Characterization of biotinylated human ACE2 and SARS-CoV-2 Omicron BA.4/5 spike protein reference materials.

Accurate diagnostic and serology assays are required for the continued management of the COVID-19 pandemic yet spike protein mutations and intellectual property concerns with antigens and antibodies used in various test kits render comparability assessments difficult. As the use of common, well-characterized reagents can help address this lack of standardization, the National Research Council Canada has produced two protein reference materials (RMs) for use in SARS-CoV-2 serology assays: biotinylated human angiotensin-converting enzyme 2 RM, ACE2-1, and SARS-CoV-2 Omicron BA.4/5 spike protein RM, OMIC-1. Reference values were assigned through a combination of amino acid analysis via isotope dilution liquid chromatography tandem mass spectrometry following acid hydrolysis, and ultraviolet-visible (UV-Vis) spectrophotometry at 280 nm. Vial-to-vial homogeneity was established using UV-Vis measurements, and protein oligomeric status, monitored by size exclusion liquid chromatography (LC-SEC), was used to evaluate transportation, storage, and freeze-thaw stabilities. The molar protein concentration in ACE2-1 was 25.3 ± 1.7 µmol L-1 (k = 2, 95% CI) and consisted almost exclusively (98%) of monomeric ACE2, while OMIC-1 contained 5.4 ± 0.5 µmol L-1 (k = 2) spike protein in a mostly (82%) trimeric form. Glycoprotein molar mass determination by LC-SEC with multi-angle light scattering detection facilitated calculation of corresponding mass concentrations. To confirm protein functionality, the binding of OMIC-1 to immobilized ACE2-1 was investigated with surface plasmon resonance and the resulting dissociation constant, KD ~ 4.4 nM, was consistent with literature values.

Characterization of a SARS-CoV-2 spike protein reference material.

Development of diagnostic testing capability has advanced with unprecedented pace in response to the COVID-19 pandemic. An undesirable effect of such speed is a lack of standardization, often leading to unreliable test results. To assist the research community surmount this challenge, the National Research Council Canada has prepared a SARS-CoV-2 spike protein reference material, SMT1-1, as a buffered solution. Value assignment was achieved by amino acid analysis (AAA) by double isotope dilution liquid chromatography-tandem mass spectrometry (LC-ID-MS/MS) following acid hydrolysis of the protein, in combination with ultraviolet-visible spectrophotometry (UV-Vis) based on tryptophan and tyrosine absorbance at 280 nm. Homogeneity of the material was established through spectrophotometric absorbance readings at 280 nm. Transportation and long-term storage stabilities were assessed by monitoring relative changes in oligomeric state by size-exclusion liquid chromatography (LC-SEC) with UV detection. The molar concentration of the spike protein in SMT1-1 was 5.68 ± 0.22 µmol L-1 (k = 2, 95% CI), with the native trimeric form accounting for ~ 94% of the relative abundance. Reference mass concentration and mass fraction values were calculated using the protein molecular weight and density of the SMT1-1 solution. The spike protein is highly glycosylated which leads to analyte ambiguity when reporting the more commonly used mass concentration. After glycoprotein molar mass determination by LC-SEC with multi-angle light scattering detection, we thus reported mass concentration values for both the protein-only portion and intact glycoprotein as 0.813 ± 0.030 and 1.050 ± 0.068 mg mL-1 (k = 2), respectively.

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

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].

Survey of peptide quantification methods and comparison of their reproducibility: A case study using oxytocin.

USP's peptide reference standards content is typically determined using an HPLC assay against an external standard for which the purity was determined by a mass balance approach. To explore the use of other analytical methods, the USP Biologics Department conducted a multi-laboratory collaborative study. The study determined the inter-laboratory variability for peptide quantitation using the following methods: HPLC assay, quantitative nuclear magnetic resonance (qNMR) spectroscopy, or amino acid analysis (AAA). The three methods were compared with regard to their suitability for quantitation of the nonapeptide oxytocin. In this study, the HPLC assay method using the same peptide bulk material as the standard showed the lowest inter-lab variability. The coefficient of variation (%CV) was calculated without counting the uncertainty associated with the purity assignment of the standard with mass balance. The proton qNMR method is a direct measurement of the peptide against an internal standard, which is not difficult to perform under common laboratory conditions. Because of the simpler operation and shorter analytical time, qNMR as a primary method for peptide reference standard value assignment deserves further exploration.

Assessing MS-based quantitation strategies for low-level impurities in peptide reference materials: application to angiotensin II.

