Mapping of transglutaminase 2 sites of human salivary small basic proline-rich proteins by HPLC high resolution ESI-MS/MS.

Due to the distinctive features of oral cavity, determination of the proteins involved in the formation of "oral protein pellicle" is demanding. The present study investigated the susceptibility of several human basic proline-rich peptides, named P-H, P-D, P-F, P-J, and II-2 as substrates of transglutaminase-2. The reactivity of P-C peptide and statherin was also investigated. Peptides purified from human whole saliva were incubated with the enzyme in the presence or in the absence of monodansyl-cadaverine. Mass spectrometry analyses of the reaction products highlighted that P-H, and P-D (P32 and A32 variants) were active substrates, II-2 was less reactive and P-F and P-J showed very low reactivity. P-C and statherin were highly reactive. All the peptides formed cyclo-derivatives, and only specific glutamine residues were involved in the cycle formation and reacted with monodansyl-cadaverine: Q29 of P-H, Q37 of P-D, Q21 of II-2, Q41 of P-C and Q37 of statherin were the principal reactive residues. One or two secondary glutamine residues of only P-H, P-D P32, P-C and statherin were hierarchically susceptible to the reaction with monodansyl-cadaverine. MS and MS/MS data were deposited to the ProteomeXchange Consortium (http://www.ebi.ac.uk/pride) via the PRIDE partner repository with the dataset identifier PXD014658.