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Proteomic Profiles of Whole Seeds, Hulls, and Dehulled Seeds of Two Industrial Hemp (Cannabis sativa L.) Cultivars

Department of Plant Production, Faculty of Agriculture and Technology, University of South Bohemia, 370 05 České Budějovice, Czech Republic
Mendel Centre of Plant Genomics and Proteomics, Central European Institute of Technology, Masaryk University, 625 00 Brno, Czech Republic
HEMP PRODUCTION CZ, Ltd., 262 72 Chraštice, Czech Republic
Department of Dairy, Fat and Cosmetics, University of Chemistry and Technology, 166 28 Prague, Czech Republic
Department of Food Biotechnology and Agricultural Products Quality, Faculty of Agriculture and Technology, University of South Bohemia, 370 05 České Budějovice, Czech Republic
Author to whom correspondence should be addressed.
Plants 2024, 13(1), 111;
Submission received: 20 November 2023 / Revised: 20 December 2023 / Accepted: 27 December 2023 / Published: 30 December 2023
(This article belongs to the Special Issue Spectra Analysis and Plants Research 2.0)


As a source of nutritionally important components, hemp seeds are often dehulled for consumption and food applications by removing the hard hulls, which increases their nutritional value. The hulls thus become waste, although they may contain valuable protein items, about which there is a lack of information. The present work is therefore aimed at evaluating the proteome of hemp (Cannabis sativa L.) at the whole-seed, dehulled seed, and hull levels. The evaluation was performed on two cultivars, Santhica 27 and Uso-31, using LC-MS/MS analysis. In total, 2833 protein groups (PGs) were identified, and their relative abundances were determined. A set of 88 PGs whose abundance exceeded 1000 ppm (MP88 set) was considered for further evaluation. The PGs of the MP88 set were divided into ten protein classes. Seed storage proteins were found to be the most abundant protein class: the averages of the cultivars were 65.5%, 71.3%, and 57.5% for whole seeds, dehulled seeds, and hulls, respectively. In particular, 11S globulins representing edestin (three PGs) were found, followed by 7S vicilin-like proteins (four PGs) and 2S albumins (two PGs). The storage 11S globulins in Santhica 27 and Uso-31 were found to have a higher relative abundance in the dehulled seed proteome (summing to 58.6 and 63.2%) than in the hull proteome (50.5 and 54%), respectively. The second most abundant class of proteins was oleosins, which are part of oil-body membranes. PGs belonging to metabolic proteins (e.g., energy metabolism, nucleic acid metabolism, and protein synthesis) and proteins related to the defence and stress responses were more abundant in the hulls than in the dehulled seeds. The hulls can, therefore, be an essential source of proteins, especially for medical and biotechnological applications. Proteomic analysis has proven to be a valuable tool for studying differences in the relative abundance of proteins between dehulled hemp seeds and their hulls among different cultivars.

1. Introduction

Hemp (Cannabis sativa L.), also known as industrial hemp, has long been cultivated as a source of solid and durable fibre in the stems, oily seeds, and active compounds useful in medicine [1,2]. Compared to Cannabis indica, most industrial hemp cultivars contain low levels of the drug tetrahydrocannabinol (THC), which has enabled an increase in its acreage in much of the world today. In addition to the valuable fibre, the seeds represent another important and economically exploitable component [3].
Hemp seeds are nutritionally very valuable; on average, whole seeds contain 25–35% fat, 20–28% protein, 25–37% carbohydrate (including fibre), 5–6% ash, and 4–8% moisture [1,3,4]. A large proportion of hemp seeds are used (mainly through cold pressing) to produce hemp oil, which is valued for its high content of polyunsaturated fatty acids. A number of papers [5,6,7,8,9] report a polyunsaturated fatty acid content of over 70% in the oil, mainly the essential n-6 linoleic acid (around 55%) and n-3 alpha-linolenic acid (12–18%) and minor levels of n-6 gamma-linolenic acid (1–3%) and n-3 stearidonic acid (<1%).
In addition to the oil, hemp seeds also contain easily digestible protein with a good amino acid composition, in which arginine plays an important role. Compared to soybean proteins, hemp seed proteins do not contain protease inhibitors in large amounts and are also considered less allergenic than proteins from other plant seeds [10]. Like the seeds of other dicotyledonous plants, hemp seed proteins are mainly represented by the globulin fraction (around 75%), with the rest being albumins [11,12].
Globulins are predominantly represented by the legumin-like 11S protein family [2,13], which, in hemp seed, is represented by edestin, which has a hexameric structure in its native state [12,14,15]. The edestin monomer has a molecular weight (MW) of 52–54 kDa and is composed of acidic (MW around 34 kDa) and basic (MW 20–25 kDa) polypeptides, which are linked together by a disulphide bond [12]. Edestin is a storage protein in the seed and is present in 60–80% of the total protein pool of hemp seeds [2,14]. The minor fraction of globulins is represented by the trimeric 7S protein (MW monomer 47-48 kDa), one of the vicilin-like proteins [4,16]. Albumin is mainly represented by a 10 kDa protein (2S albumin), which is 18% by weight of sulphur amino acids (cysteine, methionine) and, therefore, belongs to the group of sulphur-rich proteins [12].
Regarding the study of the protein profile of hemp seeds, several proteomic studies are available that deal with the analysis of proteins in whole seeds [17,18], in protein isolates [4,10], or in products after extrusion treatment [19]. Some studies [20,21,22] also present proteomic techniques as a tool suitable for verifying the authenticity of foods and their composition or assessing foods for the presence of allergens, which can be used to verify the addition of flours prepared from oilseeds (including hemp seeds) to food products. Most of the works [4,10,18,19,22] mentioned edestins or 11S globulins as major proteins in hemp seeds. Other proteins identified included proteins responsible for the biocatalysis of metabolic pathways, proteins related to nucleic acid and protein synthesis, proteins associated with membrane structures and functions, and stress proteins [17,18].
The above-mentioned studies [4,10,17,18,19] addressed protein number and identification using available databases but did not compare the relative abundance of identified items within the hemp seed protein pool. Moreover, in these studies, only one hemp cultivar was always used as the source of seed material, so information on varietal differences is lacking. Hemp seeds (more correctly, hemp achene) are dehulled for direct consumption and other food applications, which involves removing the hard, difficult-to-eat hull consisting of the pericarp and the seed coat layers, which contain primarily insoluble and indigestible fibre. On the other hand, dehulling increases the concentration of fat and protein in the dehulled seed [23]. It may thus alter the relative abundance of protein items in the dehulled seed compared to the whole (unhulled) seed. The hulls themselves can be finely ground, and the resulting powder (flour) can be used to fortify bakery, meat, or other food products with fibre and vegetable protein. However, the protein profiles of the hulls in terms of the presence and relative abundance of the different protein items have not been investigated, and information is completely lacking. Knowledge of the differences between the proteomes of the pericarp and the dehulled seed may be essential from the biological, nutritional, and technological points of view, as well as from the point of view of potential health risks and food safety.
Nowadays, the gel-free approach utilising LC-MS/MS analysis is the most efficient tool allowing the deep coverage of proteomes. The perspectives and potential of seed proteomics were recently reviewed by Smolikova et al. [24].
To increase the knowledge about the possibilities of using hemp seeds, dehulled seeds, and hulls as protein sources, the objectives of this study were (i) to perform proteomic characterisation at the level of whole seeds, dehulled seeds, and hulls; (ii) to evaluate the differences in the relative abundance of significant proteins and protein classes in these seed products in two industrial hemp cultivars.

