Abstract
Medicinal cannabis is used to relieve the symptoms of certain medical conditions, such as epilepsy. Cannabis is a controlled substance and until recently was illegal in many jurisdictions. Consequently, the study of this plant has been restricted. Proteomics studies on Cannabis sativa reported so far have been primarily based on plant organs and tissues other than buds, such as roots, hypocotyl, leaves, hempseeds and flour. As far as we know, no optimisation of protein extraction from cannabis reproductive tissues has been attempted. Therefore, we set out to assess different protein extraction methods followed by mass spectrometry-based proteomics to recover, separate and identify the proteins of the reproductive organs of medicinal cannabis, apical buds and isolated trichomes. Database search following shotgun proteomics was limited to protein sequences from C. sativa and closely related species available from UniprotKB. Our results demonstrate that a buffer containing the chaotrope reagent guanidine hydrochloride recovers many more proteins than a urea-based buffer. In combination with a precipitation with trichloroacetic acid, such buffer proved optimum to identify proteins using a trypsin digestion followed by nano-liquid chromatography tandem mass spectrometry (nLC-MS/MS) analyses. This is validated by focusing on enzymes involved in the phytocannabinoid pathway.
Highlights
Cannabis sativa plants have been cultivated and used as a food crop as well for textiles, ropes, paper and medicinal applications for millennia in Asia [1]
This experiment aims at optimising protein extraction from mature reproductive tissues of medicinal cannabis
Precipitation steps with the use of either acetone or ethanol as solvents, as well as changing in their precipitation steps with the use of either acetone or ethanol as solvents, as well as changing in their final pellet resuspension step with the use of urea- or guanidine-HCl-based buffers (Figure 1)
Summary
Cannabis sativa plants have been cultivated and used as a food crop as well for textiles, ropes, paper and medicinal applications for millennia in Asia [1]. By the end of the nineteen century, cannabis therapeutical applications had diminished due to the development of more efficient alternative therapies and analgesics, in particular opiates [2]. The beginning of the 20th century saw the surge of cannabis use as a recreational substance in Western countries, which led to the Cannabis Tax Act in 1937 and abolished its use in medicine in the U.S.A. Within the framework of this treaty, since 1967 the Australian Narcotic Drugs Act (www.odc.gov.au) has regulated the cultivation of cannabis for medicinal and related scientific purposes.
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