Date of Award

2022

Document Type

Thesis

Degree Name

MS in Chemistry

Department

Chemistry

First Advisor

ERICA Y JACOBS

Second Advisor

FRANCISCO VAZQUEZ

Third Advisor

KONA ARACHCHILA SENEVIRATHNE

Abstract

COVID-19, the 2019 disease outbreak, continues to cause severe mortality worldwide. Fast and reliable tests that identify the SARS-CoV-2 virus, which is responsible for causing the COVID-19 disease, are crucial for controlling the spread of the virus. Antigen tests are ideal for the rapid diagnosis of COVID-19. Current antigen tests for COVID-19 detect the SARS-CoV-2 nucleocapsid (N) protein. The nucleocapsid is the most abundant protein produced by the virus upon infection, which makes it a superior target for detection by an antigen test. Nucleocapsid proteins from several coronaviruses, including SARS, have previously been shown to be modified by posttranslational modification (PTM), including phosphorylation. PTMs are important mechanisms to increase the functional diversity of the virus within its host cell and modulate the antiviral response. Antigen tests require an antibody to bind to nucleocapsid protein, however, phosphorylation may differ between the nucleocapsid used to elicit the diagnostic antibody and phosphorylation on the viral protein in an infected person. In this research, his-tagged SARS-CoV-2 nucleocapsid protein was expressed in insect cells using a baculovirus expression system and analysis of purified samples using bottom-up proteomics approach followed by ESI-MS/MS along with MALDI-TOF analysis was used to localize phosphorylation sites in the SARS-CoV-2 nucleocapsid protein, which may benefit the development of better antigen tests. Results from MALDI shows that N protein is phosphorylated by an average of 13 phosphate groups. A total of 12 phosphopeptides were identified by ESI-MS/MS, of which 3 are novel sites not previously reported (T135, T265 and T329).

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