Bioinformatic software allowing the identification of peptides by confrontation of peptide fragmentation spectra obtained by mass spectrometry against targeted databases or directly by de novo sequencing are now mandatory in peptidomics/proteomics approach. The programming of the identification software requires specifying, among other things, the mass measurement accuracy of the instrument, the digestion enzyme used with the number of missed cleavages allowed. Moreover, these software are able to identify a large number of post-translational modifications (PTMs). However, peptide- and PTM-identifications are challenging in the agrofood field due to a non-specific cleavage site of physiological- or food-grade-enzymes used and the number and location of PTMs. In this study, we show the importance of adapted software programming to obtain a better peptide- and PTM-identification rate in the agrofood field. A gelatine product and one industrial gelatine hydrolysate from three different sources (beef, pork or fish), each digested by simulated gastro-intestinal digestion, by MS-grade trypsin or both were used to perform the comparisons. Two points were illustrated i) the impact of the set-up of specific enzyme- versus no specific-enzyme use and ii) the impact of a maximum 6 PTMs allowed per peptides against routinely 3. Prior knowledge of the composition of the raw proteins is an important asset for a better identification of peptide sequences.