2. Results and Discussion

2.1. Protein and Fat Contents in Original and Defatted Seed Product Samples

Protein and fat are the most valuable components of hemp seeds. As shown in Table 1, for both cultivars evaluated, Uso-31 and Santhica 27, the protein content of whole original seeds was found to be around 26–28% of dry matter (DM), and the fat content was around 29% of DM. The fat content of the whole seed corresponds to the usually reported data [1]; the protein content found is higher than or at the upper limit of the already-reported values of 20–25% [1] or 21.3–28.1% [3].
The dehulled original (non-defatted) seed has increased protein and fat contents, with values of 35% and 49%, respectively, in the two cultivars evaluated compared to the original whole seed (Table 1), which is roughly consistent with the literature reports [23,25]. In contrast, according to the literature data, significantly lower protein and fat contents were found in hulls, which have a high fibre content [25]. In the case of hulls, the effect of the cultivar on the separation of the hull from the rest of the seed seems to be more pronounced, as the reduction in the protein and fat contents of hulls relative to the whole seed does not occur in the same proportion in the two cultivars evaluated. In the case of the cultivar Uso-31, there was a reduction in the protein content of the hulls from 25.8 to 13.4% of DM (relative reduction to 51.9% content of original seed) and a reduction in the fat content from 29.4 to 6.2% (relative reduction to 21.1%). In the case of Santhica 27, there was a lower reduction in the protein and fat contents of the hulls, close to the values in the whole seed: the protein content was reduced from 27.7 to 19.2% (relative reduction to 69.3%), and the fat content was reduced from 28.7 to 15.4% (relative reduction to 53.7%).
For the defatted variants of the samples, which were subsequently subjected to detailed proteomic analysis, the fat content was reduced to below 1% of dry matter in almost all samples due to defatting with an organic solvent. The protein content increased in all samples due to defatting, with the dehulled seed samples showing an increase in protein content to above 65%, the value reported as the minimum protein content for protein concentrate products [26]. The increase in protein content is correlated with the original fat content removal. Thus, the highest growth in protein content was obtained in the dehulled seeds, from 35.4 to 71.7% of DM for the cultivar Uso-31 and from 35.6 to 68.5% of DM for the cultivar Santhica 27. Shen et al. [27] also observed increased protein content in flour and protein isolates prepared from dehulled seeds compared to the non-dehulled variants. However, they found only 41.8% protein content (Nx6.25) and 8.8% residual fat content (at 4.3% moisture content) for flour prepared from dehulled seeds after mechanical defatting, which is lower than our findings.
The results indicate that the mechanical separation of the hulls from hemp seeds is one of the two processes that significantly affect the production of hemp seed products with different primary nutrient contents. The second process should be defatting [28], either through mechanical pressing and fat extraction using an organic solvent or direct solvent use. Combining both processes (dehulling and defatting) can be essential to obtain concentrates with a higher protein content.

2.2. Characterisation of Seed Protein Pool

The essential features of the protein profiles of defatted seed, defatted hull, and dehulled seed samples are evident after one-dimensional SDS-PAGE (Figure 1). Four main zones of protein bands (A–D) are clear for all three types of samples of the two cultivars evaluated. However, the protein bands in the four zones mentioned above have different intensities. The A-zone region, with the weakest staining intensity, should represent a 7S vicilin-like protein with an MW of 47–48 kDa, similarly reported by several studies [12,29,30,31]. The most intense protein bands are those in zones B and C, which represent acidic (MW 30–35 kDa) and basic (MW 18–20 kDa) polypeptides of the 11S protein (edestin) monomer. The D zone proteins (MW 15 kDa and less) should represent the albumin fraction of the protein. The presence of edestin and the albumin fraction in SDS-PAGE gels has also been described by Liu et al. [29], Liu et al. [30], and Fang et al. [32].
Based on the evaluation of the proteomic data, 2833 protein groups (all belonging to Cannabis sativa L.) were identified, and their relative protein abundances in individual samples were determined and expressed in ppm. For further assessment of the protein pool, only protein groups (hereafter referred to as proteins or protein items) with a relative abundance greater than 1000 ppm (0.1%) in at least one of the three independent replicates for one of the three sample types (original or defatted whole seed, hulled seed, or hull) were considered. Eighty-eight of these major proteins were found (hereafter referred to as collection MP88). Their list, names, and primary data (molecular weight; the name of the corresponding protein family), including a more detailed characterisation of the proteins in the areas of biological process, molecular function, and cellular component, obtained from the UniProt KB database, are given in Table 2. Based on this detailed characterisation, the MP88 proteins were divided into ten protein classes: seed storage proteins (SP), oleosins (OL), other membrane components (MC), proteins involved in energy and metabolism (EM), translation-related proteins (TR), proteins related to DNA and RNA metabolism (DM), stress response and defence proteins (SD), proteins related to photosynthesis (PS), cytoskeleton and transport proteins (CT), and uncharacterised proteins (UP).
It should be noted that the number of proteins classified in these classes and their relative abundances in the samples of the three types reflect the condition of the maturing/mature seed. This state is associated with physiological processes involving the accumulation of di- and oligosaccharides, the synthesis of storage proteins, LEA proteins and heat-shock proteins, and the activation of antioxidant defences, in addition to the increasing dry matter content and the physical, structural changes in the cells [33]. In comparison to our experimental setup, Park et al. [17], in their study of the proteomic profiling of Cheungsam (Cannabis sativa L.) seeds using a combination of 2D PAGE and nano-LC-MS/MS, found 168 identified unique protein spots out of a total of 1102 spots resolved by the 2D PAGE separation of the extracted hemp seed protein mixture due to the lack of hemp protein sequences. Park et al. [17] used the database information of rice and Arabidopsis genomes, which had been completely sequenced at the time, to identify the proteins. The identified proteins were classified into 13 categories according to their function, as listed in the SWISS-PROT and NCBI databases.

2.2.1. Seed Storage Proteins

The SP class represents the most abundant group: seed storage proteins. The MP88 dataset included nine proteins. Three proteins (A0A7J6GWL5, A0A7J6DTA7, A0A7J6E205) were classified into the 11S family of seed storage proteins, commonly referred to as edestin in hemp seeds [12,14]; then, two proteins (A0A7J6H292, A0A7J6DXD1) were assigned to 2S albumins, and the remaining four proteins (A0A7J6H2R3, A0A7J6GLH5, A0A7J6HAT3, and A0A7J6G321) belong to the globulin family, and some of them belong to the 7S family (see Table 2). The names of these proteins do not directly imply that they are 7S proteins, but additional information found in the UniProt KB database suggests their similarity to 7S vicilin-like proteins. The occurrence of the three types of seed storage proteins mentioned above agrees with most papers that have investigated hemp seed proteins [10,16,22,34].
The main storage protein in hemp seeds is edestin. Docimo et al. [34], in their study on the molecular characterisation of the edestin gene family, found seven genes encoding seven forms of edestin, which can be divided into two types of edestin based on the sequence similarity—edestin 1 (includes four forms) and edestin 2 (involves three forms). In subsequent work [16], edestin type 3 was additionally found, which resembles type 1 more than type 2. The three above items of 11S proteins that we found are not directly identified as edestin in the UniProt KB database version used for database searching (they are named Cupin type-1 domain-containing protein) but show high sequence similarity to the edestins described in previous works [16,34]. The storage protein A0A7J6GWL5 has, according to the UniProt KB database, an MW of 109.15 kDa and is composed of a sequence of 953 amino acids, showing a close identity (99.8%) to edestin 1 (A0A090DLH8) in region 8-511, as reported in the UniProt database by, e.g., Mamone et al. [10] and Kotecka-Majchrzak et al. [22]. The second found 11S storage protein item (A0A7J6DTA7) has an MW of 52.03 kDa, is composed of 456 amino acids, and is consistent with forms of edestin 2—with item A0A090DLI7 at 98.1%, A0A090CXP9 at 97.8%, and A0A090CXP8 at 98.1%. The form of edestin 3 was not found in our experiment.

2.2.2. Proteins Involved in Energy and Metabolism

The class of proteins involved in energy and metabolism is represented by 15 proteins in the MP88 set. The most significantly represented items are those related to glycolysis, the tricarboxylic acid cycle (TCA), and other central metabolic processes, which are closely related to the physiological state of mature stored seed with preserved germination potential [33]. The importance of enzymes related to glycolysis, TCA, and the glyoxylate cycle for the metabolism of maturing oilseeds was also confirmed by Hajduch et al. [35].
Specifically, of the glycolytic enzymes, two glyceraldehyde-3-phosphate dehydrogenase items were represented in the MP88 set (items A0A7J6G6Z3 and A0A7J6HK40), in addition to triosephosphate isomerase (A0A7J6E4U9), fructose-bisphosphate aldolase (A0A7J6E5J2), and phosphopyruvate hydratase or enolase (A0A7J6GRW8). The TCA cycle is represented by two items of malate dehydrogenase (A0A7J6EZ77 and A0A7J6E8J3), and the glyoxylate cycle is represented by the enzyme malate synthase (A0A7J6FNP6).
Other important items with catalytic activity are involved in protein modification (protein disulfide-isomerase (A0A7J6EJG0) and peptidyl-prolyl cis-trans isomerase (A0A7J6EG53)), nucleotide metabolism (nucleoside-diphosphate kinase (A0A7J6HKH5)), etc. Other enzymes are related to electron and proton transfer in energy metabolism, e.g., NADH-cytochrome b5 reductase (A0A7J6HQA0) and ATP synthase (A0A0M5M1Z3). Due to their membrane localisation, these enzymes could also be classified as membrane components. Similarly, Park et al. [17] reported that the groups of proteins and enzymes related to basic metabolism and energy production in hemp seeds are the most numerous and diverse group of proteins. Their dataset also mainly lists enzymes of glycolysis, enzymes of the TCA cycle, and other important metabolic pathways.

