Ph.D. (Doctor of Philosophy)
Department of Chemistry and Biochemistry
Coronavirus disease-2019 (COVID-19), which is caused by a novel coronavirus named severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has ravaged the world for the past 3 years. Even today, there still exists a need for rapid, accurate, economical and non-invasive diagnostic testing platforms that yield high specificity and sensitivity towards the constantly mutating SARS-CoV-2. Research has consistently indicated saliva to be a more amenable specimen type for early detection of SARS-CoV-2, compared to the oral and nasopharyngeal swabs. Considering the limitations and high demand of the existing COVID-19 testing platforms, this dissertation work studies used MALDI-ToF MS (matrix-assisted laser desorption/ionization-time of flight mass spectrometry) to analyze human gargle samples and compared the resulting spectra against their COVID-19. Several protein standards, including amylase, albumin, serum immunoglobulins, controls such as pre-COVID-19 saliva and heat inactivated viruses like SARS-CoV-2, MERS-CoV, H1N1 and common human coronaviruses, 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, which included host as well as viral proteins were established thatdemonstrated high agreement with COVID-19 positive individuals. Overall, the accuracy of this developed protein profile testing platform was ≥90%. To further enhance the sensitivity of the assay, amylase, the most abundant protein found in saliva, was depleted using a rapid and inexpensive amylase depleting device. After depleting the amylase from the gargle/saliva samples, there was an enhancement in 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. MALDI-ToF results were also compared to the RT-qPCR (reverse transcriptase quantitative polymerase chain reaction) results in a 7-day study which was reported for the first time with a remarkable correlation between the two techniques. Moreover, attempts were made to detect not just the SARS-CoV-2 but also the type of variant. As mutations cause mass shifts, these could be detected using mass spectrometry. In essence, this research makes a significant contribution to the field of proteomics and mass spectrometry for the analysis of protein profiles in human saliva to elucidate novel biomarkers for COVID-19. Protein profiling of body fluids using MALDI-TOF MS could be a promising PCR-free tool for screening SARS-CoV-2 as well as various other viruses.
Chivte, Prajkta Satish, "Bioanalytical Studies of Disease Protein Profiles: MALDI-ToF MS" (2023). Graduate Research Theses & Dissertations. 7307.
Northern Illinois University
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