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.
Highlights
(RT-PCR) are the gold standard, during the current pandemic, the deployment of reverse transcription polymerase chain reaction (RT-PCR) tests has been extremely slow, and key reagents such as PCR primers and RNA extraction kits are at critical shortages
Improving targeted mass spectrometry (MS) instruments and methodologies 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.[7,8]. This approach has the potential to quantitate over 1000 proteins in a single analysis, including isoforms, and is, far superior in terms of specificity and multiplexing capability to conventional protein measurement methods, which rely on immunoaffinity [i.e., enzyme-linked immunosorbent assay (ELISA)]
To determine the feasibility of targeted MS for SARS-CoV-2 diagnostics, we developed a method for the detection and quantitation of the S and NP, which employs parallel reaction monitoring (PRM) using a high-resolution Orbitrap instrument, thereby providing very high specificity
Summary
(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. To test the ability of the PRM assay to detect S protein and NP in a relevant sample type, we spiked inactivated SARS CoV-2 virions into in vitro derived mucus.
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