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.

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

Department of Plant Production Faculty of Agriculture and Technology University of South Bohemia 370 05 České Budějovice Czech Republic

HEMP PRODUCTION CZ Ltd 262 72 Chraštice Czech Republic

Mendel Centre of Plant Genomics and Proteomics Central European Institute of Technology Masaryk University 625 00 Brno Czech Republic

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Callaway J.C. Hempseed as a nutritional resource: An overview. Euphytica. 2004;140:65–72. doi: 10.1007/s10681-004-4811-6. DOI

Burton R.A., Andres M., Cole M., Cowley J.M., Augustin M.A. Industrial hemp seed: From the field to value-added food ingredients. J. Cannabis Res. 2022;4:45. doi: 10.1186/s42238-022-00156-7. PubMed DOI PMC

Farinon B., Molinari R., Costantini L., Merendino N. The Seed of Industrial Hemp (Cannabis sativa L.): Nutritional Quality and Potential Functionality for Human Health and Nutrition. Nutrients. 2020;12:1935. doi: 10.3390/nu12071935. PubMed DOI PMC

Sun X., Sun Y., Li Y., Wu Q., Wang L. Identification and Characterization of the Seed Storage Proteins and Related Genes of Cannabis sativa L. Front. Nutr. 2021;8:678421. doi: 10.3389/fnut.2021.678421. PubMed DOI PMC

Kriese U., Schumann E., Weber W.E., Beyer M., Brühl L., Matthäus Oil content, tocopherol composition and fatty acid patterns of the seeds of 51 Cannabis sativa L. genotypes. Euphytica. 2004;137:339–351. doi: 10.1023/B:EUPH.0000040473.23941.76. DOI

Lan Y., Zha F., Peckrul A., Hanson B., Johnson B., Rao J., Chen B. Genotype x Environmental Effects on Yielding Ability and Seed Chemical Composition of Industrial Hemp (Cannabis sativa L.) Varieties Grown in North Dakota, USA. J. Am. Oil. Chem. Soc. 2019;96:1417–1425. doi: 10.1002/aocs.12291. DOI

Alonso-Esteban J.I., González-Fernández M.J., Fabrikov D., Torija-Isasa E., Sánchez-Mata M.d.C., Guil-Guerrero J.L. Hemp (Cannabis sativa L.) Varieties: Fatty Acid Profiles and Upgrading of γ-Linolenic Acid–Containing Hemp Seed Oils. Eur. J. Lipid Sci. Technol. 2020;122:1900445. doi: 10.1002/ejlt.201900445. DOI

Xu Y., Zhao J., Hu R., Wang W., Griffin J., Li Y., Sun X.S., Wang D. Effect of genotype on the physicochemical, nutritional, and antioxidant properties of hempseed. J. Agric. Food Res. 2021;3:100119. doi: 10.1016/j.jafr.2021.100119. DOI

Golimowski W., Teleszko M., Marcinkowski D., Kmiecik D., Grygier A., Kwaśnica A. Quality of Oil Pressed from Hemp Seed Varieties: ‘Ealina 8FC’, ‘Secueni Jubileu’ and ‘Finola’. Molecules. 2022;27:3171. doi: 10.3390/molecules27103171. PubMed DOI PMC

Mamone G., Picariello G., Ramondo A., Nicolai M.A., Ferranti P. Production, digestibility and allergenicity of hemp (Cannabis sativa L.) protein isolates. Food Res. Int. 2019;115:562–571. doi: 10.1016/j.foodres.2018.09.017. PubMed DOI

Miernyk J.A., Hajduch M. Seed proteomics. J. Proteom. 2011;74:389–400. doi: 10.1016/j.jprot.2010.12.004. PubMed DOI

Aluko R.E. Hemp Seed (Cannabis sativa L.) Proteins: Composition, Structure, Enzymatic Modification, and Functional or Bioactive Properties. In: Nadathur S.R., Wanasundara J.P.D., Scanlin L., editors. Sustainable Protein Sources. Academic Press; San Diego, CA, USA: 2017. pp. 121–132.

Tang C.-H., Ten Z., Wang X.-S., Yang X.-Q. Physicochemical and Functional Properties of Hemp (Cannabis sativa L.) Protein Isolate. J. Agric. Food Chem. 2006;54:8945–8950. doi: 10.1021/jf0619176. PubMed DOI

Wang Q., Xiong Y.L. Processing, Nutrition, and Functionality of Hempseed Protein: A Review. Compr. Rev. Food Sci. Food Saf. 2019;18:936–952. doi: 10.1111/1541-4337.12450. PubMed DOI

Bárta J., Bártová V., Jarošová M., Švajner J. Proteins of Oilseed Cakes, Their Isolation and Usage Possibilities. Chem. Listy. 2021;115:472–480.

