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ABSTRACT
The value of saliva as a diagnostic tool can be increased by taxonomic and functional analyses of the microbiota as recently demonstrated. In this proof-of-principle study, we compare two collection methods (Salivette® (SV) and paraffin gum (PG)) for stimulated saliva from five healthy participants and present a workflow including PG preparation which is suitable for metaproteomics.
ARTICLE HISTORY
Received 27 September 2017
Accepted 10 January 2018
KEYWORDS
Saliva; metaproteomics; human oral microbiome; whole saliva proteomics; collection method; nLC-MS/ MS
For a better understanding of the microbial species composition in health and disease of the oral cavity [1,2], saliva offers a wide range of possibilities as shown in metagenomic studies [3-5]. In addition, metaproteomics provides detailed impressions of active metabolic pathways under certain environmental conditions, which cannot be accomplished by metagenomics [6-8]. First metaproteome studies for saliva have already been performed [9-12]. Here, we conducted a comparative proofof-principle study for two saliva-stimulating collection methods (Salivette® (SV) and paraffin gum (PG)) to identify the most suitable way to perform metaproteome studies on human saliva.
We collected stimulated saliva from five healthy dental students (three men and two women) aged 20-30 years on two consecutive days. Under the supervision of an experienced dentist, the students examined each other and none of them had a probing depth of >4 mm. Based on a questionnaire we ensured that all participants met our inclusion criteria (Supplemental Table 1).
All subjects were chewing on a PG for 1 min. Within this minute all volunteers spat saliva into a sterile 50 ml Falcon tube for several times. On the next day, the participants had to chew on the SV for 1 min and the soaked cotton roll was transferred into a specific salivation vessel. Previous experiments showed that the order of the chosen saliva collection methods had no influence on the results (data not shown). Afterwards, all samples were centrifuged for 15 min at 11,500 g (4°C). Saliva collected by PG was separated into supernatant (PG_SN) and pellet (PG_P). SV samples were again centrifuged for 30 min at 17,000 g at 4°C (Salivette supernatant - SV_SN and Salivette pellet - SV_P). For SV_P only a tiny pellet was seen. Pellets were resuspended in 700 pl (PG_P) and 300 pl (SV_P) TE-Buffer....