High molecular weight glutenin subunits in some durum wheat cultivars investigated by means of mass spectrometric techniques

The primary structures of high molecular weight glutenin subunits (HMW-GS) of 5 Triticum durum Desf. cultivars(Simeto, Svevo, Duilio, Bronte, and Sant’Agata), largely cultivated in the south of Italy, and of 13 populations of the old springSicilian durum wheat landrace Timilia (Triticum durum Desf.) (accession nos. 1, 2, 3, 4, 7, 8, 9, 13, 14, 15, SG1, SG2, and SG3) wereinvestigated using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and reversedphasehigh performance liquid chromatography/nanoelectrospray ionization mass spectrometry (RP-HPLC/nESI-MS/MS).Mr ofthe intact proteins determined by MALDI mass spectrometry showed that all the 13 populations of Timilia contained the same twoHMW-GS with 75.2 kDa and 86.4 kDa, whereas the other durum wheat cultivars showed the presence of the expected HMW-GS1By8 and 1Bx7 at 75.1 kDa and 83.1 kDa, respectively. By MALDI mass spectrometry of the tryptic digestion peptides of the isolatedHMW-GS of Timilia, the 1Bx and 1By subunits were identified as the NCBInr Acc. No AAQ93629, and AAQ93633, respectively.Sequence verification for HMW-GS 1Bx and 1By both in Simeto and Timilia was obtained by MALDI mass mapping and HPLC/nESI-MSMS of the tryptic peptides. The Bx subunit of Timila presents a sequence similarity of 96% with respect to Simeto, withdifferences in the insertion of 3 peptides of 5, 9, and 15 amino acids, for a total insertion of 29 amino acids and 25 amino acidsubstitutions. These differences in the amino acidic sequence account for the determined Δm of 3294 Da between theMr of the 1Bxsubunits in Timilia and Simeto. Sequence alignment between the two By subunits shows 10 amino acid substitutions and isconsistent with the Δm of 148 Da found in the MALDI mass spectra of the intact subunits.