Tags

Type your tag names separated by a space and hit enter

Development of a Parallel Reaction Monitoring Mass Spectrometry Assay for the Detection of SARS-CoV-2 Spike Glycoprotein and Nucleoprotein.
Anal Chem. 2020 10 20; 92(20):13813-13821.AC

Abstract

There is an urgent need for robust and high-throughput methods for SARS-CoV-2 detection in suspected patient samples to facilitate disease management, surveillance, and control. Although nucleic acid detection methods such as reverse transcription polymerase chain reaction (RT-PCR) are the gold standard, during the current pandemic, the deployment of RT-PCR tests has been extremely slow, and key reagents such as PCR primers and RNA extraction kits are at critical shortages. Rapid point-of-care viral antigen detection methods have been previously employed for the diagnosis of respiratory viruses such as influenza and respiratory syncytial viruses. Therefore, the direct detection of SARS-CoV-2 viral antigens in patient samples could also be used for diagnosis of active infection, and alternative methodologies for specific and sensitive viral protein detection should be explored. Targeted mass spectrometry techniques have enabled the identification and quantitation of a defined subset of proteins/peptides at single amino acid resolution with attomole level sensitivity and high reproducibility. Herein, we report a targeted mass spectrometry assay for the detection of SARS-CoV-2 spike protein and nucleoprotein in a relevant biological matrix. Recombinant full-length spike protein and nucleoprotein were digested and proteotypic peptides were selected for parallel reaction monitoring (PRM) quantitation using a high-resolution Orbitrap instrument. A spectral library, which contained seven proteotypic peptides (four from spike protein and three from nucleoprotein) and the top three to four transitions, was generated and evaluated. From the original spectral library, we selected two best performing peptides for the final PRM assay. The assay was evaluated using mock test samples containing inactivated SARS-CoV-2 virions, added to in vitro derived mucus. The PRM assay provided a limit of detection of ∼200 attomoles and a limit of quantitation of ∼ 390 attomoles. Extrapolating from the test samples, the projected titer of virus particles necessary for the detection of SARS-CoV-2 spike and nucleoprotein detection was approximately 2 × 105 viral particles/mL, making it an attractive alternative to RT-PCR assays. Potentially, mass spectrometry-based methods for viral antigen detection may deliver higher throughput and could serve as a complementary diagnostic tool to RT-PCR. Furthermore, this assay could be used to evaluate the presence of SARS-CoV-2 in archived or recently collected biological fluids, in vitro-derived research materials, and wastewater samples.

Authors+Show Affiliations

Dynamic Omics, Antibody Discovery and Protein Engineering (ADPE), R&D AstraZeneca, Gaithersburg 20878, Maryland, United States.Dynamic Omics, Antibody Discovery and Protein Engineering (ADPE), R&D AstraZeneca, Gaithersburg 20878, Maryland, United States.Dynamic Omics, Antibody Discovery and Protein Engineering (ADPE), R&D AstraZeneca, Gaithersburg 20878, Maryland, United States.Dynamic Omics, Antibody Discovery and Protein Engineering (ADPE), R&D AstraZeneca, Gaithersburg 20878, Maryland, United States.Dynamic Omics, Antibody Discovery and Protein Engineering (ADPE), R&D AstraZeneca, Gaithersburg 20878, Maryland, United States.Dynamic Omics, Antibody Discovery and Protein Engineering (ADPE), R&D AstraZeneca, Gaithersburg 20878, Maryland, United States.Biological Therapeutics 1, Antibody Discovery and Protein Engineering (ADPE), R&D AstraZeneca, Gaithersburg 20878, Maryland, United States.Biological Therapeutics 1, Antibody Discovery and Protein Engineering (ADPE), R&D AstraZeneca, Gaithersburg 20878, Maryland, United States.Cell Therapeutics, Antibody Discovery and Protein Engineering (ADPE), R&D AstraZeneca, Gaithersburg 20878, Maryland, United States.Discovery Anti Infection, Microbial Sciences, R&D AstraZeneca, Gaithersburg 20878, Maryland, United States.Discovery Anti Infection, Microbial Sciences, R&D AstraZeneca, Gaithersburg 20878, Maryland, United States.Discovery Anti Infection, Microbial Sciences, R&D AstraZeneca, Gaithersburg 20878, Maryland, United States.Respiratory and Immunology, R&D AstraZeneca, Gaithersburg 20878, Maryland, United States.Cell Therapeutics, Antibody Discovery and Protein Engineering (ADPE), R&D AstraZeneca, Gaithersburg 20878, Maryland, United States.Dynamic Omics, Antibody Discovery and Protein Engineering (ADPE), R&D AstraZeneca, Gaithersburg 20878, Maryland, United States.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

