Top-Down Proteomics of Medicinal Cannabis

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.

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

“…In our previous experiments, a small C. sativa FASTA database retrieved from UniProt Knowledge Base was searched to identify proteins from medicinal cannabis apical buds [ 1 , 2 , 3 ]. This database corresponded to what we refer to as Uniprot515 in the present study.…”

“…The C. sativa proteins annotated in the UniProt Knowledge Base are known to carry modifications, and we have experimentally validated some of them using a top-down proteomics strategy [ 3 ]. Consequently, in this study we have included the following dynamic PTMs to the search method: N-term acetylation, acetylation and methylation of K residues, oxidation of M residues, phosphorylation of S, T and Y residues and the attachment of N-acetyl-D- glucosamine (NAG) glycogroups to N residues.…”

“…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 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 ]. These three proof-of-concept proteomics studies share one deficiency: the UniProt database searched for the purpose of protein identification was small (containing less than 500 entries) and consequently greatly underestimated the rich genetic diversity of C. sativa [ 33 ].…”

Special Issue “Top-down Proteomics: In Memory of Dr Alfred Yergey”. Alfred Linwood Yergey, III, 17 September 1941–27 May 2018

“…Al's article is followed by three firmly focused on agriculture. Vincent et al [4] have optimised top-down MS-based proteomics to study the cannabis proteome, a vastly understudied plant with a vast range of untapped medical applications whose use has recently been legislated in numerous jurisdictions. Functional foods also represent understudied proteomes and Tomazou et al [5] investigated potential anti-microbial peptides from the milk and cheese of goats and sheep, while Gutierrez et al [6] analysed saliva samples from pigs to determine early markers and mechanisms of disease-causing growth retardation.…”