2.2.3. Membrane Proteins

The representation of membrane proteins, which include two classes, namely, oleosins (OL) and other membrane components (MC), is significant. The OL are proteins involved in the structure of oil-body membranes called oleosomes [36]. In hemp seed cells, these are droplet-like structures (droplets) with a diameter of 3–5 µm, in whose membranes the membrane-specific oleosin proteins play an important structural role, with an estimated MW of ≈15 kDa [37]. In addition to oleosins, caleosins and steroleosins have also been reported as fat-body membrane proteins [38]. In the group of major proteins, three oleosin items (A0A7J6EJ89, A0A7J6F0Y4, A0A7J6H4F6) were found with MWs ranging from 15.41 to 17.36 kDa, which together represent about 8% of hemp seed proteins. The second highest relative abundance of oleosins in the hemp seed protein pool (after seed storage proteins) is thus closely related to the high fat accumulation in hemp seed tissue. Among the items of the MC class, which is represented by eight proteins, one can find a caleosin protein with peroxygenase activity (A0A7J6F280) and, e.g., aquaporin (A0A7J6GSC3), which functions as a transmembrane channel. Most of the other proteins are designated as uncharacterised proteins.

2.2.4. Proteins Involved in Stress Response and Defence

A total of 14 items were found in the MP88 dataset that can be classified as SD. These include LEA (late embryogenesis abundant) proteins (A0A7J6FL33, A0A7J6I6U6), including dehydrins (A0A7J6FXL4) as well as heat-shock proteins (A0A7J6GTA4, A0A7J6G382) and proteins associated with antioxidant protection against ROS (Reactive Oxygen Species), such as catalase (A0A7J6F3P9), peroxiredoxin (A0A7J6DT98), dehydroascorbate reductase (A0A7J6GJC9), glutaredoxin domain-containing protein (A0A7J6HTR6), and lactoylglutathione lyase or glyoxylase I (A0A7J6FPH5).
From the spectrum of stress-related proteins listed above, it is evident that the maturing/ripe seed and the developing/developed embryo must cope with progressive desiccation, temperature changes, and oxidative stress or oxygen deficiency. The occurrence of alcohol dehydrogenase in plants has been linked to oxygen deficiency.
Regarding seed defence, the occurrence of the enzyme rRNA N-glycosidase (A0A7J6DVP5) is significant. This enzyme generally belongs to the group of ribosome-inactivating proteins (RIPs), which are toxic because they inhibit proteosynthesis and are among the defence proteins [39]. Despite its toxicity, this group of proteins has the potential for possible applications in medicine or agriculture [40].
Park et al. [17] found, in their work on mature industrial hemp seeds, a similar spectrum of stress- or defence-related proteins—heat-shock proteins, glyoxylate reductase, dehydroascorbate reductase, alcohol dehydrogenase, and peroxiredoxin; in addition, they reported superoxide dismutase, which was also found in our samples, not in the MP88 set but in the set of all proteins. Aiello et al. [18] reported only Glyoxalase I and several variants of heat-shock proteins in their work focused on the proteomic characterisation of hemp seeds.

2.2.5. Other Protein Classes

Common cellular proteins include those involved in nucleic acid metabolism and subsequent protein synthesis during translation. The items included in the DM class are mainly represented by histone protein subunits (A0A7J6E9G4, A0A7J6E6Z7, A0A7J6E9K3). The TR class has the most significant number of items in the set of major proteins, as well as the EM class; in most cases (20 out of 24 items), these are ribosome subunits, e.g., two items of 60S ribosomal protein (A0A7J6GTP8, A0A7J6E9P9), as well as 40S (A0A7J6GWW0) and 50S (A0A7J6H424) ribosomal proteins and several other proteins involved in translation, e.g., elongation factor 1-alpha (A0A7J6HCW3) or EF1_GNE domain-containing protein (A0A7J6HK4).
The chloroplastic chlorophyll a-b-binding protein represents the PS class, and the CT class is represented by a protein related to intracellular sterol transport and by a protein from the actin family. Six items listed in the database had no characteristics and were therefore assigned to the UP class.

2.3. Effect of Cultivar and Dehulling on the Hemp Seed Protein Profile

The relative abundance of protein items in the MP88 set in the total protein profiles of whole seeds, hulls, and dehulled seeds with respect to the two cultivars studied (Uso-31, Santhica 27) is shown in Table 3. The percentages of the protein classes (including the group of proteins not included in the MP88 set) are expressed using pie charts in Figure 2.
The presented results (Table 3) confirmed the assumption that the hemp seed’s main class of proteins is SP, especially the 11S globulin edestin. The relative abundance of 11S globulins (summed over the three MP88 items) in whole seeds was 58.6 and 63.2% for the Santhica 27 and Uso-31 cultivars, respectively, when expressed as percentages, approximately corresponding to the literature-reported range of 60–80% [2,14]. Liu et al. [29] reported a range of relative edestin proportions of 57–76% when studying nine protein isolates prepared from seeds of different Cannabis sativa cultivars/genotypes, but this is the relative representation of edestin among the storage proteins (11S, 7S, and 2S), not the representation of edestin in the overall protein profile of hemp seeds. All three 11S protein items (A0A7J6GWL5, A0A7J6DTA7, A0A7J6E205) of the MP88 set were found to have a conclusively (except in one case) higher relative abundance in hulled seeds than in hulls. Considering the accumulation of reserve compounds in the inner seed part, this finding confirms the reported role of 11S proteins and edestins as seed reserve proteins [2,14]. The finding is also interesting concerning the potential food applications of hemp seed proteins, as edestin is their most valuable component [34].
The protein items A0A7J6H2R3, A0A7J6GLH5, A0A7J6HAT3, and A0A7J6G321, which could be classified as 7S proteins or vicilin-like proteins, represent about 3% of the seed protein pool in whole seeds of both cultivars, which is significantly less than that reported by other authors: Sun et al. [4] reported 5% and Liu et al. [29] reported a range of 9–19% (but here, as mentioned above, it is the proportion of storage proteins). In contrast to the 11S proteins, the relative abundance of 7S proteins was inconclusively higher in the hulls than in the dehulled seeds in all cases (see Table 3).
The third group of storage proteins is represented by the 2S proteins (A0A7J6H292, A0A7J6DXD1), which are classified as albumins. Collectively, their representation in the whole seed was only 2.04% (cv. Uso-31) or 1.61% (cv. Santhica 27), which is lower than the values published by other authors: Sun et al. [4] reported 13%, and Liu et al. [29] reported a range of 12–24% within a set of nine cultivars (here again, the values are proportions of storage proteins). The predominance of 2S albumins in the hulls or dehulled seeds is not at all clear.
The second most represented group of SP is the class of oleosins. Because the inner part of the seed coat accumulates more oil as a natural energy reserve, one would expect a higher representation of oleosins in dehulled seeds as key proteins that are part of the fat bodies, in which fat is stored in the cells. The results showed that the total oleosins increased with dehulling, from 8.2 (WS) to 9.4% (DS) and from 7.5 (WS) to 8.9 (DS) in Uso-31 and Santhica 27, respectively. However, this is due to the presence of the oleosin A0A7J6EJ89, which is significantly higher in the dehulled seeds than in the hulls of both cultivars. The other two proteins (A0A7J6F0Y4, A0A7J6H4F6) have no clear differences in the different parts of the seed.
Either way, the oleosin protein fraction is important in food applications as a natural emulsification system [36,37,38] or as a lipid-based delivery system in the food industry or in cosmetics and pharmaceuticals [39,40].
The protein classes EM, SD, DM, TR, and CT are collectively more abundant in the hulls (proteins of some classes are even significantly more abundant) than in the hulled seeds, which generally indicates that higher metabolic activity is taking place in the hull cells. This can be explained by the function of the hull tissues, i.e., the pericarp and seed coat layers, which form the natural barrier of the hemp achene and are thus forced to react more strongly to environmental stimuli, particularly abiotic stressors. Most of the proteins classified as SD in the MP88 dataset have a higher relative abundance in the hull than in the dehulled seed, except for dehydrin (A0A7J6FXL4), glutaredoxin domain-containing protein (A0A7J6HTR6), and dehydroascorbate reductase (A0A7J6GJC9). N-glycosidase rRNA (A0A7J6DVP5) is significantly more abundant in hulls (8-fold in cv. Santhica 27, 30-fold in cv. Uso-31) than in hulled seeds. This finding confirms the function of tRNA N-glycosidase as a defence protein [39]. The higher abundance in the hull predisposes the hull to be a potential source of this enzyme for biomedical applications. The RIP proteins have been found to have important biological properties, such as anticancer, antiviral, and neurotoxic activities [40,41]. Both chlorophyll-binding proteins (A0A7J6GUI2, A0A7J6GSM8) were significantly overrepresented in the hulls (compared to dehulled seeds) of both cultivars, which can be explained by the fact that chlorophyll is mainly found in the innermost layer of the seed coat, which is part of the hull [42].
Dehulling is an important technological process that removes the hard hull of the hemp seed, allowing the seeds to be consumed directly and used more widely in food products. Removing the hull eliminates or reduces the contents of antinutrients, improves the sensory properties of the dehulled seeds, and enhances their digestibility [42,43]. Previous work [27,43,44], and our presented results, have confirmed that dehulling can also be an important technological step in producing hemp seed protein concentrates and isolates, while this step removes big amounts of ballast substances (especially fibre components). At the same time, the proteomic results indicate the importance of hemp seed hulls as a source of a specific protein. The hulls often represent only the waste from hemp seed processing, while it could be a source of a large spectrum of proteins or as a raw material for a fibre flour product. On the other hand, the finding of a higher abundance of N-glycosidase rRNA also points to the possible risks of using products from the hulls for (direct) human consumption or the fact that the cultivar itself and its selection may play a major role in the occurrence of risk proteins.

