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Blood serum is arguably the most analyzed biofluid for disease prediction and diagnosis. Herein, we benchmarked five different serum abundant protein depletion (SAPD) kits with regard to the identification of disease‐specific biomarkers in human serum using bottom‐up proteomics. As expected, the IgG removal efficiency among the SAPD kits is highly variable, ranging from 70% to 93%. A pairwise comparison of database search results showed a 10%–19% variation in protein identification among the kits. Immunocapturing‐based SAPD kits against IgG and albumin outperformed the others in the removal of these two abundant proteins. Conversely, non‐antibody‐based methods (i.e., kits using ion exchange resins) and kits leveraging a multi‐antibody approach were proven to be less efficient in depleting IgG/albumin from samples but led to the highest number of identified peptides. Notably, our results indicate that different cancer biomarkers could be enriched up to 10% depending on the utilized SAPD kit compared with the undepleted sample. Additionally, functional analysis of the bottom‐up proteomic results revealed that different SAPD kits enrich distinct disease‐ and pathway‐specific protein sets. Overall, our study emphasizes that a careful selection of the appropriate commercial SAPD kit is crucial for the analysis of disease biomarkers in serum by shotgun proteomics.

Zodletone spring is a sulfide-rich spring in southwestern Oklahoma characterized by shallow, microoxic, light-exposed spring water overlaying anoxic sediments. Previously, culture-independent 16S rRNA gene based diversity surveys have revealed that Zodletone spring source sediments harbor a highly diverse microbial community, with multiple lineages putatively involved in various sulfur-cycling processes. Here, we conducted a metatranscriptomic survey of microbial populations in Zodletone spring source sediments to characterize the relative prevalence and importance of putative phototrophic, chemolithotrophic, and heterotrophic microorganisms in the sulfur cycle, the identity of lineages actively involved in various sulfur cycling processes, and the interaction between sulfur cycling and other geochemical processes at the spring source. Sediment samples at the spring’s source were taken at three different times within a 24-h period for geochemical analyses and RNA sequencing. In depth mining of datasets for sulfur cycling transcripts revealed major sulfur cycling pathways and taxa involved, including an unexpected potential role of Actinobacteria in sulfide oxidation and thiosulfate transformation. Surprisingly, transcripts coding for the cyanobacterial Photosystem II D1 protein, methane monooxygenase, and terminal cytochrome oxidases were encountered, indicating that genes for oxygen production and aerobic modes of metabolism are actively being transcribed, despite below-detectable levels (<1 µM) of oxygen in source sediment. Results highlight transcripts involved in sulfur, methane, and oxygen cycles, propose that oxygenic photosynthesis could support aerobic methane and sulfide oxidation in anoxic sediments exposed to sunlight, and provide a viewpoint of microbial metabolic lifestyles under conditions similar to those seen during late Archaean and Proterozoic eons.