Martin, Katherine R.Le, Ha T.Abdelgawad, AhmedYang, CanyuanLu, GuotaoKeffer, Jessica L.Zhang, XiaohuiZhuang, ZhihaoAsare-Okai, Papa NiiChan, Clara S.Batish, MonaYu, Yanbao2024-07-032024-07-032024-06-11Martin, Katherine R., Ha T. Le, Ahmed Abdelgawad, Canyuan Yang, Guotao Lu, Jessica L. Keffer, Xiaohui Zhang, et al. “Development of an Efficient, Effective, and Economical Technology for Proteome Analysis.” Cell Reports Methods 4, no. 6 (June 2024): 100796. https://doi.org/10.1016/j.crmeth.2024.100796.2667-2375https://udspace.udel.edu/handle/19716/34559This article was originally published in Cell Reports: Methods. The version of record is available at: https://doi.org/10.1016/j.crmeth.2024.100796. © 2024 The Authors. Published by Elsevier Inc. Creative Commons Attribution (CC BY 4.0) http://creativecommons.org/licenses/by/4.0/Highlights • 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.2024.100796en-USAttribution 4.0 Internationalproteomicssample preparationEmpore membraneE3technologyE4technologyon-filter digestionin-cell digestionglass beadssilica microparticlesArticle