Comparative shotgun proteomics analysis of wheat gluten proteins digested by various peptidases

The wheat flour proteome is a complex mixture of non-gluten and gluten proteins. The large number of repetitive sequences, special amino acid composition and similarity of gluten protein isoforms pose a major challenge in bottom-up proteomics. The standard peptidase used in shotgun proteomics is trypsin, which may not be suitable for all wheat proteins. In this study, flour extracts of nine common wheat genotypes were digested with the peptidases trypsin, chymotrypsin, thermolysin, pepsin and the combination of trypsin and chymotrypsin. The results showed large differences for the number of identified peptides. With 4115 peptides, trypsin resulted in by far the most peptide identifications, followed by thermolysin with 1421 peptides. However, this no longer applied at protein level. Most metabolic protein groups (673) were identified with trypsin. Among the gluten protein groups, however, around 130 were identified with each peptidase. The ω-gliadins were detected with all peptidases except trypsin. A comparison with quantitative RP-UHPLC-UV results showed that there was the greatest overlap after thermolysin digestion. Otherwise, there was no great similarity between the different peptidases, which is why their results cannot be compared with one another. The sequence coverage of gluten proteins was 51 % after thermolysin digestion, 27 % after trypsin digestion and 61 % when all peptidases were evaluated together. The results showed that digestion with various peptidases provides a more detailed picture of the wheat proteome. Some wheat protein groups could only be identified with certain peptidases, which is important if these protein groups are to be studied in more detail.