In-solution buffer-free digestion allows full-sequence coverage and complete characterization of post-translational modifications of the receptor-binding domain of SARS-CoV-2 in a single ESI-MS spectrum.

Subunit vaccines based on the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 provide one of the most promising strategies to fight the COVID-19 pandemic. The detailed characterization of the protein primary structure by mass spectrometry (MS) is mandatory, as described in ICHQ6B guidelines. In this work, several recombinant RBD proteins produced in five expression systems were characterized using a non-conventional protocol known as in-solution buffer-free digestion (BFD). In a single ESI-MS spectrum, BFD allowed very high sequence coverage (≥ 99%) and the detection of highly hydrophilic regions, including very short and hydrophilic peptides (2-8 amino acids), and the His6-tagged C-terminal peptide carrying several post-translational modifications at Cys538 such as cysteinylation, homocysteinylation, glutathionylation, truncated glutathionylation, and cyanylation, among others. The analysis using the conventional digestion protocol allowed lower sequence coverage (80-90%) and did not detect peptides carrying most of the above-mentioned PTMs. The two C-terminal peptides of a dimer [RBD(319-541)-(His)6]2 linked by an intermolecular disulfide bond (Cys538-Cys538) with twelve histidine residues were only detected by BFD. This protocol allows the detection of the four disulfide bonds present in the native RBD, low-abundance scrambling variants, free cysteine residues, O-glycoforms, and incomplete processing of the N-terminal end, if present. Artifacts generated by the in-solution BFD protocol were also characterized. BFD can be easily implemented; it has been applied to the characterization of the active pharmaceutical ingredient of two RBD-based vaccines, and we foresee that it can be also helpful to the characterization of mutated RBDs.