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.
Skyline (version 22.214.171.124) was used to build a spectral library from data processed by MaxQuant. The first set of candidate peptides was established importing UniProt SARS-CoV-2 pre-release into Skyline. Only peptides matching the library, fully digested and with no cysteine residues were included. Filtered peptides were exported into an isolation list to construct a parallel reaction monitoring (PRM) method for the mass spectrometer. Chromatographic and ion source parameters were identical to those described above. PRM data were acquired with 120,000 mass resolution (m/z 200), AGC target of 3 × 104, maximum IT of 250 ms, isolation window of m/z 1.6, and (N)CE = 27. Positive and negative samples were analyzed by the PRM method loaded into Skyline and the number of targets was reduced to the top 17 most intense ones across positive samples and absent in negative samples. A fast PRM method was achieved with a 9-min microflow chromatography separation using eight targeted peptides from nucleoprotein protein. The transition list for the selected peptides for SARS-CoV-2 was exported from the 9-min PRM Skyline method and imported into the TraceFinder Instrument Setup module.
Respiratory tract samples (nasopharyngeal swab, oropharyngeal swab, and nasal wash) were collected in virus transport media or sterile saline solution and stored at −80 °C 22. All specimens used in this study were previously analyzed by an in-house real-time RT-PCR method implemented according to WHO guidelines 24. Cross-reactivity was evaluated against specimens of other human coronaviruses (HCoV-HKU1, HCoV-229E, and HCoV-NL63), Influenza A (H1N1), respiratory syncytial virus (RSV), human metapneumovirus (HMPV), parainfluenza virus types 1 and 4, and rhinovirus/coronavirus HKU1/enterovirus coinfection previously characterized by Biofire® FilmArray® Respiratory Panel (bioMérieux, Marcy-l’Étoile, France). This study included only specimens collected as part of standard diagnostic protocols that would normally be discarded. Patient identification was not recorded or registered, and only proteins related to SARS-CoV-2 were investigated.
DDA data and search results used to build the spectrum library for this experiment are available in PRIDE with the ProteomeXchange ID PXD019119.