Proteomic Characterization of Hempseed Reveals Healthful Properties

CannaNerd Alert: Proteomics is the large-scale study of proteins, particularly their structures and functions. Proteins are vital parts of living organisms, as they are the main components of the physiological metabolic pathways of cells. The term proteomics was first coined in 1997 to make an analogy with genomics, the study of the genome. The word proteome is a portmanteau of protein and genome, and was coined by Marc Wilkins in 1994 while working on the concept as a PhD student.

The proteome is the entire set of proteins, produced or modified by an organism or system. This varies with time and distinct requirements, or stresses, that a cell or organism undergoes. Proteomics is an interdisciplinary domain that has benefited greatly from the genetic information of the Human Genome Project; it is also emerging scientific research and exploration of proteomes from the overall level of intracellular protein composition, structure, and its own unique activity patterns. It is an important component of functional genomics.

"Proteomic characterization of hempseed (Cannabis Sativa L.)

Journal of Proteomics
31 May 2016
ACCEPTED MANUSCRIPT
Aiello, Fasoli, Boschin, Lammi, Zanoni, Citterio, and Arnoldi

Abstract. This paper presents an investigation on hempseed proteome. The experimental approach, based on combinatorial peptide ligand libraries (CPLLs), SDS-PAGE separation, nLC-ESI-MS/MS identification, and database search, permitted identifying in total 181 expressed proteins. This very large number of identifications was achieved by searching in two databases: Cannabis sativa L. (56 gene products identified) and Arabidopsis thaliana (125 gene products identified). By performing a protein-protein association network analysis using the STRING software, it was possible to build the first interactomic map of all detected proteins, characterized by 137 nodes and 410 interactions.

Finally, a Gene Ontology analysis of the identified species permitted to classify their molecular functions: the great majority is involved in the seed metabolic processes (41%), responses to stimulus (8%), and biological process (7%).

Biological significance: Hempseed is an underexploited non-legume protein-rich seed. Although its protein is known for its digestibility, essential amino acid composition, and useful techno-functional properties, a comprehensive proteome characterization is still lacking. The objective of this work was to fill this knowledge gap and provide information useful for a better exploitation of this seed.

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1. Introduction
Plant foods are gaining increasing popularity, owing to the growing public awareness of their better environmental sustainability in respect to animal foods. Numerous seeds are used in human nutrition, but current research is mainly focused on protein-rich ones, such as legumes. They provide high quality proteins as well as useful health benefits, particularly in the area of cardiovascular prevention [1-4]. In the meanwhile, however, other seeds are deserving increasing attention.

Hempseed, i.e. the seed of the non-drug cultivars of industrial hemp, is certainly an underexploited protein-rich seed. Although its use as human food dates back probably to prehistory, together with the fiber utilization in textiles, the cultivation of this plant has been banned for some decades in most developed countries, owing to the morphological similarity with marijuana. In fact, hemp and marijuana are both strains of the same species Cannabis sativa L. [5], although they are characterized by very different contents of Δ9-tetrahydrocannabinol (THC). The distinction between these populations, however, is not limited to the genes underlying THC production, since these plants are significantly differentiated at genome-wide level [5]. In the last years, however, the cultivation of the non-drug cultivars of industrial hemp is legal again in many countries and the global market is rapidly increasing, since well characterized low-THC cultivars are available (THC content < 200 mg/kg) [6]. Many factors favor the reintroduction of the cultivation of this plant: it is a multipurpose crop with numerous applications in different industrial sectors [7] and it is particularly sustainable, since it rarely requires irrigation and the fast growth reduces the use of herbicides.

The great interest for hempseed depends on its nutritional content (whole seed): 35.5% oil, 24.8% protein, 20-30% carbohydrates, 27.6% total fiber (5.4% digestible and 22.2% non-digestible fiber) and 5.6% ash [8]. Moreover, the concentration of the main antinutritional factors, such as phytic acid, condensed tannins, and trypsin inhibitors, is low [9]. Currently, the main industrial interest is for the oil that has numerous applications either in food or body care products, being rich in polyunsaturated fatty acids [9]. In the meanwhile, however, there is an increasing attention for hempseed protein owing to its digestibility [10], satisfactory essential amino acid composition [10], and techno-functional properties [11-13]. Moreover, hempseed protein has also potential applications in nutraceutics and functional foods, since the treatment with suitable enzymes permits to produce hydrolyzed proteins providing useful health benefits, as hypotensive agents [14-16] and antioxidants [14, 17, 18].

Literature indicates that a major protein in hempseed is edestin, a storage protein [12]. Edestin, a hexameric 11S protein, is easily digested and contains significant amounts of all essential amino acids [8], especially sulfur amino acids [19] and arginine [12].

Hempseed proteome has been investigated only once in the Korean cultivar Cheungsam [20]: the use of conventional techniques, such as 2-dimensional electrophoresis, mass spectrometry, and search in the nr-NCBI protein database permitted to identify 168 unique protein spots [20].

However, only one was assigned to C. sativa, whereas most of them were assigned to Medicago sativa, Oryza sativa or other plants. The current improved availability of genome information about C. sativa as well as the increasing interest for this seed prompted us to investigate again hempseed proteome. This research was performed on the seeds of the French cultivar Futura.

In order to get as much as possible a comprehensive information, combinatorial peptide ligand libraries (CPLLs) were used for protein equalization [21, 22]. This is a powerful and highly sensitive technique allowing access to low- and very-low-abundance proteins, which enlarges very much the possibility of protein identification. This technique has been applied, for example, to the analysis of the proteomes of donkey milk [23] and champagne [24], or to the detection of corn allergens [25].

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4.4. Conclusion
Taking advantage of high-throughput technologies, this work has provided very innovative information on hempseed proteome, since it was possible to identify in total 181 unique gene products, 56 specific protein species identified through the C. sativa database search and 125 protein species through the A. thaliana database search. This represents a main improvement in respect to available literature were all assignments were based on other plant databases [20]. In addition, it provides the first interactomic map of C. sativa, composed by a complex grid formed by 137 nodes connected via 410 interactions, useful to explore proteins relations and to understand the C. sativa biological pathways. The relevance of this work is underlined by the increasing interest for including hempseed ingredients in regular foods and, potentially, in functional ones. In fact, recent investigations have shown that peptides deriving from the gastrointestinal simulated digestion of hempseed proteins are characterized by useful antioxidant [16] and antihypertensive properties [15]."