Tao Research Group

The outbreak of Zika virus (ZIKV) in 2016 created worldwide health emergency which demand urgent research efforts on understanding the virus biology and developing therapeutic strategies. Here, we present a time-resolved chemical proteomic strategy to track the early-stage entry of ZIKV into host cells. ZIKV was labeled on its surface with a chemical probe, which carries a photocrosslinker to covalently link virus-interacting proteins in living cells on UV exposure at different time points, and a biotin tag for subsequent enrichment and mass spectrometric identification of the receptor or other host proteins critical for virus internalization. We identified Neural Cell Adhesion Molecule (NCAM1) as a potential ZIKV receptor and further validated it through overexpression, knockout, and inhibition of NCAM1 in Vero cells and human glioblastoma cells U-251 MG. Collectively, the strategy can serve as a universal tool to map virus entry pathways and uncover key interacting proteins.

For more details: Srivastava, M., Zhang, Y., Chen, J., Sirohi, D., Miller, A., Zhang, Y., Chen, Z., Lu, H., Xu, J., Kuhn, R. J., & Andy Tao, W. (2020). Chemical proteomics tracks virus entry and uncovers NCAM1 as Zika virus receptor. Nature communications, 11(1), 3896.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been recognized as genetic risk factors for Parkinson’s disease (PD). However, compared to cancer, fewer genetic mutations contribute to the cause of PD, propelling the search for protein biomarkers for early detection of the disease. Utilizing 138 urine samples from four groups, healthy individuals (control), healthy individuals with G2019S mutation in the LRRK2 gene (non-manifesting carrier/NMC), PD individuals without G2019S mutation (idiopathic PD/iPD), and PD individuals with G2019S mutation (LRRK2 PD), we applied a phosphoproteomics strategy to determine potential diagnostic biomarkers for PD from urinary extracellular vesicles (EVs). The findings in this study demonstrate a general strategy of utilizing biofluid EV proteome/phosphoproteome as an outstanding and non-invasive source for a wide range of disease exploration.

For more details: Hadisurya, M., Li, L., Kuwaranancharoen, K., Wu, X., Lee, Z. C., Alcalay, R. N., Padmanabhan, S., Tao, W. A., & Iliuk, A. (2023). Quantitative proteomics and phosphoproteomics of urinary extracellular vesicles define putative diagnostic biosignatures for Parkinson's disease. Communications medicine, 3(1), 64.

The current workflow of mass spectrometry-based EV proteome analysis is not fully compatible in a clinical setting due to inefficient EV isolation methods and a tedious sample preparation process. To streamline and improve the efficiency of EV proteome analysis, we introduced a one-pot analytical pipeline integrating a robust EV isolation approach, EV total recovery and purification (EVtrap), with in situ protein sample preparation, to detect urinary EV proteome. By incorporating solvent-driven protein capture and fast on-bead digestion, the one-pot pipeline enabled the whole EV proteome analysis to be completed within one day. Our novel one-pot analytical pipeline demonstrated its potential for routine and robust EV proteomics in biomedical applications.

For more details: Liu, Y. K., Wu, X., Hadisurya, M., Li, L., Kaimakliotis, H., Iliuk, A., & Tao, W. A. (2023). One-Pot Analytical Pipeline for Efficient and Sensitive Proteomic Analysis of Extracellular Vesicles. Journal of proteome research, 22(10), 3301–3310.

In this study, urinary extracellular vesicular proteins are compared between healthy controls, patients with chronic kidney disease, and patients with either low grade or high-grade renal cell carcinoma (RCC). Knowing the grade of renal cell carcinoma is critical knowledge for surgeons recommending operations, but existing determination methods rely on painful and invasive tumor biopsies. The developed liquid biopsy for distinguishing low and high grade RCC utilizes urinary extracellular vesicular enrichment via EVtrap and phosphoprotein enrichment with PolyMac beads. After mass spectral analysis, upregulated phosphorylation was shown to be site specific between patients suffering from low-grade versus high-grade RCC, indicating that this technique is viable for distinguishing RCC grades even when compared to other kidney diseases or healthy patients.

For more details: Hadisurya, M., Lee, Z. C., Luo, Z., Zhang, G., Ding, Y., Zhang, H., Iliuk, A. B., Pili, R., Boris, R. S., & Tao, W. A. (2023). Data-Independent Acquisition Phosphoproteomics of Urinary Extracellular Vesicles Enables Renal Cell Carcinoma Grade Differentiation. Molecular & cellular proteomics : MCP, 22(5), 100536.

Protein kinases are major regulatory components in almost all cellular processes in eukaryotic cells. By adding phosphate groups, protein kinases regulate the activity, localization, protein–protein interactions, and other features of their target proteins. It is known that protein kinases are central components in plant responses to environmental stresses such as drought, high salinity, cold, and pathogen attack. However, only a few targets of these protein kinases have been identified. Moreover, how these protein kinases regulate downstream biological processes and mediate stress responses is still largely unknown. In this study, we introduce a strategy based on isotope-labeled in vitro phosphorylation reactions using in vivo phosphorylated peptides as substrate pools and apply this strategy to identify putative substrates of nine protein kinases that function in plant abiotic and biotic stress responses.

For more details: Wang, P., Hsu, C. C., Du, Y., Zhu, P., Zhao, C., Fu, X., Zhang, C., Paez, J. S., Macho, A. P., Tao, W. A., & Zhu, J. K. (2020). Mapping proteome-wide targets of protein kinases in plant stress responses. Proceedings of the National Academy of Sciences of the United States of America, 117(6), 3270–3280.