Abstract
BackgroundThe oral cavity is home to one of the most diverse microbial communities of the human body and a major entry portal for pathogens. Its homeostasis is maintained by saliva, which fulfills key functions including lubrication of food, pre-digestion, and bacterial defense. Consequently, disruptions in saliva secretion and changes in the oral microbiome contribute to conditions such as tooth decay and respiratory tract infections. Here we set out to quantitatively map the saliva proteome in great depth with a rapid and in-depth mass spectrometry-based proteomics workflow.MethodsWe used recent improvements in mass spectrometry (MS)-based proteomics to develop a rapid workflow for mapping the saliva proteome quantitatively and at great depth. Standard clinical cotton swabs were used to collect saliva form eight healthy individuals at two different time points, allowing us to study inter-individual differences and interday changes of the saliva proteome. To accurately identify microbial proteins, we developed a method called “split by taxonomy id” that prevents peptides shared by humans and bacteria or between different bacterial phyla to contribute to protein identification.ResultsMicrogram protein amounts retrieved from cotton swabs resulted in more than 3700 quantified human proteins in 100-min gradients or 5500 proteins after simple fractionation. Remarkably, our measurements also quantified more than 2000 microbial proteins from 50 bacterial genera. Co-analysis of the proteomics results with next-generation sequencing data from the Human Microbiome Project as well as a comparison to MALDI-TOF mass spectrometry on microbial cultures revealed strong agreement. The oral microbiome differs between individuals and changes drastically upon eating and tooth brushing.ConclusionRapid shotgun and robust technology can now simultaneously characterize the human and microbiome contributions to the proteome of a body fluid and is therefore a valuable complement to genomic studies. This opens new frontiers for the study of host–pathogen interactions and clinical saliva diagnostics.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-016-0293-0) contains supplementary material, which is available to authorized users.
Highlights
The oral cavity is home to one of the most diverse microbial communities of the human body and a major entry portal for pathogens
We showed that shotgun proteomics can readily determine 50 bacterial genera in saliva but the sequence coverage of bacterial proteins and organisms suggests that we have only scratched the surface of the oral bacterial proteome
Quantitative comparison to nextgeneration sequencing data from the Human Microbiome Project (HMP) [25] revealed excellent agreement, suggesting that proteomics could provide a valuable complement to sequencing-based measurements of the human microbiome
Summary
The oral cavity is home to one of the most diverse microbial communities of the human body and a major entry portal for pathogens. Disruptions in saliva secretion and changes in the oral microbiome contribute to conditions such as tooth decay and respiratory tract infections. Using saliva for the diagnosis of medical conditions would be attractive because it can be collected noninvasively and economically [1], but the complexity of the oral cavity and the multiple entities contributing to its homeostasis make this challenging. Promising steps towards the establishment of saliva protein biomarkers have already been undertaken [2, 3] These studies either only considered around 100 proteins with antibody-based assays or employed relatively low throughput mass spectrometry (MS)-based proteomics with extensive fractionation, which generally precluded quantification [4]. Quantitative studies would be of great help to uncover the functional connections between microbial communities and the prevalent pathologies of the oral cavity
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