Lipidomic profiling of non-mineralized dental plaque and biofilm by untargeted UHPLC-QTOF-MS/MS and SWATH acquisition.

Dental plaque is a structurally organized biofilm which consists of diverse microbial colonies and extracellular matrix. Its composition may change when pathogenic microorganisms become dominating. Therefore, dental biofilm or plaque has been frequently investigated in the context of oral health and disease. Furthermore, its potential as an alternative matrix for analytical purposes has also been recognized in other disciplines like archeology, food sciences, and forensics. Thus, a careful in-depth characterization of dental plaque is worthwhile. Most of the conducted studies focused on the screening of microbial populations in dental plaque. Their lipid membranes, on the other hand, may significantly impact substance (metabolite) exchange within microbial colonies as well as xenobiotics uptake and incorporation into teeth. Under this umbrella, a comprehensive lipidomic profiling for determination of lipid compositions of in vivo dental plaque samples and of in vitro cultivated biofilm as surrogate matrix to be used for analytical purposes has been performed in this work. An untargeted lipidomics workflow utilizing a ultra-high-performance liquid chromatography (UHPLC)-quadrupole-time-of-flight (QTOF) platform together with comprehensive SWATH (sequential window acquisition of all theoretical fragment ion mass spectra) acquisition and compatible software (MS-DIAL) that comprises a vast lipid library has been adopted to establish an extensive lipidomic fingerprint of dental plaque. The main lipid components in dental plaque were identified as triacylglycerols, followed by cholesterol, cholesteryl esters as well as diacylglycerols, and various phospholipid classes. In vivo plaque is a rare matrix which is usually available in very low amounts. When higher quantities for specific research assays are required, efficient ways to produce an appropriate surrogate matrix are mandatory. A potential surrogate matrix substituting dental plaque was prepared by cultivation of in vitro biofilm from saliva and similarities and differences in the lipidomics profile to in vivo plaque were mapped by statistical evaluation post-analysis. It was discovered that most lipid classes were highly elevated in the in vitro biofilm samples, in particular diacylglycerols, phosphatidylglycerols, and phosphatidylethanolamines (PEs). Furthermore, an overall shift from even-chain lipid species to odd-chain lipids was observed in the cultivated biofilms. On the other hand, even-chain phosphatidylcholines (PCs), lysoPCs, cholesteryl esters, and cholesterol-sulfate were shown to be specifically increased in plaque samples. Graphical abstract.

Multivariate optimization of a method for the determination of fatty acids in dental biofilm by GC-MS.

AIM: Phospholipid fatty acid methyl ester (FAME) analysis offers a simple option additionally to 16S rRNA sequencing to characterize microbial communities and to monitor changes. A method was established for the characterization of dental plaque via FAME profiles. METHODOLOGY: Fatty acids were determined as FAMEs (direct, acidic transesterification) and analyzed by GC-MS using an optimized temperature gradient. The transesterification reaction was optimized using a fractional factorial central composite face-centered design. RESULTS: Optimal conditions for the transesterification in methanol/toluene: hydrochloric acid concentration 2% (w/v), reaction time 40 min, temperature 110 °C. Method validation showed satisfactory accuracy, precision and linearity. CONCLUSION: The method provides a useful tool to characterize plaque via FAME profiles and was successfully applied to samples from ten subjects demonstrating its applicability.