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

2019 ◽  
Vol 20 (22) ◽  
pp. 5630 ◽  
Author(s):  
Delphine Vincent ◽  
Vilnis Ezernieks ◽  
Simone Rochfort ◽  
German Spangenberg
Keyword(s):  
Cannabis Sativa ◽  
Protein Extraction ◽  
Shotgun Proteomics ◽  
Reproductive Organs ◽  
Sequence Coverage ◽  
Post Translational Modifications ◽  
Apical Buds ◽  
Medicinal Cannabis ◽  
Close Relatives

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.

scholarly journals Top-Down Proteomics of Medicinal Cannabis

Proteomes ◽  
2019 ◽  
Vol 7 (4) ◽  
pp. 33 ◽  
Author(s):  
Vincent ◽  
Binos ◽  
Rochfort ◽  
Spangenberg
Keyword(s):  
Electron Transfer Dissociation ◽  
Protein Extraction ◽  
Sequence Coverage ◽  
Top Down ◽  
Post Translational Modifications ◽  
Atp Production ◽  
Denatured Protein ◽  
Apical Buds ◽  
Medicinal Cannabis

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.

scholarly journals Optimisation of Protein Extraction from Medicinal Cannabis Mature Buds for Bottom-Up Proteomics

Molecules ◽  
2019 ◽  
Vol 24 (4) ◽  
pp. 659 ◽  
Author(s):  
Delphine Vincent ◽  
Simone Rochfort ◽  
German Spangenberg
Keyword(s):  
Mass Spectrometry ◽  
Cannabis Sativa ◽  
Protein Extraction ◽  
Guanidine Hydrochloride ◽  
Shotgun Proteomics ◽  
Extraction Methods ◽  
Reproductive Organs ◽  
Apical Buds ◽  
Liquid Chromatography Tandem Mass ◽  
Medicinal Cannabis

Medicinal cannabis is used to relieve the symptoms of certain medical conditions, such as epilepsy. Cannabis is a controlled substance and until recently was illegal in many jurisdictions. Consequently, the study of this plant has been restricted. Proteomics studies on Cannabis sativa reported so far have been primarily based on plant organs and tissues other than buds, such as roots, hypocotyl, leaves, hempseeds and flour. As far as we know, no optimisation of protein extraction from cannabis reproductive tissues has been attempted. Therefore, we set out to assess different protein extraction methods followed by mass spectrometry-based proteomics to recover, separate and identify the proteins of the reproductive organs of medicinal cannabis, apical buds and isolated trichomes. Database search following shotgun proteomics was limited to protein sequences from C. sativa and closely related species available from UniprotKB. Our results demonstrate that a buffer containing the chaotrope reagent guanidine hydrochloride recovers many more proteins than a urea-based buffer. In combination with a precipitation with trichloroacetic acid, such buffer proved optimum to identify proteins using a trypsin digestion followed by nano-liquid chromatography tandem mass spectrometry (nLC-MS/MS) analyses. This is validated by focusing on enzymes involved in the phytocannabinoid pathway.

scholarly journals The Cannabis Proteome Draft Map Project

2020 ◽  
Vol 21 (3) ◽  
pp. 965 ◽  
Author(s):  
Conor Jenkins ◽  
Benjamin Orsburn
Keyword(s):  
Cannabis Sativa ◽  
Shotgun Proteomics ◽  
Protein Glycosylation ◽  
Lysine Acetylation ◽  
Proteomics Data ◽  
Phenotypic Characteristics ◽  
Plant Materials ◽  
Post Translational Modifications ◽  
Cannabis Indica ◽  
Genetic Profiles

