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.

Proteotyping SARS-CoV-2 Virus from Nasopharyngeal Swabs: A Proof-of-Concept Focused on a 3 Min Mass Spectrometry Window

“…31 Being easily adapted to 96-well plates and robotization, SP3based digestion is the method of choice for quick, highthroughput, and highly reproducible proteome digestions, as recently demonstrated. 24,32 Pathogen proteotyping by mass spectrometry has emerged in recent years as an interesting alternative to molecular biology assays because of its high specificity and speed. 12 Numerous strategies based on tandem mass spectrometry have been already proposed for the detection of pathogens from a large source of samples including environmental and clinical samples.…”

“…22 Several research groups started investigating MSbased quantification of peptides for the detection of SARS-CoV-2 in clinical samples, but most of these results are not yet published. 4,23,24 These preliminary results indicate that targeted MS, in which the mass spectrometer is programmed to precisely detect and quantify a limited number of peptides of interest, can be successfully applied to virus detection. Targeted MS is considered as the gold standard for peptide quantification due to its higher sensitivity when compared to shotgun proteomics approaches.…”

Identification of SARS-CoV-2 Proteins from Nasopharyngeal Swabs Probed by Multiple Reaction Monitoring Tandem Mass Spectrometry

“…The in silico data of the selected peptides were then compared with the literature data. 16 , 21 , 22 , 29 Peptides defined by Skyline analysis monitored for each protein are reported in the Supporting Information Table ST2 . This method contained a total of 77 peptides and 407 precursor ion–daughter ions transitions associated with the peptides selected from the defined target proteins that constitute the protein signature of CoV-2 as reported in Table ST2 .…”

A pesquisa de anticorpos e componentes virais no diagnóstico e acompanhamento de infecções causadas pelo SARS-CoV-2

“…O desafio posto pela pandemia de COVID-19 motivou diversos grupos de pesquisa em proteômica direcionada a buscarem alternativas ao teste padrão-ouro por RT-PCR. Nosso grupo foi um dos primeiros a demonstrar a possibilidade de detecção direta do vírus a partir de amostras obtidas de raspados combinados da nasofaringe e orofaringe 37 . Nesse estudo, por meio de análises proteômicas não direcionadas de amostras previamente caracterizadas por RT-PCR, foi possível detectar diversas proteínas virais, como a nucleoproteína (ou proteína do nucleocapsídeo), a glicoproteína spike, a proteína da membrana, as proteínas 3a, 7a e 8 e replicases.…”