Proteomic Identification of Hemp (Cannabis sativa) Seeds by Tandem Mass Spectrometry and Evaluation of Cytotoxic Activity

<p>Hemp (<em>Cannabis sativa</em>) has a long history of use globally as a source of traditional medicine, spiritual enlightenment, and recreational enjoyment. While it was commonly consumed for both medical and recreational purposes in the United States, it was classified as a Schedule 1 drug in 1937, severely restricting its use and research opportunities. Although hemp itself lacks psychoactive properties, <em>Cannabis sativa</em> flowers contain THC, the compound responsible for its psychotropic effects. This restriction hindered quality research into cannabis and its constituents. The study employed techniques, including top-down proteomics, size exclusion chromatography, affinity chromatography, high-resolution ESI QTOF LC/MS/MS, and De Novo protein sequencing bioinformatics tools. The process involved defatting cannabis sativa seeds in hexane and extracting protein in PBS buffer over two days at 4°C. The extracted proteins were precipitated in 80% ammonium sulfate, dialyzed, and lyophilized. Protein fractions were separated using size exclusion chromatography and blue Sepharose affinity chromatography. Separated proteins were analyzed via Tris/Glycine SDS-PAGE electrophoresis. Protein modification was carried out using performic acid oxidation, followed by Pepsin and tryptic digestion. The resulting tryptic and pepsin digests were subjected to high-resolution Q-TOF LC/MS/MS analysis, and the bioinformatics tool Peak Studio-X was utilized for the De Novo protein sequencing search against the SwissProt Viridiplantae database. The study revealed many proteins identified in the hemp proteome. Further, investigations assessed the cytotoxic activity of hemp seed crude protein extract using MCF7 human breast cancer cell lines. The results showed that hemp crude protein extract inhibited the proliferation of MCF-7 cells in a dose-dependent manner. The IC₅₀value was 154 µg/ml, suggesting relatively mild effects. Gene Ontology (GO) analysis was employed to elucidate the biological processes, molecular functions, and cellular components associated with trypsin-digested bound Blue Sepharose hemp seed proteins.</p>

Name thesis:degree_name

Master of Science (MS)

Level thesis:degree_level

Thesis

Discipline thesis:degree_discipline

Pharmaceutical Sciences

Year dc:date.available

2024

Author dc:creator

• Harris, Taran

Contributors dc:contributor

• Aftab Ahmed
• Innokentiy Maslennikov
• Keykavous Parang

• proteomics
• QTof-LCMSMS
• mass spectrometry
• gel electrophoresis
• cytotoxicity
• breast cancer
• hemp
• Other Pharmacy and Pharmaceutical Sciences

Repository record dc:identifier

https://digitalcommons.chapman.edu/pharmaceutical_sciences_theses/35

OAI identifier oai:identifier

oai:digitalcommons.chapman.edu:pharmaceutical_sciences_theses-1036