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Open AccessArticle

Top-Down Proteomics of Medicinal Cannabis

1
Agriculture Victoria Research, AgriBio, Centre for AgriBioscience, Bundoora, Victoria 3083, Australia
2
Thermo Fisher Scientific, Bio21 Institute, The University of Melbourne, 30 Flemington Rd, Parkville, Victoria 3052, Australia
*
Author to whom correspondence should be addressed.
Proteomes 2019, 7(4), 33; https://doi.org/10.3390/proteomes7040033
Received: 29 July 2019 / Revised: 6 September 2019 / Accepted: 20 September 2019 / Published: 24 September 2019
(This article belongs to the Special Issue Top-down Proteomics: In Memory of Dr. Alfred Yergey)
The revised legislation on medicinal cannabis has triggered a surge of research studies in this space. Yet, cannabis proteomics is lagging. In a previous study, we optimised the protein extraction of mature buds for bottom-up proteomics. In this follow-up study, we developed a top-down mass spectrometry (MS) proteomics strategy to identify intact denatured protein from cannabis apical buds. After testing different source-induced dissociation (SID), collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), and electron transfer dissociation (ETD) parameters on infused known protein standards, we devised three LC-MS/MS methods for top-down sequencing of cannabis proteins. Different MS/MS modes produced distinct spectra, albeit greatly overlapping between SID, CID, and HCD. The number of fragments increased with the energy applied; however, this did not necessarily translate into greater sequence coverage. Some precursors were more amenable to fragmentation than others. Sequence coverage decreased as the mass of the protein increased. Combining all MS/MS data maximised amino acid (AA) sequence coverage, achieving 73% for myoglobin. In this experiment, most cannabis proteins were smaller than 30 kD. A total of 46 cannabis proteins were identified with 136 proteoforms bearing different post-translational modifications (PTMs), including the excision of N-terminal M, the N-terminal acetylation, methylation, and acetylation of K resides, and phosphorylation. Most identified proteins are involved in photosynthesis, translation, and ATP production. Only one protein belongs to the phytocannabinoid biosynthesis, olivetolic acid cyclase. View Full-Text
Keywords: infusion; LC-MS; LC-MS/MS; SID; CID; HCD; ETD; PTMs; apical bud; cannabis intact proteins; protein standards; proteoforms; top-down sequencing; Prosight Lite; Mascot infusion; LC-MS; LC-MS/MS; SID; CID; HCD; ETD; PTMs; apical bud; cannabis intact proteins; protein standards; proteoforms; top-down sequencing; Prosight Lite; Mascot
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MDPI and ACS Style

Vincent, D.; Binos, S.; Rochfort, S.; Spangenberg, G. Top-Down Proteomics of Medicinal Cannabis. Proteomes 2019, 7, 33.

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