A Targeted LC-MRM3 Proteomic Approach for the Diagnosis of SARS-CoV-2 Infection in Nasopharyngeal Swabs

Since its first appearance, severe acute respiratory syndrome coronavirus 2 quickly spread around the world and the lack of adequate PCR testing capacities, especially during the early pandemic, led the scientific community to explore new approaches such as mass spectrometry (MS). We developed a proteomics workflow to target several tryptic peptides of the nucleocapsid protein. A highly selective multiple reaction monitoring-cubed (MRM3) strategy provided a sensitivity increase in comparison to conventional MRM acquisition. Our MRM3 approach was first tested on an Amsterdam public health cohort (alpha-variant, 760 participants) detecting viral nucleocapsid protein peptides from nasopharyngeal swabs samples presenting a cycle threshold value down to 35 with sensitivity and specificity of 94.2% and 100.0%, without immunopurification. A second iteration of the MS-diagnostic test, able to analyze more than 400 samples per day, was clinically validated on a Leiden-Rijswijk public health cohort (delta-variant, 2536 participants) achieving 99.9% specificity and 93.1% sensitivity for patients with cycle threshold values up to 35. In this manuscript, we also developed and brought the first proof of the concept of viral variant monitoring in a complex matrix using targeted MS. [Display omitted] •Injection-molded PBT swabs are a mass spectrometry friendly alternative for sample collection.•LC-MRM3 is a cheap, fast, sensitive, and selective diagnostic method for SARS-CoV-2.•Fast LC-MRM3 can detect contagious viral loads with a sensitivity greater than 95%.•Fast LC-MRM3 is easy to adapt to new targets, making it suitable for future pandemic preparedness.•LC-MRM3 is a promising tool for large-scale variant monitoring by detecting peptides with a mutation. SARS-CoV-2's rapid global spread exposed PCR testing limitations. We addressed this through mass spectrometry, developing a proteomics workflow targeting nucleocapsid protein peptides. By developing a selective MRM3 strategy, we achieved higher sensitivity compared to conventional methods. Our first approach detected viral peptides in nasopharyngeal samples with a cycle threshold (Ct) value down to 35 with sensitivity and specificity of 94.2% and 100.0% specificity without immunopurification. Subsequent mass spectrometry iterations, capable of processing 400+ samples daily, demonstrated 99.9% specificity and 93.1% sensitivity.