3. Materials and Methods

3.1. Hemp Seed Samples and Their Preparation

The seeds of two cultivars of industrial hemp (Cannabis sativa L.) with low THC content (up to 0.2%), Uso-31 (registered in EUPVP—Common Catalogue; National Listing Netherlands; variety ID: 214015) and Santhica 27 (registered in EUPVP—Common Catalogue; National Listing France; variety ID: 213998), from the 2020 harvest were obtained from Hemp Production CZ, Ltd. Three variants of samples from both cultivars were provided for analysis: whole hemp seeds, seed hulls and dehulled hemp seeds. Hemp seeds were dehulled using dehulling and separating equipment (TFYM 1000; Liaoning Qiaopai Machineries Co., Ltd., Jinzhou, China). Before analyses, the samples were disintegrated using a Grindomix GD200 knife mill (Retsch, Haan, Germany) at 10,000 rpm for 1 min. The ground samples were then subjected to defatting using the organic solvent n-hexane. To the sample powder in the plastic tube, n-hexane was added at a ratio of 1:3 (w/v), and after mixing and extraction for 2 h at room temperature (with shaking of the sample mixture every 30 min), centrifugation (rpm 4500, 10 min, 20 °C) was performed, the solvent was carefully removed from the tube, and then the whole fat extraction process was repeated twice. After defatting, the sample pellets were left free to dry in the laboratory fume hood. Three defatted sample variants—defatted whole seeds (WS), defatted hulls (H), and defatted dehulled seeds (DS)—were obtained by the above-described treatment.

3.2. Dry Matter, Protein, and Fat Contents

Dry matter, protein, and fat contents were determined for the original (with fat) and defatted versions of the hulls, whole seeds, and dehulled seeds. The dry matter content was determined gravimetrically by drying the samples at 105 °C for 3 h in an oven to constant weight. The protein content was determined using the modified Dumas combustion method using a rapid N cube (N/Protein analysis) instrument (Elementar Analysen System, Langenselbold, Germany). Each sample was analysed in triplicate, and the protein content was calculated as the nitrogen content multiplied by a factor of 6.25. The fat content was measured using the Soxhlet extraction method using an ANKOM XT 10 Extractor (ANKOM Technology, Macedon, USA), according to the manufacturer’s manual. Petroleum ether was used as an extraction reagent. The fat content was calculated from the weight differences in the sample before and after extraction.

3.3. Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis

The defatted sample variants were extracted with SDS extraction buffer (0.065 M Tris-HCl, pH 6.8; 2% (w/v) SDS; 5% (v/v) 2-sulphanylethanol) in a ratio of 1:10 (w/v) at 4 °C for 4 h. Protein separation was carried out in triplicate using cooled dual vertical slab units (SE 600; Hoefer Scientific Instruments, Holliston, MA, USA) with a discontinuous gel system (4% stacking and 12% resolving gel) in reducing conditions [45]. Protein detection was performed by using Coomassie Brilliant Blue R-250.

3.4. LC-MS/MS Analysis

Proteins for LC-MS/MS analysis were extracted in SDT buffer (4% SDS, 0.1M DTT, 0.1M Tris/HCl, pH 7.6) in a thermomixer (Eppendorf ThermoMixer C, 60 min, 95 °C, 750 rpm). After that, all samples were centrifuged (15 min, 20,000× g), and the supernatants (ca. 100 μg of total protein) were used for filter-aided sample preparation (FASP), as described elsewhere [46], using 0.75 μg of trypsin (sequencing grade; Promega). Proteins were digested overnight (18 h) at 37 °C. The resulting peptides were analysed using LC-MS/MS.
LC-MS/MS analyses of all peptides were performed using an UltiMate 3000 RSLCnano system connected to an Orbitrap Exploris 480 spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Prior to LC separation, tryptic digests were concentrated and desalted online using a trapping column (Acclaim PepMap 100 C18, 300 μm ID, 5 mm long, 5 μm particles, Thermo Fisher Scientific). After washing the trapping column with 0.1% FA, the peptides were eluted in backflush mode (flow 500 nL·min−1) from the trapping column onto an analytical column (EASY-Spray column, 75 μm ID, 250 mm long, 2 μm particles, Thermo Fisher Scientific), where peptides were separated using a 90 min gradient program (flow rate 300 nL·min−1, 3–37% of mobile phase B; mobile phase A: 0.1% FA in water; mobile phase B: 0.1% FA in 80% ACN). Both columns were heated to 40 °C.
MS data were acquired in a data-dependent strategy (cycle time 2 s). The survey scan range was set to m/z 350–2000 with a resolution of 120,000 (at m/z 200), a normalised target value of 250%, and a maximum injection time of 500 ms. HCD MS/MS spectra (isolation window m/z 1.2, 30% relative fragmentation energy) were acquired from m/z 120 with a relative target value of 50% (intensity threshold 5 × 103), a resolution of 15,000 (at m/z 200), and a maximum injection time of 50 ms. Dynamic exclusion was enabled for 45 s.

3.5. Proteomic Data Processing

For data processing, we used MaxQuant software (v2.0.3.0) [47] with an inbuilt Andromeda search engine [48]. A search was performed against protein databases of Cannabis sativa (30,194 protein sequences, version from 24 February 2022, downloaded from, accessed on 1 January 2020) and cRAP contaminants (112 sequences, version from 22 November 2018, downloaded from Modifications were set for the database search as follows: oxidation (M), deamidation (N, Q), and acetylation (Protein N-term) as variable modifications, with carbamidomethylation (C) as a fixed modification. Enzyme specificity was tryptic with two permissible missed cleavages. Only peptides and proteins with a false discovery rate threshold under 0.01 were considered. Relative protein abundance was assessed using protein intensities calculated using MaxQuant. The intensities of reported proteins were further evaluated using a software container environment (; version 4.1.3a). The processing workflow is available upon request, and it covers, in short, reverse hits and contaminant protein group (cRAP) removal, protein group intensities’ log2 transformation, and normalisation (loessF). For the purpose of this article, protein groups reported by MaxQuant are referred to as proteins or protein items.

3.6. Statistical Analysis

The program Statistica 12 (StatSoft Power Solutions Inc., Palo Alto, CA, USA) was used for the data analysis. Data were subjected to analyses of variance using the two-way ANOVA method, and the means were compared using the Tukey HSD test. Differences between the variants were considered significant at p < 0.05 unless stated otherwise.

