*2.2. Chemicals and Reagents*

All reagents and buffers were of analytical grade and purchased from Sigma-Aldrich (St. Louis, MO, USA) unless indicated otherwise.

## *2.3. Amino Acid Analysis*

Amino acid compositions of flour samples were determined according to a modified method of Spackman et al. [16]. For hydrolysis, 0.5 g of powdered seed samples were dissolved in 6 N HCl solution and heated for 24 h at 110 ◦C. The solution was evaporated under reduced pressure using a rotary evaporator (EYELA Rotary vacuum evaporator N-N SERIES, Tokyo Ridadidai Co. Ltd, Tokyo, Japan) and the residual solid was dissolved in sample dilution buffer (pH 2.2). The solution was filtered through a 0.45 μm membrane (ADVANTEC Toyo Roshi Kaisha, Japan). Amino acid analysis was performed using a Sykam (Sykam Co., Darmstadt, Germany) S7130 amino acid reagent organizer, S5200 sample injector and S2100 solvent delivery system with a cation separation column LCA K06/NA (4.6 mm × 250 mm). The flow rates of mobile phase and ninhydrin were 0.45 mL/min and 0.4 mL/min, respectively. Due to the acid hydrolysis, glutamine and asparagine were determined with glutamic acid and aspartic acid, respectively, and tryptophan was degraded and not detected.

#### *2.4. Fractionation of Te*ff *Proteins*

Three types of methods were used to sequentially extract four fractions of proteins (albumins, globulins, prolamins and glutelins) from teff seed flour. Method 1 was conducted based on a modified

protein fractionation method described by Ayoni et al. [17]. Briefly, albumins were extracted with deionized water on flour to solvent ratio of 1:10 (*w*/*v*) three times by shaking for 1 h each time and a fourth time for 30 minutes. Each extract was centrifuged at 6000× *g* for 10 min at 4 ◦C to obtain a clear supernatant, and all supernatants were combined as albumin fraction. The residue after albumin extraction was used to extract globulins using 1.25 M NaCl in a similar procedure for albumins. After washing the residue from the globulin extraction 2 times for 1 h with deionized water to remove salt, prolamins were extracted from the residue using 70% ethanol. Again, after washing with deionized water the same way as above to remove the alcohol, glutelins were extracted with 0.075 M NaOH in similar steps as above.

Method 2 was done according to a modified version of a method described by Wallace et al. [18]. Albumins and globulins were extracted exactly the same as in method 1. Prolamins were extracted using 70% ethanol containing 5% β-merkaptoethanol (βME) at RT (23 ◦C) 3 times for 1 h and a fourth extraction done overnight. Glutelins were extracted with 0.075 M NaOH containing 5% βME and 1% SDS the same way as prolamins in this method.

Method 3 was performed using a modified method by Tylor et al. [19]. Albumins and globulins were extracted exactly the same was as in Method 1 and 2. Prolamins were extracted using 60% tert-butanol containing 0.05% DTT at 23 ◦C and the fourth time extraction was done overnight. Similarly glutelins were extracted with 0.075M NaOH containing 0.05% DTT at 23 ◦C.

Each supernatant was filtered through a 0.45 μm membrane (ADVANTEC Toyo Roshi Kaisha, Japan) to remove insoluble particles and then dialyzed against distilled water with a 3.5 kDa MW cut of dialysis membrane (Spectrum Laboratories, Rancho Dominguez, CA, USA) with distilled water for 24 hours and 4 changes of water. Protein samples were freeze-dried (MCFD8512; Ilshinbiobase Co., Ltd., Gyeonggi, Korea) and stored at −20 ◦C until used for SDS-PAGE analysis.
