Isolation, Characterisation by Mass Spectrometry and Partial Amino Acid Sequencing of Avenins

Avenins were extracted with 70% (v/v) ethanol and purified by IE-HPLC followed by RP-HPLC. Ten major and one minor avenins were isolated and characterised. The amino acid compositions of the avenins were similar to those of the alcohol-extractable seed storage proteins of other cereals. They were rich in proline and glutamine and poor in basic amino acids. The exact number of cysteine residues in the avenins was determined by ESMS before and after reduction and alkylation. Free SH groups were not found in Avn-9 and Avn-3, and four disulphide bonds are therefore expected. The M rs of the avenins ranged between 20 and 28k as determined by mass spectrometry. A difference between the M r calculated from the amino acid sequence of Avn-9 and the M r measured by ESMS was indicative of an erroneous sequence, and mass spectrometric peptide mapping in combination with Edman degradation led to the identification of three sequence errors. Five of the avenins were N-terminally blocked. The N-terminal sequences of the six other avenins revealed unique highly homologous regions and exhibited short tandem repeats.