32966064

Citation

Cazares, Lisa H., et al. "Development of a Parallel Reaction Monitoring Mass Spectrometry Assay for the Detection of SARS-CoV-2 Spike Glycoprotein and Nucleoprotein." Analytical Chemistry, vol. 92, no. 20, 2020, pp. 13813-13821.
Cazares LH, Chaerkady R, Samuel Weng SH, et al. Development of a Parallel Reaction Monitoring Mass Spectrometry Assay for the Detection of SARS-CoV-2 Spike Glycoprotein and Nucleoprotein. Anal Chem. 2020;92(20):13813-13821.
Cazares, L. H., Chaerkady, R., Samuel Weng, S. H., Boo, C. C., Cimbro, R., Hsu, H. E., Rajan, S., Dall'Acqua, W., Clarke, L., Ren, K., McTamney, P., Kallewaard-LeLay, N., Ghaedi, M., Ikeda, Y., & Hess, S. (2020). Development of a Parallel Reaction Monitoring Mass Spectrometry Assay for the Detection of SARS-CoV-2 Spike Glycoprotein and Nucleoprotein. Analytical Chemistry, 92(20), 13813-13821. https://doi.org/10.1021/acs.analchem.0c02288
Cazares LH, et al. Development of a Parallel Reaction Monitoring Mass Spectrometry Assay for the Detection of SARS-CoV-2 Spike Glycoprotein and Nucleoprotein. Anal Chem. 2020 10 20;92(20):13813-13821. PubMed PMID: 32966064.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Development of a Parallel Reaction Monitoring Mass Spectrometry Assay for the Detection of SARS-CoV-2 Spike Glycoprotein and Nucleoprotein. AU - Cazares,Lisa H, AU - Chaerkady,Raghothama, AU - Samuel Weng,Shao Huan, AU - Boo,Chelsea C, AU - Cimbro,Raffaello, AU - Hsu,Hsiang-En, AU - Rajan,Sarav, AU - Dall'Acqua,William, AU - Clarke,Lori, AU - Ren,Kuishu, AU - McTamney,Patrick, AU - Kallewaard-LeLay,Nicole, AU - Ghaedi,Mahboobe, AU - Ikeda,Yasuhiro, AU - Hess,Sonja, Y1 - 2020/10/05/ PY - 2020/9/24/pubmed PY - 2020/10/29/medline PY - 2020/9/23/entrez SP - 13813 EP - 13821 JF - Analytical chemistry JO - Anal Chem VL - 92 IS - 20 N2 - There is an urgent need for robust and high-throughput methods for SARS-CoV-2 detection in suspected patient samples to facilitate disease management, surveillance, and control. Although nucleic acid detection methods such as reverse transcription polymerase chain reaction (RT-PCR) are the gold standard, during the current pandemic, the deployment of RT-PCR tests has been extremely slow, and key reagents such as PCR primers and RNA extraction kits are at critical shortages. Rapid point-of-care viral antigen detection methods have been previously employed for the diagnosis of respiratory viruses such as influenza and respiratory syncytial viruses. Therefore, the direct detection of SARS-CoV-2 viral antigens in patient samples could also be used for diagnosis of active infection, and alternative methodologies for specific and sensitive viral protein detection should be explored. Targeted mass spectrometry techniques have enabled the identification and quantitation of a defined subset of proteins/peptides at single amino acid resolution with attomole level sensitivity and high reproducibility. Herein, we report a targeted mass spectrometry assay for the detection of SARS-CoV-2 spike protein and nucleoprotein in a relevant biological matrix. Recombinant full-length spike protein and nucleoprotein were digested and proteotypic peptides were selected for parallel reaction monitoring (PRM) quantitation using a high-resolution Orbitrap instrument. A spectral library, which contained seven proteotypic peptides (four from spike protein and three from nucleoprotein) and the top three to four transitions, was generated and evaluated. From the original spectral library, we selected two best performing peptides for the final PRM assay. The assay was evaluated using mock test samples containing inactivated SARS-CoV-2 virions, added to in vitro derived mucus. The PRM assay provided a limit of detection of ∼200 attomoles and a limit of quantitation of ∼ 390 attomoles. Extrapolating from the test samples, the projected titer of virus particles necessary for the detection of SARS-CoV-2 spike and nucleoprotein detection was approximately 2 × 105 viral particles/mL, making it an attractive alternative to RT-PCR assays. Potentially, mass spectrometry-based methods for viral antigen detection may deliver higher throughput and could serve as a complementary diagnostic tool to RT-PCR. Furthermore, this assay could be used to evaluate the presence of SARS-CoV-2 in archived or recently collected biological fluids, in vitro-derived research materials, and wastewater samples. SN - 1520-6882 UR - https://www.unboundmedicine.com/medline/citation/32966064/Development_of_a_Parallel_Reaction_Monitoring_Mass_Spectrometry_Assay_for_the_Detection_of_SARS_CoV_2_Spike_Glycoprotein_and_Nucleoprotein_ L2 - https://doi.org/10.1021/acs.analchem.0c02288 DB - PRIME DP - Unbound Medicine ER -