summary
The proteome of cervical-vaginal fluid (CVF) is extremely complex, containing proteins predominantly synthesized by the endocervix and vaginal cells, and from the endometrial and tubal secretions and from the peritoneal fluid[1]. The studies of the NIH Human Microbiome Project (HMP)[15,16,17] have vastly increased the diversity of the bacteria identified in the human vagina over culture-dependent methods, and the development of a comprehensive database of 16 S rRNA sequences from common vaginal bacteria has allowed species level classification of bacteria from clinical samples[18] These molecular approaches yield detailed information on microbiome composition and the abundance of bacterial taxa present, the functional significance of diverse microbial communities and their interaction with host proteins in differing states of health and disease is lacking. To evaluate this approach for the identification of taxonomic and functional information on the vaginal microbiome, these same raw data were analyzed using a two-step method for peptide sequence matching and protein identification for metaproteomic analysis of microbial peptides present in the residual Pap test fixative from women over age 50
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