Identification and quantitation of related impurities is vital in obtaining corrected purity values for peptide certified reference materials. The sensitivity and selectivity of high-resolution mass spectrometry (MS) renders it an indispensable technique in this arena. Typical quantitation efforts involve constructing external calibration curves, although analysis of dilute peptide solutions can be complicated by analyte adsorption to vial walls, instrument tubing, etc. The standard addition method alleviates many concerns associated with this sample loss as the calibrant solutions more closely match the matrix of the samples. Yet, both strategies require acquisition of synthetic impurity peptide standards. Label-free proteomics relies on electrospray ionization (ESI)-MS signals to quantify identical peptides across multiple samples; however, peptides of differing sequence can exhibit widely disparate ESI-MS responses. This study explores the use of peak area ratios to quantitate sequence-related peptide impurities in an angiotensin II candidate certified reference material. Using synthetic standards of five abundant substances, impurity mass fractions calculated via the relative response method are in reasonable agreement with those determined from standard addition experiments, whereas external calibration measurements frequently overestimate impurity amounts. For a synthetic peptide and its related sequence impurities, the relative response method can expedite analysis and lower expenditures, and in some cases improve data quality.

Purity assignment for peptide certified reference materials by combining qNMR and LC-MS/MS amino acid analysis results: application to angiotensin II.

The purity value assignment of metrologically traceable peptide reference standards requires specialized primary methods. Conventionally, amino acid analysis by isotope dilution tandem mass spectrometry (LC-MS/MS) following peptide hydrolysis is employed as a reference method. By contrast, quantitative nuclear magnetic resonance (qNMR) spectroscopy allows for quantitation of intact peptides, thus eliminating potential bias due to hydrolysis. Both methods are susceptible to interference from related peptide impurities, which need to be accurately measured and accounted for. The mass balance approach has also been employed for peptide purity measurements, whereby the purity is defined by the sum of the mass fraction of all impurities identified. Ideally, results from these three orthogonal methods can be combined for final purity assignment of peptide reference standards. Here we report a novel strategy for correcting both LC-MS/MS and 1H-qNMR results for related peptide impurities and combining results from both methods using a Bayesian statistical approach using mass balance results as prior knowledge. The mass balance method relied on a validated 19F-qNMR method to measure the trifluoroacetic acid (TFA) counter-ion, considered an impurity in this case at nearly 25% by mass. Using a candidate certified reference material (CRM) for angiotensin II, excellent agreement was achieved with the three methods. The final purity value assignment of the candidate CRM was 691 ± 9 mg/g (k = 2).

Determination of total dissolved nitrogen in seawater by isotope dilution gas chromatography mass spectrometry following digestion with persulfate and derivatization with aqueous triethyloxonium.

In this study, we propose a novel approach for the determination of total dissolved nitrogen (TDN) in seawater combining high-precision isotope dilution GC-MS with persulfate digestion. A 2 mL sample aliquot was digested with an alkaline solution of persulfate to convert nitrogen containing compounds to nitrate. Digested samples were spiked with 15NO3- internal standard and treated with aqueous triethyloxonium to convert the analyte into volatile EtONO2. This derivative was readily separated from the matrix under gaseous form and could be sampled from the headspace before GC-MS analysis. The resulting chromatograms showed a stable flat baseline with EtONO2 as the only eluting peak (retention time 2.75 min on a DB 5.625 column). Such an approach provides specificity and obviates the shortcomings of current detection methods employed to analyze seawater samples after digestion with persulfate. In negative chemical ionization mode, the method reached a detection limit of 0.5 µmol/kg TDN (7 ng/g N) and could be applied to quantify seawater samples with 1-25 µmol/kg TDN. On the upper end of the range, quantitation could be repeated within 1%, whereas on a 6 µmol/kg TDN sample repeatability was 2.3% on eight measurements. The method was employed in two proficiency testing exercises providing results in agreement with consensus values. We investigated the impact of reagent blank and we implemented a blank-matching optimal design to account for such contribution. Finally, we performed a study on the yield of persulfate oxidation for organic and inorganic nitrogen compounds typically present in seawater. Whilst nitrite and ammonium are fully converted to nitrate, more complex organic molecules showed recoveries varying from 70% to 100%.

Determination of cyanocobalamin by isotope dilution LC-MS/MS.

Cyanocobalamin (CNCbl) is an active form of vitamin B12, commonly employed for the preparation of multivitamin supplements and fortified food. In this study, we present a novel analytical method for its determination based on stable isotope dilution liquid chromatography electrospray tandem mass spectrometry (ID LC-MS/MS). Isotopically enriched 13C15NCbl was synthesized in-house and used as internal standard. The method was validated using NIST SRM 3280 multivitamin reference material and by comparison with an independent methodology based on LC-ICPMS. The proposed method provided a detection limit of 57 pg/g and could be applied for the determination of trace level of CNCbl in multivitamin supplements with a relative standard uncertainty better than 3%. The novel ID LC-MS/MS is a primary ratio method that could become a reference for CNCbl determination in multivitamins and food supplements. The method was applied for the characterization of two NRC multivitamin tablet Certified Reference Material (CRM) candidates, VITA-1 and VITB-1 whose CNCbl levels were quantified as 2.64 ± 0.09 and 1.75 ± 0.12 µg/g, respectively.