Ponzoni E., Brambilla I.M., Galasso I. Genome-wide identification and organization of seed storage protein genes of Cannabis sativa. Biol. Plant. 2018;62:693–702. doi: 10.1007/s10535-018-0810-7. DOI

Park S.-K., Seo J.-B., Lee M.-Y. Proteomic profiling of hempseed proteins from Cheungsam. Biochim. Biophys. Acta Proteins Proteom. 2012;1824:374–382. doi: 10.1016/j.bbapap.2011.10.005. PubMed DOI

Aiello G., Fasoli E., Boschin G., Lammi C., Zanoni C., Citterio A., Arnoldi A. Proteomic characterization of hempseed (Cannabis sativa L.) J. Proteom. 2016;147:187–196. doi: 10.1016/j.jprot.2016.05.033. PubMed DOI

Leonard W., Zhang P., Ying D., Nie S., Tindal E., Fang Z. Transformation of hempseed (Cannabis sativa L.) oil cake proteome, structure and functionality after extrusion. Food Chem. 2022;384:132499. doi: 10.1016/j.foodchem.2022.132499. PubMed DOI

Stoyke M., Becker R., Brockmeyer J., Jira W., Popping B., Uhlig S., Wittke S. German Government Official Methods Board Points the Way Forward: Launch of a New Working Group for Mass Spectrometry for Protein Analysis to Detect Food Fraud and Food Allergens. J. AOAC Int. 2019;102:1280–1285. doi: 10.5740/jaoacint.19-0056. PubMed DOI

Kotecka-Majchrzak K., Sumara A., Fornal E., Montowska M. Identification of species-specific peptide markers in cold-pressed oils. Sci. Rep. 2020;10:19971. doi: 10.1038/s41598-020-76944-z. PubMed DOI PMC

Kotecka-Majchrzak K., Sumara A., Fornal E., Montowska M. Proteomic analysis of oilseed cake: A comparative study of species-specific proteins and peptides extracted from ten seed species. J. Sci. Food Agric. 2021;101:297–306. doi: 10.1002/jsfa.10643. PubMed DOI

Alonso-Esteban J.I., Pinela J., Ćirić A., Calhelha R.C., Soković M., Ferreira I.C.F.R., Barros L., Torija-Isasa E., Sánchez-Mata M.d.C. Chemical composition and biological activities of whole and dehulled hemp (Cannabis sativa L.) seeds. Food Chem. 2022;374:131754. doi: 10.1016/j.foodchem.2021.131754. PubMed DOI

Smolikova G., Gorbach D., Lukasheva E., Mavropolo-Stolyarenko G., Bilova T., Soboleva E., Tsarev A., Romanovskaya E., Podolskaya E., Zhukov V., et al. Bringing New Methods to the Seed Proteomics Platform: Challenges and Perspectives. Int. J. Mol. Sci. 2020;21:9162. doi: 10.3390/ijms21239162. PubMed DOI PMC

House J.D., Neufeld J., Leson G. Evaluating the Quality of Protein from Hemp Seed (Cannabis sativa L.) Products Through the use of the Protein Digestibility-Corrected Amino Acid Score Method. J. Agric. Food Chem. 2010;58:11801–11807. doi: 10.1021/jf102636b. PubMed DOI

Boye J.I., Barbana C. Food and Industrial Bioproducts and Bioprocessing. Wiley; Hoboken, NJ, USA: 2012. Protein Processing in Food and Bioproduct Manufacturing and Techniques for Analysis; pp. 85–113.

Shen P., Gao Z., Xu M., Rao J., Chen B. Physicochemical and structural properties of proteins extracted from dehulled industrial hempseeds: Role of defatting process and precipitation pH. Food Hydrocoll. 2020;108:106065. doi: 10.1016/j.foodhyd.2020.106065. DOI

Chen H., Xu B., Wang Y., Li W., He D., Zhang Y., Zhang X., Xing X. Emerging natural hemp seed proteins and their functions for nutraceutical applications. Food Sci. Hum. Wellness. 2023;12:929–941. doi: 10.1016/j.fshw.2022.10.016. DOI

Liu M., Toth J.A., Childs M., Smart L.B., Abbaspourrad A. Composition and functional properties of hemp seed protein isolates from various hemp cultivars. J. Food Sci. 2023;88:942–951. doi: 10.1111/1750-3841.16467. PubMed DOI

Liu M., Childs M., Loos M., Taylor A., Smart L.B., Abbaspourrad A. The effects of germination on the composition and functional properties of hemp seed protein isolate. Food Hydrocoll. 2023;134:108085. doi: 10.1016/j.foodhyd.2022.108085. PubMed DOI

