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
| Autor: | Gertrudis Rojas, Yury Valdés-Balbín, Dayana Pérez-Martínez, Vicente Verez-Bencomo, Sum Lai Lozada Chang, Isabel González, Ivan Andújar, Daniel G. Rivera, Enso Onill Torres, Luis Javier González, Gleysin Cabrera, Darielys Santana-Medero, Marika Vitikainen, Julio Palacio, Mark Emalfarb, Marta Ayala, Camila Canaán-Haden, Diamilé Roche, Lourdes Hernández, Vladimir Besada, Saloheimo Markku, Mónica Becquet, Glay Chinea, Eulogio Pimentel, Elías Nelson, Yassel Ramos, Luis Ariel Espinosa, Gerardo Guillén, Kathya Rashida de la Luz Hernández, Tamy Boggiano, Beatriz Pérez-Massón, Miladys Limonta, Alejandro Martín, Ronen Tchelet |
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| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
Spectrometry
Mass Electrospray Ionization Stereochemistry Dimer Electrospray ionization Peptide Biochemistry Analytical Chemistry law.invention RBD chemistry.chemical_compound Protein Domains Modified cysteine SDG 3 - Good Health and Well-being law Humans Hydrophilic peptides Buffer-free digestion Amino Acid Sequence Cysteine Histidine chemistry.chemical_classification SARS-CoV-2 Protein primary structure Peptide Fragments Amino acid Protein Subunits chemistry Spike Glycoprotein Coronavirus Recombinant DNA Hydrophobic and Hydrophilic Interactions Protein Processing Post-Translational Research Paper Protein Binding |
| Zdroj: | Espinosa, L A, Ramos, Y, Andújar, I, Torres, E O, Cabrera, G, Martín, A, Roche, D, Chinea, G, Becquet, M, González, I, Canaán-Haden, C, Nelson, E, Rojas, G, Pérez-Massón, B, Pérez-Martínez, D, Boggiano, T, Palacio, J, Lozada Chang, S L, Hernández, L, de la Luz Hernández, K R, Markku, S, Vitikainen, M, Valdés-Balbín, Y, Santana-Medero, D, Rivera, D G, Vérez-Bencomo, V, Emalfarb, M, Tchelet, R, Guillén, G, Limonta, M, Pimentel, E, Ayala, M, Besada, V & González, L J 2021, ' 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 ', Analytical and Bioanalytical Chemistry, vol. 413, no. 30, pp. 7559-7585 . https://doi.org/10.1007/s00216-021-03721-w Analytical and Bioanalytical Chemistry |
| ISSN: | 1432-1130 1618-2642 |
| Popis: | 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. Graphical abstract: [Figure not available: see fulltext.] |
| Databáze: | OpenAIRE |
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