4. Conclusions

In total, 2833 proteins were identified in this proteomic study of the whole seeds, hulls, and dehulled seeds of two industrial hemp (Cannabis sativa L.) cultivars. Of this number, only 88 proteins accounted for 81.5–91.4% of the relative quantity of total proteins. The proteins within this set, reflecting the physiological state of the mature and stored seeds, were classified into 10 classes according to molecular and biological functions. According to the literature, we confirmed most of all three types of storage proteins—11S (edestin), 7S (vicilin-like proteins) globulins, and 2S albumins. As expected, the 11S storage globulins were found in increased abundance, mainly in the dehulled seeds; in contrast, 7S globulins were more abundant in the hulls, and 2S albumins were ambiguously represented within the two cultivars evaluated.
The relative quantification of the data revealed that the second most abundant protein class (next to storage proteins) included oleosins as key proteins of the oil-body membranes. Metabolically important classes of proteins (e.g., proteins related to energy acquisition, nucleic acid metabolism, or protein synthesis) and proteins that are part of defence mechanisms or stress responses are more abundant in the hulls. The hulls of the hemp seed can thus be an important source of valuable proteins for use in food, medical, or biotechnological applications.
Although protein samples of two cultivars were evaluated, it became very clear that the cultivar is an important factor in the relative abundance of proteins, and the selection of a suitable cultivar will be important not only in terms of hemp cultivation as a field crop and in terms of seed-to-oil processing but also in the utilisation of the protein component of the seed.

Author Contributions

Conceptualisation, J.B. (Jan Bárta); performance of the research work, analysis, and data interpretation, J.B. (Jan Bárta), P.R., M.J., Z.Z., A.S., V.B., Z.K., J.K., F.L., J.B. (Jan Bedrníček) and P.S.; writing—original draft preparation, J.B. (Jan Bárta) and V.B.; writing—review and editing, J.B. (Jan Bárta), V.B. and F.L.; supervision, J.B. (Jan Bárta) and Z.Z.; project administration and funding acquisition, J.B. (Jan Bárta), V.Ř., V.F. and Z.Z. All authors have read and agreed to the published version of the manuscript.


This work was supported by the Ministry of Agriculture of the Czech Republic (Project No. NAZV QK 1910302). The work was also supported by the Ministry of Education, Youth and Sports of CR from the European Regional Development Fund-Project “SINGING PLANT” (No. CZ.02.1.01/0.0/0.0/16_026/0008446). CIISB, Instruct-CZ Centre of Instruct-ERIC EU consortium, funded by MEYS CR infrastructure project LM2023042, is gratefully acknowledged for the financial support of the measurements taken at the CEITEC Proteomics Core Facility. Computational resources were provided by the e-INFRA CZ project (ID:90254), supported by the Ministry of Education, Youth and Sports of the Czech Republic.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Conflicts of Interest

Authors Zlata Krejčová and Václav Říha were employed by the company HEMP PRODUCTION CZ, Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


