With the development of new vaccine technologies, such as mRNA vaccines, tissue studies are becoming increasingly important. Knowledge of the antigen expression amounts and where the antigen accumulates in the body is essential for designing safe and effective vaccines. Mammalian tissues present challenges in the detection and accurate quantification of target proteins because of their complexity and the lack of protocols that efficiently extract proteins with minimal sample loss. Here, we describe a protocol for the detection and accurate quantification of protein targets in commercially available snap-frozen lung, liver, kidney, and spleen of European domestic ferrets (Mustela putorius furo) by isotope dilution mass spectrometry (IDMS). Housekeeping proteins were chosen that range in abundance to account for different masses of tissue slices of the same organ. Target peptides used for IDMS quantification were conserved across several of the common animal model systems, including baby hamster kidney, mouse, and ferret. Hemagglutinin, the primary antigen of an influenza vaccine, was added at various concentrations to demonstrate the recovery of low-abundance proteins from the complex tissue homogenate. By using housekeeping proteins and a preparation protocol that minimizes sample loss, this study shows that IDMS can accurately quantify proteins in mammalian tissues with unmatched sensitivity and specificity.

This manuscript describes the use of an analytical assay that combines transfection of mammalian cells and isotope dilution mass spectrometry (IDMS) for accurate quantification of antigen expression. Expired mRNA COVID-19 vaccine material was stored at 4 °C, room temperature (∼25 °C), and 56 °C over a period of 5 weeks. The same vaccine was also exposed to 5 freeze-thaw cycles. Every week, the spike protein antigenic expression in mammalian (BHK-21) cells was evaluated. Housekeeping proteins, β-actin and GAPDH, were simultaneously quantified to account for the variation in cell counts that occurs during maintenance and growth of cell cultures. Data show that vaccine stored at elevated temperatures results in reduced spike protein expression. Also, maintaining the vaccine in ultracold conditions or exposing the vaccine to freeze-thaw cycles had less effect on the vaccine's ability to produce the antigen in mammalian cells. We describe the use of IDMS as an antibody-free means to accurately quantify expressed protein from mammalian cells transfected with mRNA vaccine.

The advent of mRNA vaccine technology has been vital in rapidly creating and manufacturing COVID-19 vaccines at an industrial scale. To continue to accelerate this leading vaccine technology, an accurate method is needed to quantify antigens produced by the transfection of cells with a mRNA vaccine product. This will allow monitoring of protein expression during mRNA vaccine development and provide information on how changes to vaccine components affects the expression of the desired antigen. Developing novel approaches that allow for high-throughput screening of vaccines to detect changes in antigen production in cell culture prior to in vivo studies could aid vaccine development. We have developed and optimized an isotope dilution mass spectrometry method to detect and quantify the spike protein expressed after transfection of baby hamster kidney cells with expired COVID-19 mRNA vaccines. Five peptides of the spike protein are simultaneously quantified and provide assurance that protein digestion in the region of the target peptides is complete since results between the five peptides had a relative standard deviation of less than 15 %. In addition, two housekeeping proteins, actin and GAPDH, are quantified in the same analytical run to account for any variation in cell growth within the experiment. IDMS allows a precise and accurate means to quantify protein expression by mammalian cells transfected with an mRNA vaccine.