Recently we have seen a relaxation of the historic restrictions on the use and subsequent research on the Cannabis plants, generally classified as Cannabis sativa and Cannabis indica. What research has been performed to date has centered on chemical analysis of plant flower products, namely cannabinoids and various terpenes that directly contribute to phenotypic characteristics of the female flowers. In addition, we have seen many groups recently completing genetic profiles of various plants of commercial value. To date, no comprehensive attempt has been made to profile the proteomes of these plants. We report herein our progress on constructing a comprehensive draft map of the Cannabis proteome. To date we have identified over 17,000 potential protein sequences. Unfortunately, no annotated genome of Cannabis plants currently exists. We present a method by which “next generation” DNA sequencing output and shotgun proteomics data can be combined to produce annotated FASTA files, bypassing the need for annotated genetic information altogether in traditional proteomics workflows. The resulting material represents the first comprehensive annotated protein FASTA for any Cannabis plant. Using this annotated database as reference we can refine our protein identifications, resulting in the confident identification of 13,000 proteins with putative function. Furthermore, we demonstrate that post-translational modifications play an important role in the proteomes of Cannabis flower, particularly lysine acetylation and protein glycosylation. To facilitate the evolution of analytical investigations into these plant materials, we have created a portal to host resources developed from our proteomic and metabolomic analysis of Cannabis plant material as well as our results integrating these resources.

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

Proteomes ◽  
2020 ◽  
Vol 8 (2) ◽  
pp. 13
Author(s):  
Delphine Vincent ◽  
Keith Savin ◽  
Simone Rochfort ◽  
German Spangenberg
Keyword(s):  
Search Engines ◽  
Cannabis Sativa ◽  
Protein Extraction ◽  
Shotgun Proteomics ◽  
Search Algorithms ◽  
Protein Digestion ◽  
Top Down ◽  
Model Species ◽  
Intact Proteins ◽  
Proteomics Experiment

Cannabis research has taken off since the relaxation of legislation, yet proteomics is still lagging. In 2019, we published three proteomics methods aimed at optimizing protein extraction, protein digestion for bottom-up and middle-down proteomics, as well as the analysis of intact proteins for top-down proteomics. The database of Cannabis sativa proteins used in these studies was retrieved from UniProt, the reference repositories for proteins, which is incomplete and therefore underrepresents the genetic diversity of this non-model species. In this fourth study, we remedy this shortcoming by searching larger databases from various sources. We also compare two search engines, the oldest, SEQUEST, and the most popular, Mascot. This shotgun proteomics experiment also utilizes the power of parallel digestions with orthogonal proteases of increasing selectivity, namely chymotrypsin, trypsin/Lys-C and Asp-N. Our results show that the larger the database the greater the list of accessions identified but the longer the duration of the search. Using orthogonal proteases and different search algorithms increases the total number of proteins identified, most of them common despite differing proteases and algorithms, but many of them unique as well.

scholarly journals The Cannabis Multi-Omics Draft Map Project

2019 ◽  
Author(s):  
Conor Jenkins ◽  
Ben Orsburn
Keyword(s):  
Cannabis Sativa ◽  
Shotgun Proteomics ◽  
Protein Glycosylation ◽  
Lysine Acetylation ◽  
Proteomics Data ◽  
Phenotypic Characteristics ◽  
Plant Materials ◽  
Post Translational Modifications ◽  
Cannabis Indica ◽  
Genetic Profiles

AbstractRecently we have seen a relaxation of the historic restrictions on the use and subsequent research on the Cannabis plants, generally classified as Cannabis sativa and Cannabis indica. What research has been performed to date has centered on chemical analysis of plant flower products, namely cannabinoids and various terpenes that directly contribute to phenotypic characteristics of the female flowers. In addition, we have seen many groups recently completing genetic profiles of various plants of commercial value. To date, no comprehensive attempt has been made to profile the proteomes of these plants. We report herein our progress on constructing a comprehensive draft map of the Cannabis proteome. To date we have identified over 17,000 potential protein sequences. Unfortunately, no annotated genome of Cannabis plants currently exists. We present a method by which “next generation” DNA sequencing output and shotgun proteomics data can be combined to produce annotated FASTA files, bypassing the need for annotated genetic information altogether in traditional proteomics workflows. The resulting material represents the first comprehensive annotated FASTA for any Cannabis plant. Using this annotated database as reference we can refine our protein identifications, resulting in the confident identification of 13,000 proteins with putative function. Furthermore, we demonstrate that post-translational modifications play an important role in the proteomes of Cannabis flower, particularly lysine acetylation and protein glycosylation. To facilitate the evolution of analytical investigations into these plant materials, we have created a portal to host resources we have developed from proteomic and metabolomic analysis of Cannabis plant material as well as our results integrating these resources. All data for this project is available to view or download at www.CannabisDraftMap.Org

