Karel Bezstarosti, Mart M. Lamers, Wouter A. S. Doff, Peter C. Wever, Khoa T. D. Thai, Jeroen J. A. van Kampen, Bart L. Haagmans, Jeroen A. A. Demmers
Abstract
The rapid, sensitive and specific detection of SARS-CoV-2 is critical in responding to the current COVID-19 outbreak. In this proof-of-concept study, we explored the potential of targeted mass spectrometry (MS) based proteomics for the detection of SARS-CoV-2 proteins in both research samples and clinical specimens. First, we assessed the limit of detection for several SARS-CoV-2 proteins by parallel reaction monitoring (PRM) MS in infected Vero E6 cells.
Introduction
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19), which is a severe respiratory disease [1]. The World Health Organization (WHO) has designated the ongoing pandemic of COVID-19 a Public Health Emergency of International Concern [2]. As of now, over one million deaths have been reported worldwide and this is probably an underestimation because of lack of testing capacity in large parts of the world.
SARS-CoV-2 is a positive-sense single-stranded RNA virus, which encodes several non-structural proteins such as spike, envelope, membrane and nucleocapsid protein [3]. Rapid, sensitive and specific diagnosis of SARS-CoV-2 is widely recognized to be critical in responding to this outbreak, but also for long-term improvements in patient care
Materials and methods:
Virus and cells
Vero E6 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal calf serum (FCS), HEPES, sodium bicabonate, penicillin (final concentration 100 IU/mL) and streptomycin (final concentration 100 IU/mL) at 37°C in a humidified CO2 incubator.
Organoid-derived human airway culture secretions
Organoid-derived human airway culture secretions were harvested from cultures that had been differentiated at air-liquid interphase for 3 weeks as described by Lamers et al. [13]. Secretions could be harvested by pipetting using a P1000 tip and were not diluted.
Collection and treatment of patient material samples
Nasopharyngeal swabs from COVID-19 patients were stored in universal transport medium (UTM; contains bovine serum albumin) after collection. Next, they were centrifuged at 15,000 g for 3 min to pellet down cell debris (termed ‘swab pellet’).
Discussion
We set off by analyzing the global proteome of Vero E6 cells infected with SARS-CoV-2 using standard bottom-up proteomics. Upon off-line high pH reversed-phase (RP) peptide fractionation, LC-MS was performed on an Orbitrap Lumos and RAW files were combined during data analysis.
Citation: Bezstarosti K, Lamers MM, Doff WAS, Wever PC, Thai KTD, van Kampen JJA, et al. (2021) Targeted proteomics as a tool to detect SARS-CoV-2 proteins in clinical specimens. PLoS ONE 16(11): e0259165. https://doi.org/10.1371/journal.pone.0259165
Editor: Prasenjit Mitra, Post Graduate Institute of Medical Education and Research, INDIA
Received: April 22, 2021; Accepted: October 13, 2021; Published: November 11, 2021.
Copyright: © 2021 Bezstarosti et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: Mass spectrometry data files have been deposited in the ProteomeXchange repository with identifier PXD025294 (https://www.ebi.ac.uk/pride/archive/projects/PXD025294).
Funding: The author(s) received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: PRM, parallel reaction monitoring; AUC, area under the curven; LC-MS, nanoflow liquid chromatography–mass spectrometry; Ct, threshold value; PCR, polymerase chain reaction.
