Fig. 2: Effectiveness and robustness of selective enrichment buffer and differential centrifugation approaches.

a Microscopic images of samples from the four experimental groups during the method validation phase. Supernatant (left) and debris (right) samples were obtained after different buffer treatments followed by differential centrifugation, show at 10 × and 100 × magnification, respectively. b Hierarchical clustering analysis of all samples. Samples with the same label color indicate technical replicates from identical buffer treatments, controls, baseline, or QC. c Principal component analysis (PCA) of the metaproteomic profiles generated from different sample treatment workflows. The LFQ intensities of protein groups were log-transformed, and the analysis include all protein groups identified across samples. d Pearson correlation coefficient of quantified protein groups within different groups. The analysis was conducted on the samples from the QC group, the BL group, and the supernatant samples from the CT group, as well as the four experimental groups. Protein intensities (log2-transformed) were used to calculate the correlation. All groups were compared with the QC group. Statistical significance was assessed using one-way ANOVA with Tukey’s post hoc test. Significant differences between groups are denoted by asterisks: *adjusted-p < 0.05, ****p < 0.0001, while groups with no significant differences are marked without asterisks. Sup and Deb in panels (b–d) represent supernatant and debris, respectively; labels BL, CT, GX, GT, UX, UT correspond to the experimental design as shown in Fig. 1b. QC indicates MS quality control samples.