Browsing by Author "Zhuang, Zhihao"
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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 Photo-activatable Ub-PCNA probes reveal new structural features of the Saccharomyces cerevisiae Polη/PCNA complex(Nucleic Acids Research, 2021-08-14) Shen, Siqi; Davidson, Gregory A; Yang, Kun; Zhuang, ZhihaoThe Y-family DNA polymerase η (Polη) is critical for the synthesis past damaged DNA nucleotides in yeast through translesion DNA synthesis (TLS). TLS is initiated by monoubiquitination of proliferating cell nuclear antigen (PCNA) and the subsequent recruitment of TLS polymerases. Although individual structures of the Polη catalytic core and PCNA have been solved, a high-resolution structure of the complex of Polη/PCNA or Polη/monoubiquitinated PCNA (Ub-PCNA) still remains elusive, partly due to the disordered Polη C-terminal region and the flexibility of ubiquitin on PCNA. To circumvent these obstacles and obtain structural insights into this important TLS polymerase complex, we developed photo-activatable PCNA and Ub-PCNA probes containing a p-benzoyl-L-phenylalanine (pBpa) crosslinker at selected positions on PCNA. By photo-crosslinking the probes with full-length Polη, specific crosslinking sites were identified following tryptic digestion and tandem mass spectrometry analysis. We discovered direct interactions of the Polη catalytic core and its C-terminal region with both sides of the PCNA ring. Model building using the crosslinking site information as a restraint revealed multiple conformations of Polη in the polymerase complex. Availability of the photo-activatable PCNA and Ub-PCNA probes will also facilitate investigations into other PCNA-containing complexes important for DNA replication, repair and damage tolerance.