Verification of the cDNA deduced sequence of glutenin subunit 1Dx5 and an M(r) 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.