Towards defining the whole salivary peptidome

We report the identification of 2294 peptides/proteins in whole saliva in the mass range between 700 and 16 000 Da by LC-MS and MS/MS using a MALDI-TOF/TOF mass spectrometer. Most of the identified peptides/proteins are originated from cellular debris or plasma components while only 26% (n = 607) correspond to salivary peptides/proteins species. In spite of the presence of the major salivary peptides in all samples from the six subjects analyzed, each individual presents a different pattern of fragments, many deriving from the same protein sequence. All our data, in particular the large number of fragments found, suggest high proteolytic activity insight the oral cavity. The analysis of samples by gelatin zymography showed that all saliva donors displayed multiple proteolytic bands, two identified as cathepsin D and G by MS. Analysis of the cleavage site distribution on the main peptide sequences based on contingency tables shows that the predominant cleavages occur between Gln-Gly or Tyr-Gly. These cleavages are largely associated with prohne-rich proteins peptides and with histatin 1 and P-B peptide, respectively. However, depending on the peptide class, different cleavage hits were observed suggesting the presence of a set of proteases acting in different ways according to different peptide sequences. Comparing the number of cleavages involving all residues, it is possible to observe that 44% (10%) of the observed cleavages in histatin, statherin and P-B peptide in all individuals may be explained by cathepsin D, suggesting a major role for this enzyme in oral cavity proteolysis.