Abstract
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.
Highlights
The state of Victoria in Australia was the first jurisdiction to legalise access to medicinal cannabis under the Medicinal Cannabis Act in 2016
Precursors of oxidized proteoforms were purposefully disregarded in the manual annotation step; they were included as a dynamic modification for the Mascot search
When true mass spectrometry (MS)/MS data resulting from electron transfer dissociation (ETD), collision-induced dissociation (CID), and higher-energy collisional dissociation (HCD) experiments are considered, high numbers of fragments are a requisite for proper top-down sequencing, yet it was not the MS/MS spectra with the maximum number of peaks that yielded the greatest number of matched ions in ProSight Lite (Tables 1 and 2)
Summary
The state of Victoria in Australia was the first jurisdiction to legalise access to medicinal cannabis under the Medicinal Cannabis Act in 2016 (www2.health.vic.gov.au). This work follows on from our optimization of protein extraction of mature buds from medicinal cannabis prior to bottom-up proteomics [1] In this top-down study, we used a hybrid mass spectrometer composed of a linear ion trap (LTQ or ITMS) and a Fourier-transform Orbitrap (FTMS), which we typically use for bottom-up experiments [1,30]. This powerful instrument offers high resolution (up to 240,000) and multiple modes of fragmentations (SID, CID, HCD, and ETD).
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