SARS-CoV-2 spike antigen quantification by targeted mass spectrometry of a virus-based vaccine

•The spike glycoprotein of SARS-CoV-2 mediates virus binding to host cells’ receptors and is a key target for vaccines development.•The spike induces a specific immune response. Yet, no method was developed for spike quantification.•A mass spectrometric based method for spike quantification was developed and demonstrated on spike-based vaccine.•Proof of concept of this method is shown for complex matrix samples and total spike levels were correlated with results from activity assays.•The method is simple, reliable and allows determination of antigen quantity within a sample, and can be easily implemented for any vaccine or therapeutic sample. The spike glycoprotein mediates virus binding to the host cells and is a key target for vaccines development. One SARS-CoV-2 vaccine is based on vesicular stomatitis virus (VSV), in which the native surface glycoprotein has been replaced by the SARS-CoV-2 spike protein (VSV-ΔG-spike). The titer of the virus is quantified by the plaque forming unit (PFU) assay, but there is no method for spike protein quantitation as an antigen in a VSV-based vaccine. Here, we describe a mass spectrometric (MS) spike protein quantification method, applied to VSV-ΔG-spike based vaccine. Proof of concept of this method, combining two different sample preparations, is shown for complex matrix samples, produced during the vaccine manufacturing processes. Total spike levels were correlated with results from activity assays, and ranged between 0.3−0.5 μg of spike protein per 107 PFU virus-based vaccine. This method is simple, linear over a wide range, allows quantification of antigen within a sample and can be easily implemented for any vaccine or therapeutic sample.