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Top-down proteomics of medicinal cannabis.

ABSTRACT: The revised legislation on medicinal cannabis has triggered a surge of research study in this space. Yet cannabis proteomics is lagging. In a previous study, we have optimised the protein extraction of mature buds for bottom-up proteomics. In this follow-up study, we develop a top-down proteomics strategy to identify intact denatured protein from cannabis apical buds. After testing different SID, CID, HCD and 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 produce 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 are more amenable to fragmentation than other. Sequence coverage decreases as the mass of the protein increases. Combining all MS/MS data maximised AA sequence coverage, achieving 73 percents for myoglobin. In this experiment, most cannabis proteins were smaller than 30 kD. Out of the 11,250 MS/MS spectra generated, only 213 (2 percents) were annotated amounting to 20 protein identifications. Most identified proteins are involved in photosynthesis, translation, and ATP production. Only one protein belongs to the phytocannabinoid biosynthesis, olivetolic acid cyclase. doi:10.3390/proteomes7040033

INSTRUMENT(S): RP-UPLC-ESI-LTQ-Orbitrap mass analyser

ORGANISM(S): Cannabis Sativa (marijuana)

SUBMITTER: Delphine Vincent

PROVIDER: MSV000083970 | MassIVE | Wed Jun 12 17:33:00 BST 2019

REPOSITORIES: MassIVE

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Project description:Top-down sequencing in proteomics has come of age owing to continuous progress brought to LC-MS. The quality and thoroughness of the results depend on the platform used. The latest generation of mass spectrometers displays unprecedented resolving power, very fast scanning rates, and several fragmentation modes. Unfortunately these instruments are very expensive, therefore out of reach for most laboratories. With their high resolution and broad mass range, quadrupole Time-of-Flight (Q-ToF) hybrid mass spectrometers equipped with electrospray ionisation (ESI) source and tandem MS capability by collision-induced dissociation (CID) offer a valuable alternative to analyse intact proteins and retrieve primary sequence information. As far as we know, top-down proteomics methods with such systems have only been evaluated using samples of relatively low complexity. The in source CID (IS-CID) capability of the Q-ToF instruments has been underutilised. This study aimed at optimising top-down sequencing results on intact milk proteins to achieve the greatest sequence coverage possible from samples of increasing levels of complexity. Eleven MS/MS methods varying in their IS-CID and conventional CID parameters were tested on individual and mixed protein standards and raw milk samples from two main cow breeds. Top-down sequencing results from the nine most abundant proteoforms of caseins, alpha-lactalbumin and beta-lactoglubulins were compared. Nine MS/MS methods achieved more than 70 percents sequence coverage overall to distinguish between allelic proteoforms, varying only by one or two AAs. Full 100 percent sequence information was obtained on bLGs. Two methods (Methods 8 and 10) employed high IS-CID energy failed to distinguish between allelic variations. The most optimal methods (Methods 4 and 12) utilised IS-CID on its own at low energy. Under optimal analytical conditions, sample complexity did not impact the quality and reproducibility of the results. This experiment demonstrates that Q-ToF systems remain suitable for top-down proteomics and that IS-CID should be more frequently utilised. doi:10.3390/molecules23112777

Project description:Earlier this year we published a method article aimed at optimising protein extraction from mature buds of medicinal cannabis for trypsin-based shotgun proteomics (Vincent, D., et al. Molecules 2019, 24, 659). We then developed a top-down proteomics (TDP) method (Vincent, D., et al. Proteomes 2019, 7, 33). This follow-up study aims at optimising the digestion of medicinal cannabis proteins for identification purposes by bottom-up and middle-down proteomics (BUP and MDP). Four proteases, namely a mixture of trypsin/LysC, GluC, and chymotrypsin, which target different amino acids (AAs) and therefore are orthogonal and cleave proteins more or less frequently, were tested both on their own as well as sequentially or pooled, followed by nLC-MS/MS analyses of the peptide digests. Bovine serum albumin (BSA, 66 kDa) was used as a control of digestion efficiency. With this multiple protease strategy, BSA was reproducibly 97% sequenced, with peptides ranging from 0.7 to 6.4 kD containing 5 to 54 AA residues with 0 to 6 miscleavages. The proteome of mature apical buds from medicinal cannabis was explored more in depth with the identification of 27,123 peptides matching 494 unique accessions corresponding to 229 unique proteins from Cannabis sativa and close relatives, including 130 (57%) additional annotations when the list is compared to that of our previous BUP study (Vincent, D., et al. Molecules 2019, 24, 659). Almost half of the medicinal cannabis proteins were identified with 100% sequence coverage, with peptides composed of 7 to 91 AA residues with up to 9 miscleavages and ranging from 0.6 to 10 kDa, thus falling into the MDP domain. Many post-translational modifications (PTMs) were identified, such as oxidation, phosphorylations, and N-terminus acetylations. This method will pave the way for deeper proteome exploration of the reproductive organs of medicinal cannabis, and therefore for molecular phenotyping within breeding programs. doi:10.3390/ijms20225630

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Top-down proteomics of medicinal cannabis.

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