Development and Validation of MS Based Technique, Protein Painting, to Identify Patient Antibody-Binding Epitopes on the Nucleocapsid Protein of SARS-CoV-2
The identification of antibody and epitope complexes between a target protein and a patient antibody are crucial to direct development of next-generation vaccines. Unfortunately, there are difficulties in identifying contact regions due to complexity of patient samples and burdensome available structural techniques. Protein painting allows the unbound surfaces of a protein to be masked, revealing the bound epitope. The recent coronavirus pandemic provides a case study to probe interactions between patient-derived antibodies and SARS-CoV-2 N-protein, which allows identification of immunodominant peptides at the interface region..Exploratory trials were used to optimize the technique, and adjustments were made to solve problems in carryover, low protein yields, and complexity of patient samples. Suitability of controls, altered N-protein concentration and incorporation of affinity purification of the complex were examined. Results indicate lysozyme possessed the most sequences for normalization, but the N-protein peptide spectral matches (PSMs) were low. Increasing N-protein concentration improved PSM counts subtly, but further increase was considered infeasible; subsequently, pre-purification of the N-protein and antibody complex was attempted. This resulted in low yields of N-protein. Further sample purification via SDS-PAGE will improve the purity of the N-protein sample analyzed via mass-spectrometry. Following final optimization, future directions involve utilization of additional patients which allows for identification of immunodominant regions. Prospectively, we can use this MS-based technology to examine immune responses in other pathogenic organisms.
Copyright (c) 2022 MARYAM BELLAKBIRA, Rachel Carter, Hannah Steinberg, Alessandra Luchini, Amanda Haymond
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