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Figure 1. SDS-PAGE profiles of evaluated defatted products derived from seeds of two hemp cultivars under reducing conditions (WS—defatted flour from whole seeds; H—defatted flour from milled hulls; DS—defatted flour from dehulled hemp seeds, M—protein molecular weight standard ROTI®Mark Tricolor Xtra, Carl Roth GmbH + Co. KG, Karlsruhe, Germany).
Figure 1. SDS-PAGE profiles of evaluated defatted products derived from seeds of two hemp cultivars under reducing conditions (WS—defatted flour from whole seeds; H—defatted flour from milled hulls; DS—defatted flour from dehulled hemp seeds, M—protein molecular weight standard ROTI®Mark Tricolor Xtra, Carl Roth GmbH + Co. KG, Karlsruhe, Germany).
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Figure 2. Pie charts of protein class sum proportions (in %) derived from relative abundance values in evaluated hempseed materials (WS—defatted flour from whole seeds; H—defatted flour from milled hulls; DS—defatted flour from dehulled hemp seeds).
Figure 2. Pie charts of protein class sum proportions (in %) derived from relative abundance values in evaluated hempseed materials (WS—defatted flour from whole seeds; H—defatted flour from milled hulls; DS—defatted flour from dehulled hemp seeds).
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Table 1. Dry matter, protein, and fat contents in evaluated hemp seed samples (mean ± standard deviation).
Table 1. Dry matter, protein, and fat contents in evaluated hemp seed samples (mean ± standard deviation).
MaterialCultivarVariantDry Matter Content (% FM)Protein Content (% DM)Fat Content (% DM)
OriginalUso-31whole seeds92.01 ± 0.07 b25.79 ± 1.68 e29.40 ± 0.87 b
hulls91.57 ± 0.13 bc13.40 ± 0.16 h6.19 ± 0.19 d
dehulled seeds94.84 ± 0.07 a35.35 ± 0.07 d49.07 ± 0.41 a
Santhica 27whole seeds91.39 ± 0.05 bc27.74 ± 1.70 e28.65 ± 0.63 b
hulls91.39 ± 0.86 bc19.20 ± 0.62 g15.43 ± 0.92 c
dehulled seeds93.87 ± 0.62 a35.61 ± 0.31 cd49.34 ± 0.79 a
DefattedUso-31whole seeds89.96 ± 0.17 d37.85 ± 0.83 c1.18 ± 0.26 e
hulls91.19 ± 0.09 bc17.08 ± 0.18 g0.05 ± 0.09 e
dehulled seeds91.38 ± 0.25 bc71.74 ± 0.15 a0.27 ± 0.15 e
Santhica 27whole seeds89.76 ± 0.06 d35.83 ± 0.89 cd0.64 ± 0.13 e
hulls90.63 ± 0.20 cd21.79 ± 0.21 f0.23 ± 0.19 e
dehulled seeds90.09 ± 0.25 d68.50 ± 0.44 b0.04 ± 0.04 e
DM—dry matter; FM—fresh matter; Different letters in columns indicate the statistically significant difference at p < 0.05 (Tukey HSD test).
Table 2. Description of evaluated proteins (information from UniProt KB database) included in MP88 collection.
Table 2. Description of evaluated proteins (information from UniProt KB database) included in MP88 collection.
AccessionProtein NameMol. Mass (Da)Protein FamiliesBiological Process—Molecular Function—Cellular Component
Seed storage proteins (SP)
A0A7J6GWL5Cupin type-1 domain-containing protein109,15111S seed storage protein—globulins familyx—nutrient reservoir activity—x
A0A7J6DTA7Cupin type-1 domain-containing protein52,03111S seed storage protein—globulins familyx—nutrient reservoir activity—x
A0A7J6E205Cupin type-1 domain-containing protein62,38911S seed storage protein—globulins familyx—nutrient reservoir activity—membrane
A0A7J6H2R3Cupin type-1 domain-containing protein108,332 x—x—membrane
A0A7J6GLH5Cupin type-1 domain-containing protein108,083 x—x—membrane
A0A7J6HAT3Cupin type-1 domain-containing protein 25,314 x—x—x
A0A7J6G321Cupin type-1 domain-containing protein55,360 x—x—x
A0A7J6H292Bifunctional inhibitor/plant lipid transfer protein/seed storage helical domain-containing protein16,7522S seed storage albumins family; Plant LTP familyx—lipid binding; nutrient reservoir activity—x
A0A7J6DXD1Bifunctional inhibitor/plant lipid transfer protein/seed storage helical domain-containing protein17,5582S seed storage albumins family; Plant LTP familyx—lipid binding; nutrient reservoir activity—x
Oleosins (OL)
A0A7J6EJ89Oleosin 15,410Oleosin familyreproductive process; post-embryonic development—x—membrane; monolayer-surrounded lipid storage body
A0A7J6F0Y4Oleosin 17,355Oleosin familyreproductive process; post-embryonic development—x—membrane; monolayer-surrounded lipid storage body
A0A7J6H4F6Oleosin 16,884Oleosin familyreproductive process; post-embryonic development—x—membrane; monolayer-surrounded lipid storage body
Other membrane components (MC)
A0A7J6I425Verticillium wilt resistance-like protein124,317Receptor-like protein familyx—x—plasma membrane
A0A7J6F280Peroxygenase27,243Caleosin familyx—x—membrane
A0A7J6G6U3Uncharacterized protein 71,299 x—oxidoreductase activity—membrane
A0A7J6DNR1Uncharacterized protein 64,236 x—x—membrane
A0A7J6DPR7Uncharacterized protein 20,132 x—x—membrane
A0A7J6ENC9Uncharacterized protein 19,908 x—transmembrane transporter activity—membrane
A0A7J6GSC3Aquaporin TIP3-227,504MIP/aquaporin (TC 1.A.8) familyx—channel activity—membrane
A0A7J6I7S9Uncharacterized protein 41,884 x—x—membrane
Proteins involved in energy and metabolism (EM)
A0A7J6G6Z3Glyceraldehyde 3-phosphate dehydrogenase NAD(P) binding domain-containing protein 31,874Glyceraldehyde-3-phosphate dehydrogenase familyx—NAD binding; oxidoreductase activity, acting on the aldehyde or oxo group of donors, NAD or NADP as acceptor —x
A0A7J6E4U9Triose-phosphate isomerase30,435Triosephosphate isomerase familyglycolytic process—triose-phosphate isomerase activity—x
A0A7J6EFG0Uncharacterized protein 46,262 x—oxidoreductase activity—x
A0A7J6EJG0Protein disulfide-isomerase 56,660Protein disulfide isomerase familyx—protein disulfide isomerase activity—endoplasmic reticulum lumen
A0A7J6E5J2Fructose-bisphosphate aldolase 38,370Class I fructose-bisphosphate aldolase familyglycolytic process—fructose-bisphosphate aldolase activity—x
A0A7J6EZ77Malate dehydrogenase 36,570LDH/MDH superfamily, MDH type 1 familymalate metabolic process; tricarboxylic acid cycle—L-malate dehydrogenase activity—x
A0A7J6HK40Glyceraldehyde 3-phosphate dehydrogenase NAD(P) binding domain-containing protein31,950Glyceraldehyde-3-phosphate dehydrogenase familyx—NAD binding; oxidoreductase activity, acting on the aldehyde or oxo group of donors, NAD or NADP as acceptor—x
A0A7J6GRW8Phosphopyruvate hydratase 46,384Enolase familyglycolytic process—magnesium ion binding; phosphopyruvate hydratase activity—phosphopyruvate hydratase complex
A0A7J6EG53Peptidyl-prolyl cis-trans isomerase 18,171Cyclophilin-type PPIase familyprotein folding; protein peptidyl-prolyl isomerization—peptidyl-prolyl cis-trans isomerase activity—x
A0A7J6FNP6Malate synthase 63,098Malate synthase familyglyoxylate cycle; tricarboxylic acid cycle—malate synthase activity—glyoxysome
A0A7J6E8J3Malate dehydrogenase 36,478LDH/MDH superfamily, MDH type 2 familymalate metabolic process; tricarboxylic acid cycle; malate metabolic process; tricarboxylic acid cycle—L-malate dehydrogenase activity—membrane
A0A7J6DST1NADP-dependent oxidoreductase domain-containing protein38,559Aldo/keto reductase familyx—oxidoreductase activity—x
A0A7J6HKH5Nucleoside-diphosphate kinase 16,290NDK familyCTP biosynthetic process; GTP biosynthetic process; UTP biosynthetic process—nucleoside diphosphate kinase activity—x
A0A7J6HTX3Tyrosinase copper-binding domain-containing protein66,851Tyrosinase familypigment biosynthetic process—catechol oxidase activity; metal ion binding—x
A0A7J6HQA0NADH-cytochrome b5 reductase 31,100Flavoprotein pyridine nucleotide cytochrome reductase familyx—cytochrome-b5 reductase activity, acting on NAD(P)H—membrane
Stress response and defence proteins (SD)
A0A7J6DVP5rRNA N-glycosylase 28,760Ribosome-inactivating protein familydefence response; negative regulation of translation—rRNA N-glycosylase activity; toxin activity—x
A0A7J6FXL4Dehydrin29,312Plant dehydrin familyresponse to abscisic acid; response to cold; response to water deprivation—metal ion binding—x
A0A7J6I6U6Uncharacterized protein 59,963LEA type 4 familyx—x—x
A0A7J6G382Uncharacterized protein 17,523Small heat shock protein (HSP20) familyresponse to stress—x—x
A0A7J6HZ01Annexin 36,065Annexin familyresponse to stress—calcium ion binding; calcium-dependent phospholipid binding—x
