Browsing by Author "Keffer, Jessica L."
Now showing 1 - 2 of 2
Results Per Page
Sort Options
Item Development of an efficient, effective, and economical technology for proteome analysis(Cell Reports: Methods, 2024-06-11) Martin, Katherine R.; Le, Ha T.; Abdelgawad, Ahmed; Yang, Canyuan; Lu, Guotao; Keffer, Jessica L.; Zhang, Xiaohui; Zhuang, Zhihao; Asare-Okai, Papa Nii; Chan, Clara S.; Batish, Mona; Yu, YanbaoHighlights • Rapid, robust, and cost-effective alternative to proteomics sample preparation • Versatile filter devices can meet a wide range of proteomics analysis needs • On-filter in-cell digestion facilitates low-input proteomics • Ready-to-go E3 and E4 filter devices are available Motivation Conventional proteomics sample processing methods often have high technical barriers to broad biomedical scientists, leading to difficulties for quick adoption and standardization. Existing protocols are also typically associated with costly reagents and accessories, making them less feasible for resource-limited settings as well as for clinical proteomics and/or core facilities where large numbers of samples are usually processed. Thus, there is a strong unmet need for an easy-to-use, reliable, and low-cost approach for general proteomics sample preparation. Summary We present an efficient, effective, and economical approach, named E3technology, for proteomics sample preparation. By immobilizing silica microparticles into the polytetrafluoroethylene matrix, we develop a robust membrane medium, which could serve as a reliable platform to generate proteomics-friendly samples in a rapid and low-cost fashion. We benchmark its performance using different formats and demonstrate them with a variety of sample types of varied complexity, quantity, and volume. Our data suggest that E3technology provides proteome-wide identification and quantitation performance equivalent or superior to many existing methods. We further propose an enhanced single-vessel approach, named E4technology, which performs on-filter in-cell digestion with minimal sample loss and high sensitivity, enabling low-input and low-cell proteomics. Lastly, we utilized the above technologies to investigate RNA-binding proteins and profile the intact bacterial cell proteome. Graphical abstract available at: https://doi.org/10.1016/j.crmeth.2024.100796Item Iron Oxidation by a Fused Cytochrome-Porin Common to Diverse Iron-Oxidizing Bacteria(mBio, 2021-07-27) Keffer, Jessica L.; McAllister, Sean M.; Garber, Arkadiy I.; Hallahan, Beverly J.; Sutherland, Molly C.; Rozovsky, Sharon; Chan, Clara S.Iron (Fe) oxidation is one of Earth’s major biogeochemical processes, key to weathering, soil formation, water quality, and corrosion. However, our understanding of microbial contribution is limited by incomplete knowledge of microbial iron oxidation mechanisms, particularly in neutrophilic iron oxidizers. The genomes of many diverse iron oxidizers encode a homolog to an outer membrane cytochrome (Cyc2) shown to oxidize iron in two acidophiles. Phylogenetic analyses show Cyc2 sequences from neutrophiles cluster together, suggesting a common function, though this function has not been verified in these organisms. Therefore, we investigated the iron oxidase function of heterologously expressed Cyc2 from a neutrophilic iron oxidizer Mariprofundus ferrooxydans PV-1. Cyc2PV-1 is capable of oxidizing iron, and its redox potential is 208 ± 20 mV, consistent with the ability to accept electrons from Fe2+ at neutral pH. These results support the hypothesis that Cyc2 functions as an iron oxidase in neutrophilic iron-oxidizing organisms. The results of sequence analysis and modeling reveal that the entire Cyc2 family shares a unique fused cytochrome-porin structure, with a defining consensus motif in the cytochrome region. On the basis of results from structural analyses, we predict that the monoheme cytochrome Cyc2 specifically oxidizes dissolved Fe2+, in contrast to multiheme iron oxidases, which may oxidize solid Fe(II). With our results, there is now functional validation for diverse representatives of Cyc2 sequences. We present a comprehensive Cyc2 phylogenetic tree and offer a roadmap for identifying cyc2/Cyc2 homologs and interpreting their function. The occurrence of cyc2 in many genomes beyond known iron oxidizers presents the possibility that microbial iron oxidation may be a widespread metabolism.