Cov²MS: an automated matrix-independent assay for mass spectrometric detection and measurement of SARS-CoV-2 nucleocapsid protein
- Organism: SARS-CoV-2
- Instrument: Xevo TQ-XS,TripleTOF 6600
SARS-CoV-2, Covid-19, Mass Spectrometry, Proteomics, Viral proteins, Peptides, Diagnostics
Lab head: Maarten Dhaenens
Submitter: Bart Van Puyvelde
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 to use liquid chromatography coupled to mass spectrometry (LC-MS) and describe a method developed to allow accurate, high throughput measurement of SARS-CoV-2 nucleocapsid (NCAP) protein.
Methods. We use Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) technology to enrich and quantify proteotypic peptides of the NCAP protein from trypsin-digested samples from COVID-19 patients.
Results. This makes the Cov²MS assay compatible with most matrices including nasopharyngeal swabs, saliva and blood plasma, while increasing the sensitivity into the attomole range, up to a 1000-fold increase compared to direct detection in matrix. In addition, there is a strong positive correlation between the SISCAPA antigen assay and qPCR detection up to a quantification cycle (Cq) of 30. The automatable “addition only” sample preparation and digestion protocol, the peptide enrichment and the reduced dependency upon LC together allow analysis of up to 500 samples per day per MS instrument. Importantly, peptide enrichment allowed detection of NCAP protein in a pooled sample containing a single PCR positive patient mixed with 31 PCR negative samples, without loss in sensitivity. We also propose novel target peptides for Influenza A and B.
Conclusion. Since the Cov²MS assay is insensitive to matrix or pooling and easily multiplexed, it can be used to test for many different pathogens and could provide longitudinal epidemiological monitoring of large numbers of pathogens within a population, applied as an early warning system.
Each sample was prepared using the same workflow, namely 180 µL of sample (for plasma only 60 µL) was precipitated by adding 7 volumes of ice-cold acetone (-20°C). After spinning at 16.000g and 0°C, the supernatant was discarded and 1µg of Trypsin/Lys-C mix (Promega, Madison, USA) in 150 µL 100 mM ABC was added. Prior to an incubation step of 30minutes at 37°C, to facilitate trypsin digestion, the samples were transferred from Protein LoBind® tubes into a 96-well sample collection plate (Waters Corporation). To inhibit further digestion, 50 µL of a 0.22 mg/mL TLCK (Sigma-Aldrich, Saint-Louis, USA) in 10mM HCl solution is added to each sample, followed by mixing at 1000 rpm for 5 minutes at room temperature. Note that each sample was spiked with 100 fmol of the Cov-MS QconCAT standard (Polyquant, Bad Abbach, Germany) before acetone crash. Afterwards, the antibody-coupled magnetic bead immunoadsorbents were resuspended fully by vortex mixing. Equal volumes of the six bead suspensions were mixed and 60 µL of the mixture was added to the tryptic digested samples, once tryptic digestion activity had been neutralized. Plates were first shaken at 1400 rpm for 3 minutes to ensure that beads were fully resuspended prior to a one hour incubation at 1100 rpm at room temperature. After incubation, the plates were placed on a custom-made magnet array (SISCAPA Assay Technologies Cat. No. MP001 ) for 1 minute and once the beads had been drawn to the sides of each well, the supernatant (approximately 260 µL) was removed. The beads were then washed through the addition of 150 µl wash buffer (0.03% CHAPS, 1xPBS) to each sample followed by resuspending the beads at 1400 rpm for 1 minute. The sample plates were then again placed on the magnet array and the supernatant was removed. The washing step was then performed a second time. Subsequently, the beads were resuspended in 50 µl elution buffer (1 % formic acid, 0.03% CHAPS) and