Cov 2 MS: an automated matrix-independent assay for mass spectrometric detection and measurement of SARS-CoV-2 nucleocapsid protein in infectious patients
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Abstract
INTRODUCTION
The pandemic readiness toolbox needs to be extended, providing diagnostic tools that target different biomolecules, using orthogonal experimental setups and fit-for-purpose specification of detection. Here we build on a previous Cov-MS effort that used liquid chromatography-mass spectrometry (LC-MS) and describe a method that allows accurate, high throughput measurement of SARS-CoV-2 nucleocapsid (N) protein.
MATERIALS and METHODS
We used Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) technology to enrich and quantify proteotypic peptides of the N protein from trypsin-digested samples from COVID-19 patients.
RESULTS
The Cov 2 MS assay was shown to be compatible with a variety of sample matrices including nasopharyngeal swabs, saliva and blood plasma and increased the sensitivity into the attomole range, up to a 1000-fold increase compared to direct detection in matrix. In addition, a strong positive correlation was observed between the SISCAPA antigen assay and qPCR detection beyond a quantification cycle (Cq) of 30-31, the level where no live virus can be cultured from patients. The automatable “addition only” sample preparation, digestion protocol, peptide enrichment and subsequent reduced dependency upon LC allow analysis of up to 500 samples per day per MS instrument. Importantly, peptide enrichment allowed detection of N protein in a pooled sample containing a single PCR positive sample mixed with 31 PCR negative samples, without loss in sensitivity. MS can easily be multiplexed and we also propose target peptides for Influenza A and B virus detection.
CONCLUSIONS
The Cov 2 MS assay described is agnostic with respect to the sample matrix or pooling strategy used for increasing throughput and can be easily multiplexed. Additionally, the assay eliminates interferences due to protein-protein interactions including those caused by anti-virus antibodies. The assay can be adapted to test for many different pathogens and could provide a tool enabling longitudinal epidemiological monitoring of large numbers of pathogens within a population, applied as an early warning system.
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SciScore for 10.1101/2022.02.09.22270547: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: Samples: Residual Covid-19 nasopharyngeal patient samples were obtained from the AZ Delta hospital, Roeselare, Belgium with approval of the University Hospital Ghent ethics committee (BC-09263). Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources This process allows selection of highly specific, high affinity anti-peptide antibodies capable of binding low abundance peptides from solution and retaining them through extensive washing steps designed to minimize non-specific background. anti-peptidesuggested: NoneExperimental Models: Cell Lines Sent… SciScore for 10.1101/2022.02.09.22270547: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: Samples: Residual Covid-19 nasopharyngeal patient samples were obtained from the AZ Delta hospital, Roeselare, Belgium with approval of the University Hospital Ghent ethics committee (BC-09263). Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources This process allows selection of highly specific, high affinity anti-peptide antibodies capable of binding low abundance peptides from solution and retaining them through extensive washing steps designed to minimize non-specific background. anti-peptidesuggested: NoneExperimental Models: Cell Lines Sentences Resources Phuket/3073/2013): MDCK cells were cultured, seeded and infected according to the protocol outlined above. MDCKsuggested: CLS Cat# 602280/p823_MDCK_(NBL-2, RRID:CVCL_0422)Software and Algorithms Sentences Resources The SARS-CoV-2 sample preparation protocol was automated using the Andrew Alliance Pipette+ and Shaker+ connected device (Waters Corporation, Milford, MA, USA) both operated via the OneLab platform. OneLabsuggested: (OneLab, RRID:SCR_005545)PCR amplification was performed using a CFX96 real-time thermal cycler (Bio-Rad Laboratories) and data were analysed with the SARS-CoV-2 Viewer (Seegene). Bio-Rad Laboratoriessuggested: (Bio-Rad Laboratories, RRID:SCR_008426)Skyline (version 21.1) was used to process the raw LC-MS data using a template file containing the six target peptides. Skylinesuggested: (Skyline, RRID:SCR_014080)Results from OddPub: Thank you for sharing your data.
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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