Ultrafast Tyrosinase-Mediated Biotinylation of Living Cell Surface Analysis Reveals Novel Cell Surface Proteins Responsible for Influenza A Virus Entry

Cell surface proteins are integral to a myriad of biological processes, including cell-cell interactions, signal transduction, and cell adhesion. Notably, these proteins also serve as key receptors for numerous pathogens. However, a comprehensive analysis of the surfaceome remains a significant challenge, primarily due to the high hydrophobicity and low abundance of these proteins. Here, we developed a novel cell surface profiling approach, tyrosinase-mediated cell surface labeling (TYRCSL). Tyrosinase can mildly oxidize a phenol or catechol to the corresponding <i>o</i>-quinone, which rapidly reacts with available protein nucleophiles. Leveraging the fast-labeling kinetics (within 1 min) and minimal toxicity of this method, we explored surfaceome dynamics during early stages of influenza A virus (IAV) entry. Our findings reveal that cell surface proteins PODXL2, CNTNAP1, and GPR39 play a crucial role in virus binding and internalization, providing valuable insights into the molecular mechanisms of IAV entry. TYRCSL shows the potential to be applied to the system-wide study of the entry process across a spectrum of pathogens and other instantaneous surfaceome changes under various stimuli.