High-Throughput Quantification of Spike Protein Expression by LC-MS for mRNA-LNP Evaluation and Immune Response...

The COVID-19 pandemic has accelerated interest in immuno-multiple reaction monitoring (immuno-MRM) for peptide quantification, with early efforts focusing on SARS-CoV-2 biomarker detection in clinical nasopharyngeal swabs. However, the emergence of mRNA vaccines has created a new and pressing need for robust methods to quantify antigen expression. Here, we present an optimized immuno-MRM method targeting the SARS-CoV-2 spike fusion peptide SFIEDLLFNK, designed and validated to quantify antigen expression following mRNA or plasmid transfection. This method offers high sensitivity, precision, and linearity across a broad dynamic range, enabling an accurate assessment of protein translation in vitro. In addition to measuring antigen levels, analysis of the flow-through provides insight into host cell proteomic responses, supporting the comprehensive characterization of mRNA vaccine efficacy and safety. This dual-function workflow serves as a powerful tool for vaccine development, quality control, and regulatory evaluation of RNA-based therapeutics.