Direct Detection of Glycated Human Serum Albumin and Hyperglycosylated IgG3 in Serum, by MALDI-ToF Mass Spectrometry, as a Predictor of COVID-19 Severity

Abstract

The prefusion spike protein of SARS-CoV-2 binds advanced glycation end product (AGE)-glycated human serum albumin (HSA) and a higher mass (hyperglycosylated/glycated) immunoglobulin (Ig) G3, as determined by matrix assisted laser desorption mass spectrometry (MALDI-ToF). We set out to investigate if the total blood plasma of patients who had recovered from acute respiratory distress syndrome (ARDS) as a result of COVID-19, contained more glycated HSA and higher mass (glycosylated/glycated) IgG3 than those with only clinically mild or asymptomatic infections. A direct serum dilution, and disulphide bond reduction, method was developed and applied to plasma samples from SARS-CoV-2 seronegative (n = 30) and seropositive (n = 31) healthcare workers (HCWs) and 38 convalescent plasma samples from patients who had been admitted with acute respiratory distress (ARDS) associated with COVID-19. Patients recovering from COVID-19 ARDS had significantly higher mass AGE-glycated HSA and higher mass IgG3 levels. This would indicate that increased levels and/or ratios of hyper-glycosylation (probably terminal sialic acid) IgG3 and AGE glycated HSA may be predisposition markers for the development of COVID-19 ARDS as a result of SARS-CoV2 infection. Furthermore, rapid direct analysis of serum/plasma samples by MALDI-ToF for such humoral immune correlates of COVID-19 presents a feasible screening technology for the most at risk; regardless of age or known health conditions.

Keywords: COVID-19; MALDI-ToF MS; glycated albumin; glycovariant/glycated IgG3; plasma.

Figure 1

Mass spectra profile, 40,000 m/z to 85,000 m/z, of human reduced plasma samples (treated with Tris(2-carboxyethyl)phosphine (TCEP)) in order to reveal immunoglobulin heavy chains (IgM Hc ‘75,000 m/z, IgA ~56,000 m/z not found, IgG3 54,000 m/z and unconfirmed IgX, thought to be IgG4 Hc, at 48,000 m/z. HSA resolved at 66,4000+ m/z and transferrin at 79,000 m/z.

Figure 2

Box and whisker plots of relative intensitities and variance in peak apex molecular mass of IgG1 heavy chains (IgG1 Hc), IgG3 heavy chains (IgG3 Hc) and human serum albumin (HSA) for the different sample groups: Blue represents data from SARS-CoV-2 seronegative HCWs, Orange from SARS-CoV-2 seropositive HCWs having recovered from COVID-19 with mild symptoms and Red sample data from convalescent patients recovering from COVID-19 ARDS. The table to the right is numeric data from the plots detailing the mean and median values of peak intensity (arbitrary units (AU)) and molecular mass (m/z) of IgG1 Hc, IgG3 Hc and HSA for the respective groups.

Figure 3

Diagrammatic representation of IgG1 (left) and IgG3 (right) illustrating the differences in heavy chain structure with special reference to the larger neck domain and the O-linked glycosylation sites found there. Affinity binding: –, none; +,weak; ++, strong; and +++, very strong.

Figure 4

Spike protein sialic acid glycan binding (S1 subunit NTD-domain) role in (A) mucosal epithelial attachment and infection of respiratory cells and (B) potential role in binding IgG3 at the neck region and AGE glycated HSA, during viremia, thereby aiding immune evasion and deposition of reactive complex that may give rise to vascular pathologies.