Fig. 1: Schematic illustration of the iterative optimization strategy for developing the OSaMPle workflow.

a Proof-of-principle validation of the two-component processing workflow. Pelleted salivary aliquots were treated with one of four different buffers, followed by differential centrifugation to effectively isolate and enrich microbes from host debris. “PBS”, “U”, “T”, and “U + T” in panel (a) refer to “PBS”, “6 M urea in PBS”, “0.5% Tween 80 in PBS”, and “6 M urea, 0.5% Tween 80 in PBS”, respectively. b Experimental design and workflow for optimizing the selective enrichment buffer. Saliva samples from nine healthy human subjects were pooled and aliquoted into 18 portions for method optimization. These were divided into six groups, including a Baseline group (BL), a Control group (CT), and four experimental groups (GX, GT, UX and UT), with three technical replicates per group. The ‘+’ and ‘−’ signs indicate the presence (+) or absence (−) of specific buffer components in each group. In the BL group, sample pellets were suspended and washed in PBS but were not subjected to differential centrifugation. In the CT group and the four experimental groups, the sample pellets were treated with PBS or a combination of a detergent and a denaturant, followed by differential centrifugation (the detailed workflow of which is illustrated in the box within panel a). After pretreatment, all samples were washed by PBS buffer, followed by cell lysis, protein extraction, and digestion, and subsequently analyzed by LC-MS/MS. Logos for MetaLab and eHOMD are included in Fig. 1b, which originate from MetaLab software and https://www.homd.org, respectively.