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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.
Pubmed ID: 31554318
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Collection of data of protein sequence and functional information. Resource for protein sequence and annotation data. Consortium for preservation of the UniProt databases: UniProt Knowledgebase (UniProtKB), UniProt Reference Clusters (UniRef), and UniProt Archive (UniParc), UniProt Proteomes. Collaboration between European Bioinformatics Institute (EMBL-EBI), SIB Swiss Institute of Bioinformatics and Protein Information Resource. Swiss-Prot is a curated subset of UniProtKB.
View all literature mentionsCentral repository for collection of functional information on proteins, with accurate and consistent annotation. In addition to capturing core data mandatory for each UniProtKB entry (mainly, the amino acid sequence, protein name or description, taxonomic data and citation information), as much annotation information as possible is added. This includes widely accepted biological ontologies, classifications and cross-references, and experimental and computational data. The UniProt Knowledgebase consists of two sections, UniProtKB/Swiss-Prot and UniProtKB/TrEMBL. UniProtKB/Swiss-Prot (reviewed) is a high quality manually annotated and non-redundant protein sequence database which brings together experimental results, computed features, and scientific conclusions. UniProtKB/TrEMBL (unreviewed) contains protein sequences associated with computationally generated annotation and large-scale functional characterization that await full manual annotation. Users may browse by taxonomy, keyword, gene ontology, enzyme class or pathway.
View all literature mentionsA software package and server used to identify and characterize proteins from primary sequence databases using mass spectrometry data. Mascot integrates peptide mass fingerprinting, sequence querying, and MS/MS ion searching in order to search for proteins in databases like SwissProt, NCBInr, EMBL EST divisions, contaminants, and cRAP. If a license is purchased, users may: search data sets that exceed the 1200 spectrum limit of the free version; set up automated, high throughput work; add and edit proteins and quantification methods; and search a preferred collection of sequence databases. The software package works with instruments from AB Sciex, Agilent, Bruker, Jeol, Shimadzu, Thermo Scientific, and Waters.
View all literature mentionsA software which acquires and processes data sets, primarily through the Xcalibur system.
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