Protein-level evidence of specific IGHV chain usage in immune responses to SARS-CoV-2 using an undepleted proteomic profiling workflow

Antibodies play a critical role in directing responses of the adaptive immune system towards infectious organisms. They exhibit great diversity as a consequence of genetic rearrangements and somatic hypermutation in the antibody binding sites which affects their functionality in vivo. Convergent responses have been identified to a number of pathogens using transcriptomics and isolated which identified convergent usage of certain variable heavy chain mutations. However, protein level evidence for such variants can be challenging to obtain. In a pilot study we have compared depleted and undepleted proteomics workflows using less than 10 µL of plasma from two patients with a severe current SARS-CoV-2 infection and two convalescent patients. Proteomics analysis was performed using a rapid 15-minute gradient with a high-sensitivity cyclic ion mobility mass spectrometer. Progenesis QI for proteomics and R 4.3.1 were used to analyse mass spectral data. To validate this workflow, the relative normalized abundance of CRP was compared against hospital measurements and the relative proportion of abundant proteins in the undepleted workflow was compared against expected proportions in plasma. While more low abundance and non-secretory proteins were identified in the depleted workflow, immunoglobulin variable regions could only be consistently identified in the undepleted samples. Of the 306 functional immunoglobulin heavy variable region sequences present in the International ImMunoGeneTics Information System, 62 were identified. This included two proteoforms with neutralising activity in COVID-19 patients identified using RNA-Seq workflows, VH3-53 and VH1-2. VH1-2 was found in all patients, whilst VH3-53 was found in both patients with a severe current infection. This approach has provided protein-level evidence of specific variable heavy chain usage in COVID-19. Further analysis of the ManARTS COVID-19 cohort of 184 patients will provide further insights into the presence and level of expression of particular immunoglobulin isoforms which can be linked to patient phenotypes.