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Open AccessArticle

Optimisation of Protein Extraction from Medicinal Cannabis Mature Buds for Bottom-Up Proteomics

Agriculture Victoria Research, AgriBio, Centre for AgriBioscience, Bundoora, Victoria 3083, Australia
Author to whom correspondence should be addressed.
Academic Editor: Makoto Tsunoda
Molecules 2019, 24(4), 659;
Received: 25 January 2019 / Revised: 11 February 2019 / Accepted: 11 February 2019 / Published: 13 February 2019
(This article belongs to the Special Issue Biological Sample Analysis by Liquid Chromatography)
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. View Full-Text
Keywords: urea; guanidine-HCl; trypsin digestion; nLC-MS/MS; apical bud; trichome urea; guanidine-HCl; trypsin digestion; nLC-MS/MS; apical bud; trichome
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Vincent, D.; Rochfort, S.; Spangenberg, G. Optimisation of Protein Extraction from Medicinal Cannabis Mature Buds for Bottom-Up Proteomics. Molecules 2019, 24, 659.

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