Matix-assisted laser desorption/ionization mass spectrometric peptide mapping of high molecular weight glutenin subunits 1Bx7 and 1Dy10 in Cheyenne cultivar

This study describes the verification of the cDNA-deduced amino acid sequen ces of high molecular weight glutenin subunits 1Dy10 and 1Bx7 in Cheyenne c ultivar by direct matrix-assisted laser desorption/ionization time-of-fligh t mass spectrometry (MALDI-TOFMS) analysis of their tryptic fragments omitt ing chromatographic pre-separation. These polypeptides have a conserved str ucture consisting of a long central repetitive domain that prevents the app lication of conventional sequencing procedures such as Edman degradation. T he published sequence of subunit 1Dy10 contains 7 Lys and 13 Arg residues; thus the production of 21 tryptic peptides is expected. The cDNA-deduced se quence for 1Bx7 subunit includes 5 Lys and 15 Arg residues, but the presenc e of three Arg-Pro bonds, which are normally not cleaved by trypsin, predic ts only 19 tryptic peptides. Three different matrices (DHB, SA and HCCA) in combination with the most compatible sample preparation procedures were us ed in order to obtain the maximum 1Dy10 and 1Bx7 sequence coverage. MALDI a nalysis of the 1Dy10 tryptic digest resulted in the identification of all 2 1 expected peptides. In the case of 1Bx7 MALDI analysis resulted in the ide ntification of 17 of the 19 expected peptides, giving a sequence coverage o f 99.3%. These results were sufficient to rule out glycosylation of the 1Dy 10 and 1Bx7 proteins and to assess the absence of any other post-translatio nal modification, to within the detection limits of the method. Copyright ( C) 2001 John Wiley & Sons, Ltd.