Verification of the cDNA deduced sequence of glutenin subunit 1Dx5 and an Mr 58 000 repetitive peptide by matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS)

MALDI-MS was used to confirm the cDNA derived amino acid sequence of HMW subunit 1Dx5 and an E. coli-expressed repetitive M-r 58 000 peptide based on residues 102-643. Analysis of the M-r 58 000 peptide and of tryptic peptides separated by RP-HPLC showed that the experimentally determined M-r are consistent, with the calculated values, within the experimental error. This indicates a substantial correctness of the gene derived sequence. In the same way, analysis of tryptic peptides from 1Dx5 allowed about 80% of the sequence to be confirmed, covering residues 1-669 with the exception of two short peptides. There was also good agreement with sequence of the M-r 58 000 peptide in the region of identity. The results, giving evidence for a substantial correctness of a large part of the sequence. confirm the absence of extensive glycosylation in subunit 1Dx5 and demonstrate the value of MALDI-MS for direct sequence analysis of HMW subunits and similar proteins. (C) 2000 Academic Press.