A0A7J6I4E918 kDa seed maturation protein15,710LEA type 1 familyembryo development ending in seed dormancy—x—x
A0A7J6FL33Late embryogenesis abundant protein D-2930,417 x—x—x
A0A7J6GTA4SHSP domain-containing protein 17,897Small heat shock protein (HSP20) familyx—x—x
A0A7J6FPH5Lactoylglutathione lyase 32,764Glyoxalase I familyx—lactoylglutathione lyase activity; metal ion binding—x
A0A7J6DT98Peroxiredoxin 24,133Peroxiredoxin family, Prx6 subfamilyx—thioredoxin-dependent peroxiredoxin activity—x
A0A7J6GJC9Dehydroascorbate reductase23,719GST superfamily, DHAR familyascorbate glutathione cycle—glutathione dehydrogenase (ascorbate) activity; glutathione transferase activity—x
A0A7J6F3P9Catalase 57,402Catalase familyhydrogen peroxide catabolic process; response to oxidative stress—catalase activity; heme binding; metal ion binding—x
A0A7J6HTR6Glutaredoxin domain-containing protein 14,424Glutaredoxin family, CPYC subfamilyx—x—x
A0A7J6FRN5Alcohol dehydrogenase41,112Zinc-containing alcohol dehydrogenase familyx—oxidoreductase activity; zinc ion binding—x
Proteins related to DNA and RNA metabolism (DM)
A0A7J6EYT6PPC domain-containing protein 30,708 x—minor groove of adenine-thymine-rich DNA binding—x
A0A7J6E9G4Histone H4 11,409Histone H4 familyx—DNA binding; protein heterodimerization activity; structural constituent of chromatin—nucleosome; nucleus
A0A7J6E6Z7Histone H2A 15,148Histone H2A familyx—DNA binding; protein heterodimerization activity; structural constituent of chromatin—nucleosome; nucleus
A0A7J6E9K3Histone H2B 16,235Histone H2B familyx—DNA binding; protein heterodimerization activity; structural constituent of chromatin —nucleosome; nucleus
A0A7J6HBN5ATP-dependent RNA helicase33,486DEAD box helicase familyx—ATP binding; hydrolase activity; RNA binding; RNA helicase activity—x
Translation-related proteins (TR)
A0A7J6HCW3Elongation factor 1-alpha 49,259TRAFAC class translation factor GTPase superfamily, Classic translation factor GTPase family, EF-Tu/EF-1A subfamilyx—GTP binding; GTPase activity; translation elongation factor activity—x
A0A7J6GTP860S acidic ribosomal protein P0 33,920Universal ribosomal protein uL10 familyribosome biogenesis—x—ribonucleoprotein complex; ribosome
A0A7J6HHK4Translation elongation factor EF1B beta/delta subunit guanine nucleotide exchange domain-containing protein25,124EF-1-beta/EF-1-delta familyx—translation elongation factor activity—eukaryotic translation elongation factor 1 complex
A0A7J6F1D8KH type-2 domain-containing protein 28,920Universal ribosomal protein uS3 familytranslation—RNA binding; structural constituent of ribosome—ribonucleoprotein complex; ribosome
A0A7J6HVI4Ribosomal_L28e domain-containing protein 16,563Eukaryotic ribosomal protein eL28 familytranslation—structural constituent of ribosome—ribonucleoprotein complex; ribosome
A0A7J6E6C3Ribosomal protein L730,458Universal ribosomal protein uL30 familymaturation of LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA)—structural constituent of ribosome—cytosolic large ribosomal subunit
A0A7J6I4S9Ribosomal protein L6 N-terminal domain-containing protein25,942Eukaryotic ribosomal protein eL6 familytranslation—structural constituent of ribosome—ribonucleoprotein complex; ribosome
A0A7J6I08360S acidic ribosomal protein P116,784Eukaryotic ribosomal protein P1/P2 familytranslational elongation—structural constituent of ribosome—ribonucleoprotein complex6; ribosome
A0A7J6F1J940S ribosomal protein S1416,389Universal ribosomal protein uS11 familytranslation—structural constituent of ribosome—ribonucleoprotein complex; ribosome
A0A7J6GA02Ribosomal_S7 domain-containing protein 22,343Universal ribosomal protein uS7 familytranslation—RNA binding; structural constituent of ribosome—small ribosomal subunit
A0A7J6G6F960S ribosomal protein L22-2; peroxidase14,052Eukaryotic ribosomal protein eL22 family; Peroxidase familytranslation; hydrogen peroxide catabolic process; response to oxidative stress—structural constituent of ribosome; heme binding; lactoperoxidase activity—ribonucleoprotein complex; ribosome; extracellular region
A0A7J6I79660S ribosomal protein L1217,790Universal ribosomal protein uL11 familytranslation—structural constituent of ribosome—ribonucleoprotein complex; ribosome
A0A7J6HT6640S ribosomal protein S9-223,142Universal ribosomal protein uS4 familytranslation—rRNA binding; structural constituent of ribosome—small ribosomal subunit
A0A7J6ENY3RRM domain-containing protein 11,250 x—nucleic acid binding—x
A0A7J6FAW940S ribosomal protein S1817,635Universal ribosomal protein uS13 familytranslation—rRNA binding; structural constituent of ribosome—ribonucleoprotein complex; ribosome
A0A7J6I9M7Ribosomal_L18e/L15P domain-containing protein 20,840Eukaryotic ribosomal protein eL18 familytranslation—mRNA binding; structural constituent of ribosome—ribonucleoprotein complex; ribosome
A0A7J6FX59KOW domain-containing protein 16,767Universal ribosomal protein uL24 familytranslation—RNA binding; structural constituent of ribosome—large ribosomal subunit
A0A7J6F744Ribosomal_L14e domain-containing protein 15,331Eukaryotic ribosomal protein eL14 familytranslation—RNA binding; structural constituent of ribosome—ribosome
A0A7J6E9P960S ribosomal protein L27 15,794Eukaryotic ribosomal protein eL27 familytranslation—structural constituent of ribosome—ribonucleoprotein complex; ribosome
A0A7J6H42450S ribosomal protein L23, chloroplastic 17,400Universal ribosomal protein uL23 family; DHBP synthase familytranslation; riboflavin biosynthetic process—mRNA binding; rRNA binding; structural constituent of ribosome; 3,4-dihydroxy-2-butanone-4-phosphate synthase activity; GTP binding—ribonucleoprotein complex; ribosome
A0A7J6EFK040S ribosomal protein S1716,185Eukaryotic ribosomal protein eS17 familytranslation—structural constituent of ribosome—ribonucleoprotein complex; ribosome
A0A7J6G5C760S ribosomal protein L2314,997Universal ribosomal protein uL14 familytranslation—structural constituent of ribosome—ribonucleoprotein complex; ribosome
A0A7J6GWW040S ribosomal protein S2614,763Eukaryotic ribosomal protein eS26 familytranslation—structural constituent of ribosome; nucleotidyltransferase activity—ribonucleoprotein complex; ribosome
A0A7J6E4Y660S acidic ribosomal protein P311,903Eukaryotic ribosomal protein P1/P2 familytranslational elongation—structural constituent of ribosome—ribonucleoprotein complex; ribosome
Proteins related to photosynthesis (PS)
A0A7J6GUI2Chlorophyll a-b binding protein, chloroplastic 28,276Light-harvesting chlorophyll a/b-binding (LHC) protein familyphotosynthesis; light harvesting—chlorophyll binding—chloroplast thylakoid membrane; photosystem I; photosystem II
A0A7J6GSM8Chlorophyll a-b binding protein, chloroplastic 28,429Light-harvesting chlorophyll a/b-binding (LHC) protein familyphotosynthesis, light harvesting—chlorophyll binding—chloroplast thylakoid membrane; photosystem I; photosystem II
Cytoskeleton and transport proteins (CT)
A0A7J6I6X2MD-2-related lipid-recognition domain-containing protein16,057 intracellular sterol transport—sterol binding—x
A0A7J6I828Actin41,726Actin familyx—ATP binding—cytoplasm; cytoskeleton
A0A0M5M1Z3ATP synthase subunit alpha 55,324ATPase alpha/beta chains familyactin filament bundle assembly; actin filament capping—actin filament binding; ATP binding; proton-transporting ATP synthase activity, rotational mechanism—mitochondrial inner membrane; proton-transporting ATP synthase complex, catalytic core F(1)
Uncharacterised proteins (UP)
A0A7J6FR97Uncharacterized protein 35,774 x—x—x
A0A7J6FP03Uncharacterized protein 16,800 x—x—x
A0A7J6I334Uncharacterized protein 15,922 x—x—x
A0A7J6GUW7Uncharacterized protein 13,786 x—x—x
A0A7J6HWC7Uncharacterized protein 17,135 x—x—x
x—information in UniProt KB database is not available.
Table 3. Relative abundance of the MP88 collection proteins (in ppm) in defatted hemp seed products.
Table 3. Relative abundance of the MP88 collection proteins (in ppm) in defatted hemp seed products.
AccessionProtein NameUso-31Santhica 27
Whole SeedHullsDehulled SeedsWhole SeedHullsDehulled Seeds
A0A7J6GWL5Cupin type-1 domain-containing protein292,953 abc249,256 cd301,796 ab265,551 bcd225,575 d316,781 a
