Production and Characterization of a SARS-CoV-2 Nucleocapsid Protein Reference Material

Abstract

Rapid antigen tests have become a widely used COVID-19 diagnostic tool with demand accelerating in response to the highly contagious SARS-CoV-2 Omicron variant. Hundreds of such test kits are approved for use worldwide, predominantly reporting on the presence of the viral nucleocapsid (N) protein, yet the comparability among manufacturers remains unclear and the need for reference standards is recognized. To address this lack of standardization, the National Research Council Canada has developed a SARS-CoV-2 nucleocapsid protein reference material solution, NCAP-1. Reference value determination for N protein content was realized by amino acid analysis (AAA) via double isotope dilution liquid chromatography-tandem mass spectrometry (LC-ID-MS/MS) following acid hydrolysis of the protein, in conjunction with UV spectrophotometry based on tryptophan and tyrosine absorbance at 280 nm. The homogeneity of the material was established through spectrophotometric absorbance readings at 280 nm. The molar concentration of the N protein in NCAP-1 was 10.0 ± 1.9 μmol L^-1 (k = 2, 95% confidence interval). Reference mass concentration and mass fraction values were subsequently calculated using the protein molecular weight and density of the NCAP-1 solution. Changes to protein higher-order structure, probed by size-exclusion liquid chromatography (LC-SEC) with UV detection, were used to evaluate transportation and storage stabilities. LC-SEC revealed nearly 90% of the N protein in the material is present as a mixture of hexamers and tetramers. The remaining low molecular weight species (<30 kDa) were interrogated by top-down mass spectrometry and determined to be autolysis products homologous to those previously documented for N protein of the original SARS-CoV [Biochem. Biophys. Res. Commun.2008t, 377, 429-433].

Figure 1

Normalized protein content in 15 randomly selected NCAP-1 units. Data points and error bars represent averages and standard deviations, respectively, of triplicate technical replicates. Dotted line is the average molar concentration determined by A₂₈₀ and dashed lines indicate expanded uncertainty (k = 2, 95% CI).

Figure 2

Size exclusion UV chromatograms of (A) molecular weight standard protein mix and (B) NCAP-1. Void volume was determined from elution of blue dextran. Standard protein mix consists of thyroglobulin (Tg), ferritin (Fer), aldolase (Ald), conalbumin (ConA), and ovalbumin (Ov). Panel (C) shows molecular weight calibration of the SEC column and calculated values for sample peaks in (B). Red dashed lines in panel (B) result from Gaussian decomposition and correspond to hexamers (N₆) and tetramers (N₄). K_av values calculated using eq 2.

Figure 3

Normalized amount of (A) total nucleocapsid oligomer (hexamers and tetramers) and (B) low molecular weight species in NCAP-1 under various storage conditions. Values result from triplicate measurements of two units per condition. Data points and error bars represent averages and standard deviations, respectively.

Figure 4

Freeze–thaw effects on protein size heterogeneity in NCAP-1. Left axis: Relative amount of N protein oligomer as a function of freeze–thaw cycle number. Right axis: Relative hexamer contribution to total N oligomer signal. Two NCAP-1 units were used for each condition and analyzed in triplicate. Data points and error bars represent averages and standard deviations, respectively. The average N oligomer values of the control samples (zero F/T cycles) ± 1% are indicated by the dotted and dashed lines, respectively, and are for visualization purposes only.

Figure 5

Denaturing LC-MS of NCAP-1 unit stored at −80 °C. (A) LC-MS chromatogram with main elution peak cropped to observe minor species. Peak numbering corresponds to that of Table 1. (B) Deconvoluted mass spectrum resulting from integration of main elution peak in panel (A). Peak labeling corresponds to N protein residue numbers. Inset shows zoom-in on intact N peak.