Chemicals and reagents.

Solvents and reagents used for the present studies were analytical quality or better, unless described
differently. For the preparation of HPLC solvents, trifluoroacetic acid (TFA) of sequencing grade (Fluka 91699) and acetonitrile
(ACN) of gradient grade (Merck 100030) or of preparative grade (Merck 113358) were used. Bovine serum albumin (BSA
standard) was purchased from Serva (11920), and propyl-4-hydroxybenzoate from Fluka (54790).

Solvents. A1: 0.4 mol/L of NaCl + 0.067 mol/L of HKNaPO 4 (pH 7.6); A2: 0.4 mol/L of NaCl. B1: 60% (v/v) ethanol (EtOH); B2:
50% (v/v) 1-propanol (1-PrOH); B3: 50% (v/v) 2-propanol (2-PrOH). C1: 50% (v/v) 1-PrOH + 2 mol/L of urea + 0.05 mol/L of
Tris-HCl (pH 7.5) + 1% (w/v) dithioerythritol (DTE) under nitrogen; C2: 50% (v/v) 2-PrOH + 0.08 mol/L of Tris-HCl (pH 8.0) +
1% (w/v) DTE under nitrogen.

RP-HPLC. Routine RP-HPLC of the filtered protein fractions extracted from flour and the different protein standards was
performed using a Beckman instrument (solvent module 126, System Gold software); Nucleosil 300-5 C8 column material
(Machery-Nagel 71265) packed into a 4.6- × 240-mm column by an HPLC packing instrument (Shandon) under a pressure of 450
bar (which was much cheaper than a commercially available column); column temperature was 50°C; sample loop was 2 mL;
injection was 200 mL of albumins and globulins, 50 or 80 mL of gliadins, and 100 or 150 mL of glutenin subunits; 0.1% (v/v) TFA
(500 mL) was injected before and after sample injection. Elution system I: A) TFA (0.1%, v/v); B) ACN (preparative grade) + TFA
(99.9/0.1%, v/v). Linear gradients: 0 min 20% B, 20 min 60% B (albumins-globulins); 0 min 28% B, 30 min 56% B (gliadins,
glutenin subunits, gliadin standard, LMW standard, HMW standard); 0 min 39.5% B, 20 min 41.0% B (BSA standard). Flow rate
was 1.0 mL/min; detection was by UV absorbance at 210 nm. After each run, the column was cleaned with 90% B (5 min) and
equilibrated with the starting B concentration (10 min).

For the analysis of HMW subunits, changes included column temperature of 53°C and injection of 250 mL of the glutenin extract.
Elution system II: A) ACN (15%, v/v) + 2-PrOH (10%, v/v) + TFA (0.1%, v/v); B) ACN (30%, v/v) + 2-PrOH (8%, v/v) + TFA
(0.1%, v/v). Linear gradients: 0 min 46% B, 60 min 71% B, 61–65 min 100% B. Flow rate was 0.8 mL. Other conditions tested
included: Nucleosil 300-5 C 18 column material (Machery-Nagel 71252); gradient grade ACN; column temperature 70°C.
Gradients: 0 min 24% B, 50 min 56% B, or 0 min 27% B, 20 min 55% B (gliadins, glutenin subunits). UV absorbance was at 220
nm. The integration procedure was automatically handled by the software. The baseline was adjusted to zero when injector was
moved to inject position. Integration was suppressed for the first part of the chromatograms (negative and positive solvent peaks).
The elution borders of the protein groups were set to absorbance minima between specific peaks. T he absorbance spectra of eluted
proteins were determined by a diode-array detector (Beckman module 168). Column quality was tested with propyl-4-
hydroxybenzoate (1.5 mg dissolved in 30 mL of methanol) eluted with 50% B of elution system I or II; peak width at half peak
height was taken as quality criterion.