Highly sensitive quantification of proteins from the SARS-CoV-2 antigen in nasopharyngeal swab samples

Using the SCIEX Triple Quad™ 7500 LC-MS/MS System – QTRAP® Ready  

Catherine S. Lane1, Bart Van Puyvelde2, Katleen Van Uytfanghe2, Andrea-Bhangu-Uhlmann3, Maarten Dhaenens2
SCIEX, UK, 2Ghent University, Belgium, 3PolyQuant GmbH, Germany


Targeted peptide quantification assay has been developed to investigate the potential use of nasopharyngeal swabs in a saline matrix for the quantification of SARS-CoV-2 coronavirus proteins using LC-MS/MS. The gains in sensitivity attained by moving the assay onto the next-generation SCIEX Triple Quad 7500 LC-MS/MS System and reducing matrix effects by changing swab storage medium were characterized.

12617 7500


Research into the epidemiology of the SARS-CoV-2 virus necessitates wide-scale sampling of large populations. The majority of assays for viral testing target detection of the SARS-CoV-2 nucleic acid (RNA), however, antigen tests targeting viral protein are generating interest globally amongst researchers in this field. The use of mass spectrometry for the detection of proteins from biological matrices is well established. However, the desire for increased sensitivity is always a goal in any quantitative assay and, thus far, in this case, mass spectrometry has struggled to match the sensitivities achieved by the more widely used reverse-transcriptase polymerase chain reaction (RT-PCR)-based tests. Here, the sensitivity of peptide detection in swab matrices is explored in order to compare with the sensitivity typical in RNA based detection strategies.

The SCIEX 7500 System shows significant gains in sensitivity relative to the previous generation of instruments because of improvements in both the generation and the sampling of ions.1,2 Furthermore, the preservation medium utilized for the collection of swab samples can have a considerable impact on the sensitivity of the LC-MS/MS assay.3 Here, building on the method developed previously4, the MRM assay is applied to the analysis of nasopharyngeal swab samples preserved in eSwab medium (saline matrix), using the SCIEX 7500 System to quantify the increased sensitivity (Figure 1).

Figure 1. Quantification of NCAP peptide. Calibration line and extracted ion chromatograms (XICs) for  KQQTVTLLPAADLDDFSK from SARS-CoV-2 NCAP protein spiked into nasopharyngeal swab samples (XICs shown are for 2.5 ng and 10 ng protein in 250 µL swab samples).  

Key features of the quantification of SARS-CoV-2 proteins using the SCIEX 7500 System

  • Fast, robust peptide quantification targeting detection of two of the viral proteins from the SARS-CoV-2 coronavirus particle4 has been extended, improved and applied to the analysis of nasopharyngeal swab samples, in eSwab matrix, using the SCIEX 7500 System
  • Stable-isotope-labeled internal standards, generated via use of the Cov-MS QconCAT construct, provide enhanced quantitative robustness and also allow monitoring of swab sampling efficiency through inclusion of peptide markers of tissue damage
  • Analysis time was reduced from 7 min to 4.5 min per sample
  • Detection limits of 0.07-0.22 fmol protein per µL eSwab matrix

The work described here was carried out as part of the Cov-MS consortium3, a community group established by Maarten Dhaenens at Ghent University, Belgium, with the aim of increasing applicability, accessibility, sensitivity and robustness of the detection of SARS-CoV-2 by electrospray mass spectrometry, thus increasing awareness of the technique as an important research tool. The consortium is made up of both academic laboratories and industrial partners. As such, the samples analyzed here were also examined by multiple other collaborators.