A Multiple Protease Strategy to Optimise the Shotgun Proteomics of Mature Medicinal Cannabis Buds

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.

The Power of Three in Cannabis Shotgun Proteomics: Proteases, Databases and Search Engines

“…Individual mature apical buds of medicinal cannabis were sampled in triplicate (labelled “bud1”, “bud2” and “bud3” hereafter) and proteins were extracted using a trichloroacetic acid/acetone precipitation followed by resuspension in a guanidine-HCl buffer as detailed in [ 1 , 2 ]. A plant protein content of 100 µg was digested using either a trypsin/Lys-C protease mixture (TL, Mass Spectrometry Grade, 100 μg, Promega), or chymotrypsin (C, Sequencing Grade, 25 μg, Promega) or rAsp-N (A, Sequencing Grade, 10 μg, Promega).…”

“…A plant protein content of 100 µg was digested using either a trypsin/Lys-C protease mixture (TL, Mass Spectrometry Grade, 100 μg, Promega), or chymotrypsin (C, Sequencing Grade, 25 μg, Promega) or rAsp-N (A, Sequencing Grade, 10 μg, Promega). Digestions with trypsin/Lys-C and chymotrypsin have been described in [ 2 ]. For the digestion using rAsp-N, 50 mM Ammonium bicarbonate (pH 7.8) was added to the dithiothreitol (DTT)-reduced and iodoacetamide (IAA)-alkylated proteins in order to drop the guanidine-HCl resuspension buffer molarity below 1 M. The protease was carefully solubilised in 0.1 mL of ddH 2 O.…”

“…To achieve a 1:50 ratio of protease:proteins, as instructed by the manufacturer, a 20 µL aliquot of rAsp-N solution was added and gently mixed with the protein extracts. The mixture was incubated at 37 °C in the dark for 1 h. All digests were cleaned up using solid phase extraction (SPE) cartridges followed by evaporation, as explained in [ 1 , 2 ]. Peptides digests were analysed using nLC-MS/MS exactly as described in [ 1 , 2 ].…”

“…Bottom-up proteomics (BUP) refers to the characterization of proteins by analysis of their peptides released through proteolysis. When BUP is performed on a mixture of proteins it is called shotgun proteomics [ 1 , 2 , 3 , 4 ]. Large-scale or high-throughput analyses of highly complex samples are commonly accomplished using a BUP strategy [ 5 ].…”

“…In a first step, we optimized the extraction of cannabis proteins, demonstrating the superiority of guanidine-hydrochloride over urea [ 1 ]. In a second step, we optimized the digestion of cannabis proteins using a multiprotease (four enzymes) and multidigestion (single, double and triple digestion) approach, questioning the rationale behind limiting the number of missed cleavages allowed [ 2 ]. In a third step, we developed a top-down proteomics strategy to analyse intact proteins and discovered that cannabis proteins are predominantly methylated [ 3 ].…”