Abstract
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.
Highlights
The recent revised legislation on medicinal cannabis has triggered a surge of medical and clinical research studies evaluating the effect of major cannabis components on human health
We developed a top-down proteomics (TDP) strategy complementary to bottom-up proteomics (BUP) which allowed the detection of unreported post-translational modifications (PTMs) of the identified intact cannabis proteins, such as the excision of the N-terminus M, and the presence of methylations, acetylations, and phosphorylations [12]
The proteome of mature apical buds was satisfactorily covered as assessed by the identification of all the enzymes involved in the biosynthesis of phytocannabinoids, along with many other enzymes from cannabis secondary metabolism
Summary
The recent revised legislation on medicinal cannabis has triggered a surge of medical and clinical research studies evaluating the effect of major cannabis components on human health. Guo and colleagues have employed seven proteases of low specificity (chymotrypsin and elastase), medium specificity (trypsin) and high specificity (GluC, LysC, AspN, and ArgC) in a complex combinatorial design of single-, double-, and triple-enzyme digests to devise the optimum sample digestion and proteome coverage of HeLa cell lysates [54] While they report an increase in both protein identifications and mean sequence coverage, they further conclude that dynamic range rather than enzyme bias is the most limiting factor to proteome exploration. We chose three orthogonal digestions, namely chymotrypsin (of low specificity targeting hydrophobic residues Y, F, W, and to a lesser extent L), a ready-to-use mixture of trypsin/LysC (of medium specificity targeting positively charged residues R and K), and GluC (high specificity targeting negatively charged residues mostly E and occasionally D under our experimental conditions) These enzymes were carefully selected based on their specificity to yield peptides spanning from 0.5 to 10 kDa as to cover both BUP and MDP ranges. The results are discussed in terms of reproducibility, number of identified peptides, missed cleavages, PTM detection, AA sequence and proteome coverages
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