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. We developed a high-throughput targeted proteomics assay to detect SARS-CoV-2 proteins directly from nasopharyngeal and oropharyngeal swabs. Sample preparation was fully automated by using a modified magnetic particle-based proteomics approach implemented on a robotic liquid handler. The use of turbulent flow chromatography included four times multiplexed on-line sample cleanup and liquid chromatography separation. Mass spectrometry detection of nucleoprotein peptides was achieved within 2.5 min, enabling the analysis of more than 500 samples per day. The method was validated qualitatively (Tier 3) and quantitatively (Tier 1) using 855 specimens previously analyzed by real-time RT-PCR and was able to detect up to 84% of positive cases with up to 97% of specificity. The strategy here presented has high sample stability and should be considered as an option for testing in large populations.
Skyline (version 188.8.131.52) 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 (combined materials from nasopharyngeal and oropharyngeal swabs) were collected in virus transport media45 or sterile saline solution and stored at −80 °C . All specimens used in this study were previously analyzed by an in-house real-time RT-PCR method implemented according to WHO guidelines 46. 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.