Ultra-deep and quantitative saliva proteome reveals dynamics of the oral microbiome

Grassl et al. Genome Medicine (2016) 8:44 DOI 10.1186/s13073-016-0293-0 RESEARCH Open Access Ultra-deep and quantitative saliva proteome reveals dynamics of the oral microbiome Niklas Grassl1, Nils Alexander Kulak1,2, Garwin Pichler1,2, Philipp Emanuel Geyer1,3, Jette Jung4, Sören Schubert4, Pavel Sinitcyn5, Juergen Cox5 and Matthias Mann1,3* Abstract Background: The 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. Methods: We 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 interindividual 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. Results: Microgram 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. Conclusion: Rapid 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. * Correspondence: 1 Department of Proteomics and Signal Transduction, Max-Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany 3 Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark Full list of author information is available at the end of the article © 2016 Grassl et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Grassl et al. Genome Medicine (2016) 8:44 Background Using saliva for the diagnosis of medical conditions would be particularly 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. In addition to the secretions of oral grands, saliva contains cells shed from the epithelium of the oral cavity and harbors the oral microbiome. Promising steps towards the establishment of saliva protein biomarkers have already been undertaken [2, 3]. However, 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]. Further interest in saliva has recently been fueled by the discovery that the oral microbiome and the gut microbiome are the most diverse ones of the human body and that they correlate well with each other [5]. There is now compelling evidence for a link between the human microbiome and conditions such as obesity, allergies, and even autoimmune diseases like multiple sclerosis [6–8]. In addition, tooth decay and other diseases of the oral cavity are known to be caused by bacteria but turn out to be insufficiently explained by one species alone [9, 10]. Therefore, first metagenomics and then metaproteomics studies have already aimed to relate bacterial composition to caries incidence [10, 11]. However, reproducible identification and consistent quantification of bacteria remain challenging. Dynamic, quantitative studies would be of great help to uncover the functional connections between microbial communities and the prevalent pathologies of the oral cavity. During the past few years, our laboratory has focused on simplifying and streamlining the proteomics workflow, with the aim of bringing the technology closer to clinical applications. Here we set out to characterize the saliva proteome at the greatest depth possible while still minimizing steps that could compromise quantification. We also developed a rapid single-run analysis workflow, starting from standard clinical cotton swabs and delivering results in a few hours, while retaining a quantification depth of thousands of proteins. This allowed us to investigate changes in the saliva proteome upon perturbation in a healthy cohort. We also analyzed interindividual differences in the saliva proteome and quantitatively addressed the long-standing question of the degree to which the plasma and saliva proteomes are correlated. Finally, we asked if our in-depth workflow can characterize the oral microbiome and its dynamics and confirmed detected species by the established method of culturing followed by Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) as well as data from next-generation sequencing projects. Page 2 of 13 Methods Experimental design We collected saliva at two different time points from four female and four male, healthy, non-smoking individuals aged 24 to 40 years with Caucasian backgrounds. All subjects were asymptomatic, did not take any drugs or antiseptics, visited the dentist regularly, and showed no signs of inflammation, bleeding, or infection as judged by a medical student (N.G.). The study was approved by the ethics committee of the Max Planck Society and all donors provided their written informed consent to participate in this (...truncated)