The therapeutic success of in vitro-transcribed (IVT) mRNA depends on its stability and efficient translation. However, IVT mRNA is highly susceptible to immune-mediated degradation, limiting its efficacy. To investigate the binding protein profiles of IVT mRNA, we transfected HUVECs with 4SU-modified mRNA for 6h. LC-MS/MS was performed to identify the binding proteins of IVT mCherry mRNA.