Establishing a mass spectrometry-based system for rapid detection of SARS-CoV-2 in large clinical sample cohorts

Abstract

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is pressing public health systems around the world, and large population testing is a key step to control this pandemic disease. Here, we develop a high-throughput targeted proteomics assay to detect SARS-CoV-2 nucleoprotein peptides directly from nasopharyngeal and oropharyngeal swabs. A modified magnetic particle-based proteomics approach implemented on a robotic liquid handler enables fully automated preparation of 96 samples within 4 hours. A TFC-MS system allows multiplexed analysis of 4 samples within 10 min, enabling the processing of more than 500 samples per day. We validate this method qualitatively (Tier 3) and quantitatively (Tier 1) using 985 specimens previously analyzed by real-time RT-PCR, and detect up to 84% of the positive cases with up to 97% specificity. The presented strategy has high sample stability and should be considered as an option for SARS-CoV-2 testing in large populations.

Fig. 1. Test development workflow.

Design and optimization: peptide selection was performed by analyzing tryptic extracts from patient samples previously defined as positive or negative for SARS-CoV-2 by real-time RT-PCR. Data-dependent acquisitions (DDA) were achieved by microflow chromatography coupled to hybrid quadrupole-orbitrap tandem mass spectrometry. Skyline was used to generate the isolation list for parallel reaction monitoring (PRM). Several PRMs were performed before selection of the final two peptides that were exported as selected reaction monitoring (SRM) coordinates to a triple quadrupole tandem mass spectrometry. Automated sample prep/rapid LC-MS/MS (liquid chromatography coupled to tandem mass spectrometry) analysis: sample preparation was optimized, aiming for simplicity and speed and was fully implemented in a robotic liquid handler. Multiplexed four-channel analysis resulted in a 2.5-min acquisition time per sample.

Fig. 2. Schematic illustration of turbulent flow chromatography (TFC) setup in Transcend TLX-4 system coupled to TSQ Altis triple quadrupole mass spectrometer.

The TLX-4 system has four independent channels (channel 1: blue, channel 2: orange, channel 3: green and channel 4: red) that can be used simultaneously. The analytical run in each channel has three steps. The first step consists of sample injection, loading into the TFC column and cleanup. The sample is injected into the sample loop by autosampler and transferred to the valve interface module using a quaternary loading pump with high flow rate. For first dimension chromatography, the sample is loaded and washed into TFC column by a turbulent flow, flushing away unwanted matrix and large molecules while targeted analytes are retained in particles interaction sites. In the second step, the first and second columns are connected, and analytes are eluted from the TFC column by the solvent loop, transferred and focused on the analytical column head. Third step includes TFC column washing with stronger organic solvents, solvent loop refilling and ultraperformance chromatographic separation by an organic multistep linear gradient. As soon as the target analytes are eluted, the selecting valve directs the current channel flow path to the mass spectrometer (MS) to achieve data acquisition. Lastly, columns are re-equilibrated to initial conditions before starting the next run. For the present method, MS acquisition window has 2.5 min enabling multiplex analysis of up to four samples within ten minutes. Aria software coordinated multiplexed sequence logic to maximize analysis throughput.

Fig. 3. Selected reaction monitoring chromatograms of SARS-CoV-2-positive clinical respiratory tract specimen showing targeted peptides.

a Qualitative (Tier 3) assay; b Quantitative (Tier 1) assay. The first three residues of each peptide are used to label peptide peaks: DGI (DGIIWVATEGALNTPK) and IGM (IGMEVTPSGTWLTYTGAIK) from nucleoprotein; HSG (HSGFEDELSEVLENQSSQAELK) from ¹⁵N-labeled surrogate standard chromogranin A (in Tier 3) and SYE (SYELPDGQVITIGNER) from human beta actin (in Tier 1).

Fig. 4. Overview of automated sample preparation for SARS-CoV-2 SP3-based extraction on a Hamilton Robotics Microlab STARlet liquid handling system.

a Patient samples were collected with swabs placed in virus transport medium or sterile saline solution and stored at −80 °C before automated sample processing. The highlighted nucleocapsid peptides were monitored by turbulent flow chromatography coupled to tandem mass spectrometry. b Schematic overview showing the steps and Hamilton desk setup for automated SP3 sample processing: (1) viral particle and internal standard precipitation on magnetic bead using ethanol (EtOH) to a final concentration of 50% (v/v); (2) one-step viral lysis using a sodium dodecyl sulfate-based (SDS) buffer and dithiothreitol (DTT) for 5 min at 65 °C followed by dilution and bead precipitation; (3) magnetic separation and three consecutive wash steps with ethanol 80% (v/v); (4) protein elution from magnetic bead followed by digestion with trypsin in triethylamonium bicarbonate buffer (TEAB) for 2 h at 37 °C, post-digestion acidification with trifluoroacetic acid (TFA), and recovery of peptides in a new sample plate.