<p>A significant
development in mass spectrometry instrumentation and software in the past
decade has led to its application in solving complex biological problems. One
of the emerging areas is Chemical Proteomics that involves design and use of
chemical reagents to probe protein functions in ‘a live cell’ environment. Another
aspect of Chemical Proteomics is the identification of target proteins of a
drug or small molecule. This is assisted by photoreactive groups, which on exposure
to UV light, covalently link the target proteins that can be purified by
affinity-based enrichment followed by mass-spectrometric identification. This
phenomenon of Photoaffinity labeling (PAL) has been widely used in a broad
range of applications. Herein, we have designed chemical tools to study Zika
endocytosis and phosphoproteomics.</p>
<p>Zika virus
has attracted the interest of researchers globally, following its outbreak in
2016. While a significant development has been made in understanding the structure
and pathogenesis, the actual mechanism of Zika entry into host cells is largely
unknown. We designed a chemical probe to tag the live virus, leading to the
identification of the virus receptors and other host factors involved in viral
entry. We further validated neural cell adhesion molecule (NCAM1) as a host
protein involved in early phase entry of Zika virus into Vero cells.</p>
<p>The second aspect is the development of the DIGE
(Difference Gel Electrophoresis) technology for phosphoproteomics. Phosphoproteins
are known to be involved in various signaling pathways and implicated in
multiple diseased states. We designed chemical reagents composed of titanium
(IV) ion, diazirine and a fluorophore, to covalently label the phosphoproteins.
Cyanine3 and cyanine5 fluorophores were employed to reveal the difference in
phosphorylation between samples for the comparative proteomics. Thus far, we have
successfully demonstrated the labeling of standard phosphoproteins in both simple
and complex protein mixtures, and the future efforts are towards applying the
technology to identify phosphoproteins in a cell lysate.</p>
| Identifer | oai:union.ndltd.org:purdue.edu/oai:figshare.com:article/9108128 |
| Date | 16 August 2019 |
| Creators | Mayank Srivastava (5930294) |
| Source Sets | Purdue University |
| Detected Language | English |
| Type | Text, Thesis |
| Rights | CC BY 4.0 |
| Relation | https://figshare.com/articles/DEVELOPMENT_OF_CHEMICAL_PROTEOMIC_APPROACHES_TO_STUDY_VIRAL_ENDOCYTOSIS_AND_PHOSPHOPROTEOMICS/9108128 |
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