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RSS FeedsIJMS, Vol. 20, Pages 5630: A Multiple Protease Strategy to Optimise the Shotgun Proteomics of Mature Medicinal Cannabis Buds (International Journal of Molecular Sciences)

 
 

11 november 2019 15:02:29

 
IJMS, Vol. 20, Pages 5630: A Multiple Protease Strategy to Optimise the Shotgun Proteomics of Mature Medicinal Cannabis Buds (International Journal of Molecular Sciences)
 


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.


 
179 viewsCategory: Biochemistry, Biophysics, Molecular Biology
 
IJMS, Vol. 20, Pages 5628: RgsA Attenuates the PKA Signaling, Stress Response, and Virulence in the Human Opportunistic Pathogen Aspergillus fumigatus (International Journal of Molecular Sciences)
IJMS, Vol. 20, Pages 5626: Epinephrine Infiltration of Adipose Tissue Impacts MCF7 Breast Cancer Cells and Total Lipid Content (International Journal of Molecular Sciences)
 
 
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