Deep metaproteomic mapping of gingival crevicular fluid reveals distinct microbial community at prepubertal and circumpubertal stages.

BACKGROUND: Orthodontic diagnosis and treatment planning are closely associated with the pubertal growth spurt. Previously, we developed a simplified MS-based protocol for deep quantitative analysis of human gingival crevicular fluid (GCF) proteome for skeletal maturity indicators. The purpose of this study is to perform an in-depth and comparative analysis of the GCF metaproteome at prepubertal and circumpubertal stages to aid oral health and skeletal maturity evaluation. METHODS: Based on our previously obtained and published LC-MS data, the GCF metaproteome of 45 children (24 subjects from prepubertal group and 21 subjects from circumpubertal group) were analyzed by searching against the Human Oral Microbiome Database using FragPipe software. Differentially expressed bacterial proteins between two groups were analyzed using Wilcoxon rank sum test. Differentially abundant taxa between two groups were evaluated using linear discriminant effect size (LEfSe) analysis. RESULTS: A total of 192 genera were identified in GCF. Neisseria (Neisseriales), Comamonadaceae, Burkholderiales, Proteobacteria and Betaproteobacteria were the most abundant bacterial taxa at prepubertal stage. Firmicutes, Saccharibacteria_TM7_[G-1] (Saccharibacteria TM7), Bacterium_HMT349, Bacilli and Alphaproteobacteria were the most abundant bacterial taxa of GCF microbiota at circumpubertal stage. Compared to that in the prepubertal group, enrichment of Firmicutes (Bacillus) was observed in the circumpubertal group. CONCLUSIONS: Based on our developed and already published single-pot, solid-phase enhanced sample-preparation (SP3)-based liquid chromatography (LC) - high-field asymmetric waveform ion mobility spectrometry (FAIMS)-MS protocol for deep quantitative analysis of human GCF metaproteome, we were able to generate the largest dataset of the human GCF metaproteome (14376 bacterial proteins) to date and revealed distinct microbial community at prepubertal and circumpubertal stages. The proposed protocol and findings will be useful to aid oral health and skeletal maturity evaluation for orthodontic diagnosis and treatment planning.