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Targeted proteomics as a tool to detect SARS-CoV-2 proteins in clinical specimens

ABSTRACT: The rapid, sensitive and specific detection of SARS-CoV-2 is critical in responding to the current COVID-19 outbreak. Here, we explore the potential of targeted mass spectrometry based proteomics for the detection of SARS-CoV-2 proteins in both research and clinical samples. First, we assessed the limit of detection for several SARS-CoV-2 proteins by parallel reaction monitoring (PRM) mass spectrometry. For Nucleocapsid the limit of detection was found to be in the mid-attomole range (0.9 x 10-12 g). Next, this PRM assay is applied to the detection of viral proteins in in vitro mucus substitutes, as well as in various clinical specimens such as nasopharyngeal swabs and sputum. In this proof-of-concept study SARS-CoV-2 proteins could unambiguously be detected in various clinical samples, suggesting that the sensitivity of this technology may be sufficiently high to further explore its potential role in diagnostics.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Cercopithecus Aethiops (green Monkey) (grivet) Homo Sapiens (human)

TISSUE(S): Saliva

DISEASE(S): Covid-19

SUBMITTER: Jeroen Demmers

LAB HEAD: Jeroen Demmers

PROVIDER: PXD025294 | Pride | 2021-10-26

REPOSITORIES: Pride

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			Action	DRS
	2181_3_E_15april20_50ng.raw	Raw
	2181_3_E_15april20_50ng_PRM.raw	Raw
	2181_3_E_16april20_cells_virus_PRM_100pg.raw	Raw
	2181_3_E_16april20_cells_virus_PRM_10ng.raw	Raw
	2181_3_E_16april20_cells_virus_PRM_1ng.raw	Raw

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Project description:There is an urgent need for robust and high-throughput methods for SARS-CoV-2 detection in suspected pa-tient samples to facilitate disease management, surveillance, and control. Although nucleic acid detection methods such as 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 detec-tion methods have been previously employed for the diagnosis of respiratory viruses such as influenza and respiratory syn-cytial viruses. Therefore, the direct detection of SARS-CoV-2 viral antigens in patient samples could also be used for diagno-sis 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 pro-teins/peptides at single amino acid resolution with attomole level sensitivity and high reproducibility. Herein we report a tar-geted mass spectrometry assay for the detection of SARS- CoV-2 spike protein and nucleoprotein in a relevant biologi-cal matrix. Recombinant full-length spike protein and nucleoprotein were digested and prototypic peptides were selected for parallel reaction monitoring (PRM) quantitation using a high resolution Orbitrap instrument. A spectral library, which con-tained 7 proteotypic peptides (4 from spike protein and 3 from nucleoprotein) and the top 3 to 4 transitionsMS2 spectra, was generated and evaluated. From the original spectral library, we selected 2 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 de-rived mucus. The PRM assay provided a limit of detection (LOD) of ~200 attomoles and a limit of quantitation (LOQ) of ~ 390 attomoles. Extrapolating from the test samples, the projected titer of virus particles necessary for detection of SARS-CoV-2 spike and nucleoprotein detection was approximately 2E5 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 pres-ence of SARS-CoV-2 in archived or recently collected biological fluids, in-vitro derived research materials, and wastewater samples

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Targeted proteomics as a tool to detect SARS-CoV-2 proteins in clinical specimens

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