A0A7J6DTA7Cupin type-1 domain-containing protein336,178 ab290,488 bc358,906 a319,042 abc278,002 c350,150 a
A0A7J6E205Cupin type-1 domain-containing protein2991 b734 c5698 a1767 bc1546 bc2397 b
A0A7J6H2R3Cupin type-1 domain-containing protein6299 ab7324 a6399 ab5745 b6426 ab6214 ab
A0A7J6GLH5Cupin type-1 domain-containing protein3410 a3532 a3127 a2708 a2851 a2780 a
A0A7J6HAT3Cupin type-1 domain-containing protein 5750 bc7593 a5372 c5890 bc6958 ab5461 c
A0A7J6G321Cupin type-1 domain-containing protein16,117 b18,916 a15,978 b10,262 d13,227 c10,338 d
A0A7J6H292Bifunctional inhibitor/plant lipid transfer protein/seed storage helical domain-containing protein19,306 a21,984 a17,488 a15,188 a12,731 a15,075 a
A0A7J6DXD1Bifunctional inhibitor/plant lipid transfer protein/seed storage helical domain-containing protein1052 ab817 bc1297 a929 bc681 c909 bc
A0A7J6EJ89Oleosin 16,729 bc7800 d26,828 a12,215 cd10,393 cd21,992 ab
A0A7J6F0Y4Oleosin 40,499 a41,904 a38,200 a38,126 a44,509 a38,168 a
A0A7J6H4F6Oleosin 25,020 a25,250 a29,762 a24,470 a28,268 a28,554 a
A0A7J6I425Verticillium wilt resistance-like protein988 a1458 a1525 a1547 a1874 a1371 a
A0A7J6F280Peroxygenase4675 ab5415 a4283 b4015 b4225 b4176 b
A0A7J6G6U3Uncharacterized protein 1205 a1115 ab1164 a749 c898 bc721 c
A0A7J6DNR1Uncharacterized protein 145 b81 b52 b13,567 a16,616 a95 b
A0A7J6DPR7Uncharacterized protein 1549 ab1264 bc1966 a703 d775 d968 cd
A0A7J6ENC9Uncharacterized protein 1498 c2198 ab1177 c1561 bc2410 a1395 c
A0A7J6GSC3Aquaporin TIP3-22236 a2728 a2585 a3037 a3023 a2988 a
A0A7J6I7S9Uncharacterized protein 2546 abc824 d3336 a1959 bc1464 cd2655 ab
A0A7J6G6Z3Glyceraldehyde 3-phosphate dehydrogenase NAD(P) binding domain-containing protein3130 bc4596 a2123 c3207 bc4012 ab2765 c
A0A7J6E4U9Triose-phosphate isomerase2120 a2489 a1862 a1912 a2233 a2080 a
A0A7J6EFG0Uncharacterized protein 2577 b3347 a2398 b1993 cd2455 b1774 d
A0A7J6EJG0Protein disulfide-isomerase 948 bc1218 ab823 c1315 ab1597 a1239 ab
A0A7J6E5J2Fructose-bisphosphate aldolase 1637 cd2756 b1158 d2346 b3594 a1745 c
A0A7J6EZ77Malate dehydrogenase 999 bc1648 a732 c1154 b1552 a836 bc
A0A7J6HK40Glyceraldehyde 3-phosphate dehydrogenase NAD(P) binding domain-containing protein926 ab1276 a684 b898 ab1277 a702 b
A0A7J6GRW8Phosphopyruvate hydratase 701 bc1043 a638 c807 b1142 a560 c
A0A7J6EG53Peptidyl-prolyl cis-trans isomerase 4223 b6554 a3436 b3861 b6019 a3035 b
A0A7J6FNP6Malate synthase 816 bc1225 a591 d878 b1284 a667 cd
A0A7J6E8J3Malate dehydrogenase 995 bc1289 ab730 c1129 bc1792 a939 bc
A0A7J6DST1NADP-dependent oxidoreductase domain-containing protein7132 a5649 ab7532 a1840 b5212 ab7102 a
A0A7J6HKH5Nucleoside-diphosphate kinase 1096 b1555 a539 c1050 b1602 a802 bc
A0A7J6HTX3Tyrosinase copper-binding domain-containing protein266 d1265 b1 e611 c1660 a10 e
A0A7J6HQA0NADH-cytochrome b5 reductase 959 bc1153 ab340 d1244 ab1389 a607 cd
A0A7J6DVP5rRNA N-glycosylase 485 bc2044 a60 d331 c584 b71 d
A0A7J6FXL4Dehydrin1169 a983 a1413 a1457 a1723 a1265 a
A0A7J6I6U6Uncharacterized protein 877 b1277 a711 bc840 bc1297 a634 c
A0A7J6G382Uncharacterized protein 5578 b7698 a4057 c2919 de3976 cd2535 e
A0A7J6HZ01Annexin 1282 bc1481 b1040 cd1193 cd1791 a961 d
A0A7J6I4E918 kDa seed maturation protein3226 abc3796 a2444 bc3612 a3344 ab2104 c
A0A7J6FL33Late embryogenesis abundant protein D-292567 b2793 b1986 b2815 b3770 a2076 b
A0A7J6GTA4SHSP domain-containing protein 1359 b2012 a1083 c693 de929 cd536 e
A0A7J6FPH5Lactoylglutathione lyase 2462 ab2739 a1947 c2097 bc2472 ab1897 c
A0A7J6DT98Peroxiredoxin 3473 bc4054 b2623 c4150 b6328 a3130 bc
A0A7J6GJC9Dehydroascorbate reductase868 a1052 a703 ab480 b733 ab724 ab
A0A7J6F3P9Catalase 600 bc1393 a323 d777 b1421 a399 cd
A0A7J6HTR6Glutaredoxin domain-containing protein 2690 ab1809 b3306 a2262 ab2009 ab2969 ab
A0A7J6FRN5Alcohol dehydrogenase843 b1160 a620 cd690 bc1129 a452 d
A0A7J6EYT6PPC domain-containing protein 1681 b1234 b2057 b14,310 a1344 b2149 b
A0A7J6E9G4Histone H4 4793 cd9218 a3183 e5008 c7431 b3664 de
A0A7J6E6Z7Histone H2A 1523 cd3063 a829 d1950 bc2576 ab1207 cd
A0A7J6E9K3Histone H2B 1251 bc1921 a1166 bc1116 bc1558 ab858 c
A0A7J6HBN5ATP-dependent RNA helicase686 d1147 b464 e821 c1293 a655 d
A0A7J6HCW3Elongation factor 1-alpha 2695 bc3321 ab2066 c2668 bc3676 a2345 c
A0A7J6GTP860S acidic ribosomal protein P0 882 cd1325 b749 d1117 bc1698 a825 cd
A0A7J6HHK4Translation elongation factor EF1B beta/delta subunit guanine nucleotide exchange domain-containing protein811 abc946 ab593 c932 ab1065 a749 bc
A0A7J6F1D8KH type-2 domain-containing protein 696 de899 bc515 e973 b1239 a727 cd
A0A7J6HVI4Ribosomal_L28e domain-containing protein 1077 bc1341 ab728 c1301 abc1864 a1283 abc
A0A7J6E6C3Ribosomal protein L7691 cd1031 ab530 d854 bc1163 a618 d
A0A7J6I4S9Ribosomal protein L6 N-terminal domain-containing protein620 cd815 b562 d759 bc1061 a636 bcd
A0A7J6I08360S acidic ribosomal protein P1753 d977 bc803 cd1045 ab1204 a854 bcd
A0A7J6F1J940S ribosomal protein S141583 bc1859 b1190 c1582 bc2413 a1300 c
A0A7J6GA02Ribosomal_S7 domain-containing protein 804 ab893 ab593 b703 b1360 a683 b
A0A7J6G6F960S ribosomal protein L22-2; peroxidase1520 bc1897 ab1152 c1664 bc2307 a1241 c
A0A7J6I79660S ribosomal protein L12869 bc1133 ab685 c948 bc1279 a785 c
A0A7J6HT6640S ribosomal protein S9-2915 b1336 a544 c961 b1286 a728 bc
A0A7J6ENY3RRM domain-containing protein 397 b1022 a403 b1068 a1321 a525 b
A0A7J6FAW940S ribosomal protein S18733 b1031 ab673 b1000 ab1212 a806 b
A0A7J6I9M7Ribosomal_L18e/L15P domain-containing protein886 bcd1093 b663 d936 bc1458 a793 cd
A0A7J6FX59KOW domain-containing protein 982 ab955 ab815 b804 b1453 a953 ab
A0A7J6F744Ribosomal_L14e domain-containing protein 1065 b1480 a683 c1047 b1470 a929 bc
A0A7J6E9P960S ribosomal protein L27 835 b1374 a767 b1211 a1216 a951 b
A0A7J6H42450S ribosomal protein L23, chloroplastic 782 ab767 ab662 b784 ab1018 a777 ab
A0A7J6EFK040S ribosomal protein S17607 bc793 b545 c726 bc1017 a589 bc
A0A7J6G5C760S ribosomal protein L23651 b603 bc371 c805 ab1065 a647 b
A0A7J6GWW040S ribosomal protein S26 880 bc1124 ab722 c1100 ab1460 a826 bc
A0A7J6E4Y660S acidic ribosomal protein P3540 a570 a588 a759 a1068 a627 a
A0A7J6GUI2Chlorophyll a-b binding protein, chloroplastic 434 bc2459 a54 c864 b2696 a155 bc
A0A7J6GSM8Chlorophyll a-b binding protein, chloroplastic 1449 c6038 a203 d2560 b5606 a522 d
A0A7J6I6X2MD-2-related lipid-recognition domain-containing protein1140 a1477 a1082 a1430 a1696 a1194 a
A0A7J6I828Actin973 a1218 a928 a1019 a1255 a806 a
A0A0M5M1Z3ATP synthase subunit alpha 549 bc1081 a377 d629 b1035 a405 cd
A0A7J6FR97Uncharacterized protein 1215 a1071 a1257 a1047 a1123 a1174 a
A0A7J6FP03Uncharacterized protein 12,415 ab14,264 a9720 b13,312 ab16,039 a10,073 b
A0A7J6I334Uncharacterized protein 1235 bc1552 a852 d1149 cd1479 ab948 cd
A0A7J6GUW7Uncharacterized protein 786 ab856 ab643 b777 ab1061 a692 b
A0A7J6HWC7Uncharacterized protein 1506 cd2038 ab1028 d1570 bc2260 a1045 d
Different lowercase letters in the rows indicate a statistically significant difference at the p < 0.05 (Tukey HSD test) among the defatted hemp seed products of two cultivars (for each protein separately).
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Bárta, J.; Roudnický, P.; Jarošová, M.; Zdráhal, Z.; Stupková, A.; Bártová, V.; Krejčová, Z.; Kyselka, J.; Filip, V.; Říha, V.; et al. Proteomic Profiles of Whole Seeds, Hulls, and Dehulled Seeds of Two Industrial Hemp (Cannabis sativa L.) Cultivars. Plants 2024, 13, 111.

AMA Style

Bárta J, Roudnický P, Jarošová M, Zdráhal Z, Stupková A, Bártová V, Krejčová Z, Kyselka J, Filip V, Říha V, et al. Proteomic Profiles of Whole Seeds, Hulls, and Dehulled Seeds of Two Industrial Hemp (Cannabis sativa L.) Cultivars. Plants. 2024; 13(1):111.

Chicago/Turabian Style

Bárta, Jan, Pavel Roudnický, Markéta Jarošová, Zbyněk Zdráhal, Adéla Stupková, Veronika Bártová, Zlatuše Krejčová, Jan Kyselka, Vladimír Filip, Václav Říha, and et al. 2024. "Proteomic Profiles of Whole Seeds, Hulls, and Dehulled Seeds of Two Industrial Hemp (Cannabis sativa L.) Cultivars" Plants 13, no. 1: 111.

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