Cannabis sativa has been dubbed “the plant of the thousand and one molecules” owing to its propensity to produce a plethora of phytochemicals with myriad biological activities as well as fibrous components. Out of the 500 compounds that have been described thus far, more than 90 are phytocannabinoids including cannabidiolic acid (CBDA) and delta 9-tetrahydrocannabinolicacid (THCA) accumulating within the trichomes of mature buds. One of the key phytocannabinoid-related enzymes is olivetolic acid (OLA) cyclase (OAC) acting in succession to convert hexanoyl-CoA into OLA.
Cannabis is a controlled substance and until recently was illegal in many jurisdictions. The recent revised legislation on medicinal cannabis has triggered a surge of medical and clinical research studies evaluating the effect of major cannabis components on human health. Medicinal cannabis is used to relieve the symptoms of certain medical conditions, such as epilepsy.
The state of Victoria in Australia was the first jurisdiction to legalise access to medicinal cannabis under the Medicinal Cannabis Act in 2016. In this context, Agriculture Victoria Research (AVR) have controlled access to medicinal cannabis material grown to full maturity in the state-of-the-art Victorian government medicinal cannabis cultivation facility.
A comprehensive systems biology approach including genome sequencing, transcriptomics, proteomics and metabolomics was undertaken by AVR to fully characterise the various medicinal cannabis cultivars developed in house.
In this study, we present three complementary proteomics strategies, namely bottom-up proteomics (BUP) through the use of the most commonly used protease trypsin1, middle-down proteomics (MDP) by exploiting alternative orthogonal proteases2, and top-down proteomics (TDP) which required innovative data mining methods and revealed numerous proteoforms3. We use OAC to illustrate the power of proteomics in cannabis research, with respect to sequence coverage and post-translational modifications (PTMs).