Fast and low-cost detection of SARS-CoV-2 peptides by tandem mass spectrometry in clinical samples

Abstract

The current outbreak of severe acute respiratory syndrome associated with 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. Real-time reverse-transcription PCR (real-time RT-PCR) is the gold standard test for virus detection but the soaring demand for this test resulted in shortage of reagents and instruments, severely limiting its applicability to large-scale screening. To be used either as an alternative, or as a complement, to real-time RT-PCR testing, we developed a high-throughput targeted proteomics assay to detect SARS-CoV-2 proteins directly from clinical respiratory tract samples. Sample preparation was fully automated by using a modified magnetic particle-based proteomics approach implemented on a robotic liquid handler, enabling a fast processing of samples. The use of turbulent flow chromatography included four times multiplexed on-line sample cleanup and UPLC separation. MS/MS detection of three peptides from SARS-CoV-2 nucleoprotein and a 15N-labeled internal global standard was achieved within 2.5 min, enabling the analysis of more than 500 samples per day. The method was validated using 562 specimens previously analyzed by real-time RT-PCR and was able to detect over 83% of positive cases. No interference was found with samples from common respiratory viruses, including other coronaviruses (NL63, OC43, HKU1, and 229E). The strategy here presented has high sample stability and low cost and should be considered as an option to large population testing.

A rapid and sensitive method to detect SARS-CoV-2 virus using targeted-mass spectrometry

“…The major advantage of our method is its short run time with a 2.3 min gradient which is the fastest reported till date for detection of SARS-CoV-2 peptides. Cardozo et al (2020) reported a MRM with a gradient of 10.5 min that could detect SARS-CoV-2 peptides with a sensitivity of 83%. Interestingly, they used fully automated sample preparation protocol using robotic liquid handler and turbulent flow chromatography, multiplexed for online sample clean-up and UPLC separation that enabled them to run 4 samples within the 10.5 min run.…”

“…They also shortlisted several peptides for targeted mass spectrometric studies (Gouveia et al 2020). Cardozo et al have reported the detection of three peptides using a 10.5 min run time with 83% sensitivity and 96% specificity (Cardozo et al 2020). Ihling et al used a gargle solution of 3 infected patients and report the identification of 1 peptide from nucleoprotein using a 180 min gradient (IIhling et al 2020).…”

A rigorous evaluation of optimal peptide targets for MS-based clinical diagnostics of Coronavirus Disease 2019 (COVID-19)

“…For establishing workflows to evaluate virus-specific peptides, three published cell culture datasets [22][23][24] which used SARS-COV2 infected Vero cell lines were chosen, along with five clinical datasets [25][26][27][28][29] .…”

“…In order to evaluate the most robustly detectable SARS-CoV-2 peptides, and make the detection of these viral peptides in human samples in a clinical setting all the more feasible, we set out to examine proteomic datasets from three cell culture-based studies [22][23][24] and five clinical studies [25][26][27][28][29] . We utilized automated workflows implemented in the Galaxy platform and made accessible via the European Galaxy public instance to first identify as many SARS-CoV-2 peptides possible in all samples, creating a master list of SARS-CoV-2 peptides identified across the samples.…”

The separation sciences, the front end to proteomics: An historical perspective

“…The TFC comprised a four‐time multiplexed online sample clean‐up and ultra‐performance liquid chromatography separation. The method was validated using 562 specimens that had been previously analyzed by RT‐PCR and detected over 83% of the positive cases (Cardozo et al, 2020). In another example, a number of different methods were assessed for quantitative measurement of metallodrug–protein interactions.…”