scholarly journals The Cannabis Proteome Draft Map Project

2020 ◽  
Author(s):  
Conor Jenkins ◽  
Ben Orsburn
Keyword(s):  
Cannabis Sativa ◽  
Shotgun Proteomics ◽  
Protein Glycosylation ◽  
Lysine Acetylation ◽  
Proteomics Data ◽  
Phenotypic Characteristics ◽  
Plant Materials ◽  
Post Translational Modifications ◽  
Cannabis Indica ◽  
Genetic Profiles

Recently we have seen a relaxation of the historic restrictions on the use and subsequent research on the Cannabis plants, generally classified as Cannabis sativa and Cannabis indica. What research has been performed to date has centered on chemical analysis of plant flower products, namely cannabinoids and various terpenes that directly contribute to phenotypic characteristics of the female flowers. In addition, we have seen many groups recently completing genetic profiles of various plants of commercial value. To date, no comprehensive attempt has been made to profile the proteomes of these plants. We report herein our progress on constructing a comprehensive draft map of the Cannabis proteome. To date we have identified over 17,000 potential protein sequences. Unfortunately, no annotated genome of Cannabis plants currently exists. We present a method by which “next generation” DNA sequencing output and shotgun proteomics data can be combined to produce annotated FASTA files, bypassing the need for annotated genetic information altogether in traditional proteomics workflows. The resulting material represents the first comprehensive annotated protein FASTA for any Cannabis plant. Using this annotated database as reference we can refine our protein identifications, resulting in the confident identification of 13,000 proteins with putative function. Furthermore, we demonstrate that post-translational modifications play an important role in the proteomes of Cannabis flower, particularly lysine acetylation and protein glycosylation. To facilitate the evolution of analytical investigations into these plant materials, we have created a portal to host resources we have developed from proteomic and metabolomic analysis of Cannabis plant material as well as our results integrating these resources. All data for this project is available to view or download at www.CannabisDraftMap.Org

scholarly journals Long-Term Follow-Up of a Female Patient Treated with Olaparib—Hope for a Long Life without Relapse?

2021 ◽  
Vol 18 (7) ◽  
pp. 3430
Author(s):  
Mateusz Kozłowski ◽  
Katarzyna Nowak ◽  
Aneta Cymbaluk-Płoska
Keyword(s):  
Ovarian Cancer ◽  
Parp Inhibitor ◽  
Brca1 Gene ◽  
High Specificity ◽  
Reproductive Organs ◽  
Platinum Based Chemotherapy ◽  
Long Term Follow Up ◽  
History Of ◽  
Advanced Stages

Ovarian cancer is one of the most common cancers of the reproductive organs. As there are no symptoms in the early stages, it is mainly detected in the advanced stages. Even then, the symptoms are non-specific and include, for example, abdominal pain, early satiety, or changes in bowel habits. Both biochemical marker levels and imaging studies are used in the initial diagnosis. However, it should be emphasized that they are not characterized by high specificity. Treatment is multistage, and usually first-line debulking surgery is used followed by platinum-based chemotherapy. Here we present a clinical case of a 56-year-old female, a carrier of a mutation in the BRCA1 gene, with a history of breast cancer and with recurrent epithelial ovarian cancer. The patient was qualified for treatment with a PARP inhibitor and is currently undergoing treatment with olaparib. In the patient’s follow up of 50 months to date, there has been no recurrence of cancer. Few side effects have been observed, and the most serious one that can be effectively treated is anemia. On the basis of the described case, the authors concluded that olaparib treatment is effective, relatively safe, and does not significantly affect daily functioning.