Potin F., Lubbers S., Husson F., Saurel R. Hemp (Cannabis sativa L.) Protein Extraction Conditions Affect Extraction Yield and Protein Quality. J. Food Sci. 2019;84:3682–3690. doi: 10.1111/1750-3841.14850. PubMed DOI

Fang B., Gu Z., Ohm J.-B., Chen B., Rao J. Reverse micelles extraction of hemp protein isolate: Impact of defatting process on protein structure, functionality, and aromatic profile. Food Hydrocoll. 2023;135:108158. doi: 10.1016/j.foodhyd.2022.108158. DOI

Angelovici R., Galili G., Fernie A.R., Fait A. Seed desiccation: A bridge between maturation and germination. Trends Plant Sci. 2010;15:211–218. doi: 10.1016/j.tplants.2010.01.003. PubMed DOI

Docimo T., Caruso I., Ponzoni E., Mattana M., Galasso I. Molecular characterization of edestin gene family in Cannabis sativa L. Plant Physiol. Biochem. 2014;84:142–148. doi: 10.1016/j.plaphy.2014.09.011. PubMed DOI

Hajduch M., Matusova R., Houston N.L., Thelen J.J. Comparative proteomics of seed maturation in oilseeds reveals differences in intermediary metabolism. Proteomics. 2011;11:1619–1629. doi: 10.1002/pmic.201000644. PubMed DOI

Nikiforidis C.V. Structure and functions of oleosomes (oil bodies) Adv. Colloid. Interface Sci. 2019;274:102039. doi: 10.1016/j.cis.2019.102039. PubMed DOI

Garcia F.L., Ma S., Dave A., Acevedo-Fani A. Structural and Physicochemical Characteristics of Oil Bodies from Hemp Seeds (Cannabis sativa L.) Foods. 2021;10:2930. doi: 10.3390/foods10122930. PubMed DOI PMC

Lopez C., Novales B., Rabesona H., Weber M., Chardot T., Anton M. Deciphering the properties of hemp seed oil bodies for food applications: Lipid composition, microstructure, surface properties and physical stability. Food Res. Int. 2021;150:110759. doi: 10.1016/j.foodres.2021.110759. PubMed DOI

Puri M., Kaur I., Perugini M.A., Gupta R.C. Ribosome-inactivating proteins: Current status and biomedical applications. Drug Discov. Today. 2012;17:774–783. doi: 10.1016/j.drudis.2012.03.007. PubMed DOI

Bolognesi A., Bortolotti M., Maiello S., Battelli M.G., Polito L. Ribosome-Inactivating Proteins from Plants: A Historical Overview. Molecules. 2016;21:1627. doi: 10.3390/molecules21121627. PubMed DOI PMC

Citores L., Iglesias R., Ferreras J.M. Antiviral Activity of Ribosome-Inactivating Proteins. Toxins. 2021;13:80. doi: 10.3390/toxins13020080. PubMed DOI PMC

Kim E.-S., Kwon T.-H., Park S.-H. Structural Characteristics of Shells in a Fibrous Cultivar of Cannabis sativa L. J. Nat. Fibers. 2023;20:2216951. doi: 10.1080/15440478.2023.2216951. DOI

Shen P., Gao Z., Xu M., Ohm J.-B., Rao J., Chen B. The impact of hempseed dehulling on chemical composition, structure properties and aromatic profile of hemp protein isolate. Food Hydrocoll. 2020;106:105889. doi: 10.1016/j.foodhyd.2020.105889. DOI

Sharma S., Tamilselvan T., Shakeb M., Prabhasankar P. Hydrothermal treatment of hemp seeds (Cannabis sativa L.): Impact on its dehulling yield, fatty acid profile and nutritional characteristics. J. Sci. Food Agric. 2023;103:2681–2689. doi: 10.1002/jsfa.12328. PubMed DOI

Laemmli U.K. Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature. 1970;227:680–685. doi: 10.1038/227680a0. PubMed DOI

Wiśniewski J.R., Zougman A., Nagaraj N., Mann M. Universal sample preparation method for proteome analysis. Nat. Methods. 2009;6:359–362. doi: 10.1038/nmeth.1322. PubMed DOI

Cox J., Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 2008;26:1367–1372. doi: 10.1038/nbt.1511. PubMed DOI

Cox J., Neuhauser N., Michalski A., Scheltema R.A., Olsen J.V., Mann M. Andromeda: A Peptide Search Engine Integrated into the MaxQuant Environment. J. Proteome Res. 2011;10:1794–1805. doi: 10.1021/pr101065j. PubMed DOI

Proteomic Profile of Flaxseed (Linum usitatissimum L.) Products as Influenced by Protein Concentration Method and Cultivar