Saliva and Saliva Extracellular Vesicles for Biomarker Candidate Identification-Assay Development and Pilot Study in...

Saliva is gaining increasing attention as a source of biomarkers due to non-invasive and undemanding collection access. Extracellular vesicles (EVs) are nano-sized, cell-released particles that contain molecular information about their parent cells. In this study, we developed methods for saliva biomarker candidate identification using EV-isolation and proteomic evaluation. We used pooled saliva samples for assay development. EVs were isolated using membrane affinity-based methods followed by their characterization using nanoparticle tracking analysis and transmission electron microscopy. Subsequently, both saliva and saliva-EVs were successfully analyzed using proximity extension assay and label-free quantitative proteomics. Saliva-EVs had a higher purity than plasma-EVs, based on the expression of EV-proteins and albumin. The developed methods could be used for the analysis of individual saliva samples from amyotrophic lateral sclerosis (ALS) patients and controls (n = 10 each). The starting volume ranged from 2.1 to 4.9 mL and the amount of total isolated EV-proteins ranged from 5.1 to 42.6 µg. Although no proteins were significantly differentially expressed between the two groups, there was a trend for a downregulation of ZNF428 in ALS-saliva-EVs and an upregulation of IGLL1 in ALS saliva. In conclusion, we have developed a robust workflow for saliva and saliva-EV analysis and demonstrated its technical feasibility for biomarker discovery.