scholarly journals Mass Spectrometry Based-Proteomic Analysis of Anisakis spp.: A Preliminary Study towards a New Diagnostic Tool

Genes ◽  
2020 ◽  
Vol 11 (6) ◽  
pp. 693 ◽  
Author(s):  
Valeria Marzano ◽  
Stefania Pane ◽  
Gianluca Foglietta ◽  
Stefano Levi Mortera ◽  
Pamela Vernocchi ◽  
...  
Keyword(s):  
Diagnostic Tool ◽  
Protein Extraction ◽  
Shotgun Proteomics ◽  
Diagnostic Tools ◽  
Routine Practice ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Proteomics Approach ◽  
Anisakis Spp ◽  
Tof Ms

Anisakiasis is nowadays a well-known infection, mainly caused by the accidental ingestion of Anisakis larvae, following the consumption of raw or undercooked fishes and cephalopods. Due to the similarity of symptoms with those of common gastrointestinal disorders, this infection is often underestimated, and the need for new specific diagnostic tools is becoming crucial. Given the remarkable impact that MALDI–TOF MS biotyping had in the last decade in clinical routine practice for the recognition of bacterial and fungi strains, a similar scenario could be foreseen for the identification of parasites, such as nematodes. In this work, a MALDI–TOF MS profiling of Anisakis proteome was pursued with a view to constructing a first spectral library for the diagnosis of Anisakis infections. At the same time, a shotgun proteomics approach by LC–ESI–MS/MS was performed on the two main fractions obtained from protein extraction, to evaluate the protein species enriched by the protocol. A set of MALDI–TOF MS signals associated with proteins originating in the ribosomal fraction of the nematode extract was selected as a potential diagnostic tool for the identification of Anisakis spp.

scholarly journals Generation of a Comprehensive Transcriptome Atlas and Transcriptome Dynamics in Medicinal Cannabis

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Shivraj Braich ◽  
Rebecca C. Baillie ◽  
Larry S. Jewell ◽  
German C. Spangenberg ◽  
Noel O. I. Cogan
Keyword(s):  
Gene Expression ◽  
Gene Expression Analysis ◽  
Developmental Stages ◽  
Cannabis Sativa ◽  
Glandular Trichomes ◽  
Present Knowledge ◽  
Differential Gene Expression Analysis ◽  
Medicinal Cannabis ◽  
Differential Gene ◽  
Different Tissues

Abstract Cannabinoids are the main medicinal compounds of interest in the plant Cannabis sativa, that are primarily synthesised in the glandular trichomes; found on female floral buds. The content, composition and yield of secondary metabolites (cannabinoids and terpenoids) is influenced by the plant’s genetics and environment. Some initial gene expression experiments have been performed from strains of this plant species that contrasted in cannabinoid production, however the present knowledge about detailed trichome transcriptomics in this species is limited. An extensive transcriptome atlas was generated by RNA sequencing using root, shoot, flower and trichome tissues from a female plant strain (Cannbio-2) and was enhanced with the addition of vegetative and reproductive tissues from a male cannabis plant. Differential gene expression analysis identified genes preferentially expressed in different tissues. Detailed trichomics was performed from extractions specifically from glandular trichomes as well as female floral tissues at varying developmental stages, to identify stage-specific differentially expressed genes. Candidate genes involved in terpene and cannabinoid synthesis were identified and the majority were found to have an abundant expression in trichomes. The comprehensive transcriptome is a significant resource in cannabis for further research of functional genomics to improve the yield of specialised metabolites with high pharmacological value.

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