More than a year after the COVID-19 pandemic was declared, the need still exists for accurate, rapid, inexpensive and non-invasive diagnostic methods that yield high specificity and sensitivity towards the current and newly emerging SARS-CoV-2 strains. Compared to the nasopharyngeal (NP) swabs, several studies have established saliva as a more amenable specimen type for early detection of SARS-CoV-2. Considering the limitations and high demand for COVID-19 testing, we employed MALDI-ToF mass spectrometry in the analysis of 60 gargle samples from human donors and compared the resultant spectra against COVID-19 status. Several standards, including isolated human serum immunoglobulins, and controls, such as pre-COVID-19 saliva and heat inactivated SARS-CoV-2 virus, were simultaneously analyzed to provide a relative view of the saliva and viral proteome as they would appear in this workflow. Five potential biomarker peaks were established that demonstrated high concordance with COVID-19 positive individuals. Overall, the agreement of these results with RT-qPCR testing on NP swabs was ≥90% for the studied cohort, which consisted of young and largely asymptomatic student athletes. From a clinical standpoint, the results from this pilot study suggest that MALDI-ToF could be used to develop a relatively rapid and inexpensive COVID-19 assay.

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Human saliva contains a plethora of proteins whose presence and concentration can be monitored for diagnosis and progression of disease. Saliva has been extensively probed for the diagnosis of several systemic and infectious diseases because of the ease with which it can be collected. However, amylase, the most abundant protein found in saliva can obscure the detection of low-abundance proteins by MALDI-ToF MS (matrix-assisted laser desorption/ionization-time of flight mass spectrometry) and diminish the diagnostic utility of this specimen type. In the present study, we used a device to deplete salivary amylase from water-gargle samples through affinity adsorption. After depletion, profiling of the saliva proteome was performed by MALDI-ToF MS on gargle samples from subjects whose COVID-19 (coronavirus disease 2019) status was confirmed by NP (nasopharyngeal) swab RT-qPCR (reverse transcription polymerase chain reaction). Amylase depletion led to the enhancement of signal intensities of various peaks as well as the detection of previously unobserved peaks in the MALDI-ToF spectra. The overall specificity and sensitivity after amylase depletion was 100% and 85.17% respectively for detecting COVID-19. Our simple, rapid and inexpensive technique to deplete salivary amylase can be used to unmask spectral diversity in saliva by MALDI-ToF MS, reveal low-abundant proteins and aid in the establishment of novel biomarkers for diseases.