KTH Uhlen Lab - SARS-CoV-2 SISCAPA

Rapid and Sensitive Detection of SARS-CoV-2 Infection Using Quantitative Peptide Enrichment Analysis
Data License: CC BY 4.0 | ProteomeXchange: PXD026366 | doi:10.6069/8ba1-8n77
  • Organism: Homo sapiens, SARS-CoV-2
  • Instrument: Xevo TQ-XS
  • SpikeIn: Yes
  • Keywords: LC-MS, MRM, SARS-Cov-2, NCAP, RT-PCR, digest, peptide, SISCAPA, affinity
  • Lab head: Fredrik Edfors Submitter: Andreas Hober
Abstract
Reliable, robust, large-scale molecular testing for SARS-CoV-2 is essential for monitoring the ongoing Covid-19 pandemic. We have developed a scalable analytical approach to detect viral proteins based on peptide immunoaffinity enrichment combined with liquid chromatography - mass spectrometry (LC-MS). This is a multiplexed strategy, based on targeted proteomics analysis and read-out by LC-MS, capable of precisely quantifying and confirming the presence of SARS-CoV-2 in PBS swab media from combined throat/nasopharynx/saliva samples. The results reveal that the levels of SARS-CoV-2 measured by LC-MS correlate well with their corresponding RT-PCR readout. The analytical workflow shows similar turnaround times as regular RT-PCR instrumentation with quantitative readout of viral proteins corresponding to cycle thresholds (Ct) equivalents ranging from 20 to 35. Using RT-PCR as a reference, we demonstrate that the LC-MS-based method has 100% specificity and 86% sensitivity when analyzing clinical samples collected from asymptomatic patients. These results suggest that a scalable analytical method based on LC-MS has a place in future pandemic preparedness centers to complement current virus detection technologies.
Experiment Description
Antibody-coupled magnetic bead immune adsorbents corresponding to four SIL peptides (ADETQALPQR-13C615N4, AYNVTQAFGR-13C615N4, DGIIWVATEGALNTPK-13C615N2, and NPANNAAIVLQLPQGTTLPK-13C615N2) were resuspended fully by vortex mixing. The suspension of each anti-peptide antibody tube was mixed together in 1:1 ratio and 40 µL of the mixture was added to each digest. The plate was mixed at 1400 rpm to ensure that beads were resuspended and thereafter incubated for one hour at 800 rpm at room temperature. After one hour incubation, the plate was placed on a magnet array. As soon as the beads had settled on the sides of each well (typically one min), the supernatant was removed. 150 µL of wash buffer (0.03% CHAPS, 1xPBS) was added to each sample and the beads were fully resuspending at 1400 rpm for 30 s and 450 rpm for another 30 s. The plate was placed on the magnet array again and the supernatant was removed. This step was repeated three times. The beads were subsequently resuspended in 50 µL elution buffer (0.5 % formic acid, 0.03% CHAPS, 1xPBS) and incubated for five min at room temperature. The beads were discarded by transferring the eluent to a QuanRecovery plate (Waters Corporation) for LC-MS analysis. Chromatography was performed on an ACQUITY UPLC I-Class FTN system, with Binary Solvent Manager and column heater (Waters Corporation). 20 µL of the enriched sample was injected onto an ACQUITY Premier Peptide BEH C18, 2.1 mm x 50 mm, 1.7 µm, 300 Å column (Waters Corporation) and separated using a gradient elution of mobile phase A containing laboratory 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. The gradient elution was performed at 0.6 ml/min with initial inlet conditions at 5% B, increasing to 28% B over 4.5 min, followed by a column wash at 90% B for 0.6 min and a return to initial conditions at 5% B. The total run time was 5.7 min, with a 6.5 min injection-to-injection cycle time.
Sample Description
A three-point collection (throat, nasal, saliva) was performed by participants using a self-sampling collection kit (Sansure Biotech, Changsha, China) containing 1xPBS buffer. All tests were self-sampled by a three-point collection procedure (throat, nasopharynx, saliva). Clinical samples collected by swabs were dipped into the sample collection tube and transported to the laboratory within eight hours. All samples were heat-inactivated to ensure that the core temperature of the vial reached at least 56°C for 30 min.
Created on 10/5/21, 9:19 PM
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sars-cov-2_siscapa_2021-09-30_16-56-52.sky.zip2021-10-05 21:19:071124356