mixed at 1400 rpm for 5 min at room temperature. Finally, after placing the plates on the magnetic plate, the eluents containing the peptides were transferred to a QuanRecovery plate (Waters Corporation) for LC-MS analysis. Liquid chromatography was performed on an ACQUITY UPLC I-Class FTN system, with Binary Solvent Manager and column heater (Waters Corporation). Ten microliters of the enriched sample were injected onto an ACQUITY Premier Peptide BEH C18 column (2.1 mm x 30 (or 50) mm, 1.7 µm, 300 Å) column (Waters Corporation). Peptide separation was performed using a gradient elution of mobile phase A containing LC-MS grade de-ionised water with 0.1% (v/v) formic acid, and mobile phase B containing LC-MS grade acetonitrile with 0.1% (v/v) formic acid. For the 1min run, gradient elution was performed at 1 mL/min with initial inlet conditions at 7% B, increasing to 40% B over 0.3 min, followed by a column wash at 90% B for 0.25 min and a return to initial conditions at 7% B. The total run time was 0.8 min. For the 2min run, gradient elution was performed at 0.8 mL/min with initial inlet conditions at 5% B, increasing to 15% B from 0.15 to 0.35 min and retain a steady state for 0.25min. Subsequently, over 0.4 min, gradient B is increased to 25%, followed by a column wash at 90% B for 0.25 min and returning to initial conditions at 5% B. The total run time was 1.8 min. For the 8min run, gradient elution was performed at 0.6 mL/min with initial inlet conditions at 5% B, increasing to 33% B over 5.5 min, followed by a column wash at 90% B for 1.4 min and a return to initial conditions at 5% B. The total run time was 8 min.
A Xevo TQ-XS tandem MS (Waters Corporation, Wilmslow, UK) operating in positive electrospray ionization (ESI+) was used for the detection and quantification of the peptides. The instrument conditions were as follows: capillary voltage 0.5 kV, source temperature 150°C, desolvation temperature 600°C, cone gas flow 150 L/h, and desolvation gas flow 1000 L/h. The MS was calibrated at unit mass resolution for MS1 and MS2.
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). These were analysed by the clinical laboratory of the AZ Delta hospital using the Allplex 2019-nCoV RT-PCR assay from Seegene Inc. as described by De Smet et al (15). In short, RNA was extracted from nasopharyngeal swabs using STARMag 96 x 4 Viral DNA/RNA 200 C Kit (Seegene Technologies) on the Hamilton STARlet workstation, followed by real-time PCR using the Allplex SARS-CoV-2 assay. PCR amplification was run on a CFX96 real-time thermal cycler (Bio-Rad Laboratories), and data were analysed with the SARS-CoV-2 Viewer (Seegene). The presence or absence of SARS-CoV-2 RNA was determined by RT-PCR combined with multiplexed fluorescent probing, in which three different SARS-CoV-2 genes i.e. E-gen (FAM), RdRP (Cal Red 610) and N gene (Quasar 670), and an internal control (HEX) were targeted.
Lyophilised recombinant NCAP protein from Sino Biological was reconstituted to a concentration of 0.1µg/µL in 100 mM ammonium bicarbonate (ABC). Then, a 50000 amol/µL calibration standard was prepared for each medium i.e. Pure (100mM ABC), Copan UTM, Bioer UTM, Sigma Virocult, eSwab, PBS, Plasma, Synthetic saliva and Patient saliva, by spiking a certain volume of the NCAP solution into SARS-CoV-2 negative nasopharyngeal pools. A serial dilution was then performed using the SARS-CoV-2 negative nasopharyngeal pools to obtain a dilution series with following concentrations: 10000, 2000, 400, 80, 16, 4, 2 and 0 amol/µL.
An equimolar dilution series of recombinant NCAP protein from Influenza A (Victoria/2570/2019, Influenza B (Phuket/3073/2013) and SARS-CoV-2 (Wuhan strain) was created in 100mM ABC. The dilution series contained the following calibrators: 10000, 2000, 400, 80, 16, 4, 2 and 0 amol/µL.
Created on 2/2/22, 10:16 PM