*Article* **What You Extract Is What You Get: Different Methods of Protein Extraction from Hemp Seeds**

**Annalisa Givonetti †, Chiara Cattaneo \*,† and Maria Cavaletto**

Dipartimento di Scienze e Innovazione Tecnologica-DiSIT, Università del Piemonte Orientale, 13100 Vercelli, Italy; annalisa.givonetti@uniupo.it (A.G.); maria.cavaletto@uniupo.it (M.C.)

**\*** Correspondence: chiara.cattaneo@uniupo.it

† These authors contributed equally to the work.

**Abstract:** *Cannabis sativa* L. seeds are rich in essential polyunsaturated fatty acids and highly digestible proteins, with a good nutritional value. Proteomics studies on hempseed reported so far have mainly been conducted on processed seeds and, to our knowledge, no optimization of protein extraction from hemp seeds has been performed. This study investigates the SDS-PAGE profile of hempseed proteins comparing different methods of extraction, (Osborne sequential extraction, TCA/acetone, MTBE/methanol, direct protein solubilization of defatted hempseed flour), two conditions to keep low temperature during seed grinding (liquid nitrogen or ice) and two solubilization buffers (urea-based or Laemmli buffer). Among the tested conditions, the combination of liquid nitrogen + TCA/acetone + Laemmli buffer was not compatible with SDS-PAGE of proteins. On the other hand, urea-based buffer achieved more reproducible results if combined with all the other conditions. TCA/acetone, MTBE/methanol, and direct protein solubilization of defatted hempseed flour demonstrated a good overview of protein content, but less abundant proteins were poorly represented. The Osborne sequential separation was helpful in diluting abundant proteins thus enhancing the method sensitivity.

**Keywords:** protein extraction; hempseed; seed storage proteins; SDS-PAGE
