Sulfate-reducing bacteria (SRB) are widespread in marine and terrestrial environments, where they often form syntrophic associations with bacteria, archaea, and eukaryotes. Among the most intimate of these are multipartite symbioses in gutless marine oligochaete worms, which host SRB and sulfur-oxidizing endosymbionts that exchange sulfur compounds syntrophically. To investigate this symbiosis, we generated 2D-LC-MS/MS metaproteomes of holobiont homogenates. Single Olavius algarvensis worms were pooled (n=50), homogenized and the symbionts were separated from each other and host debris by differential pelleting adapted from Hinzke et al., 2018. Homogenates were centrifuged for 2 minutes at 4 °C and 4000 x g (sample P1), the supernatant was transferred to a clean Eppendorf tube (sample S1), sterile filtered seawater (SFW) was added to sample P1; both P1 and S1 were centrifuged again with the same settings. The second supernatant from P1 was saved as S2 and again SFW was added to P1; S1 supernatant was transferred to a new tube (sample S3). P1 and S2 were centrifuged again for 2 minutes at 4 °C and 4000 x g. The resulting supernatant from P1 was discarded; the resulting supernatant from S2 was transferred to a new Eppendorf tube (sample S4). Samples S3 and S4 were centrifuged a final time for 7 minutes at 4 °C and 21000 x g and the resulting supernatant of S3 was saved in a new Eppendorf tube sample S5. Two of the resulting pellets (P1 most enriched in Cand. Thiosymbion, S3 most enriched in smaller symbionts (including Gamma3, Delta1, Delta4, Delta3, and the spirochete endosymbionts) and the final supernatant fraction (S5) were stored at -80 °C until further processing. Triplicates of the P1, S3, and S5 sample set were processed for proteomics, each resulting in 11 raw MS files corresponding to the different steps in the pH gradient. All 11 files were imported as sample fractions to Proteome Discoverer resulting in single results files for each sample.