RP-HPLC-ESI-MS evidenced that salivary cystatin B is detectable in adult human whole saliva mostly as S-modified derivatives: S-Glutathionyl, S-cysteinyl and S-S 2-mer.

An HPLC-ESI-MS analysis of adult human whole saliva evidenced three protein masses (M average 11,487±2, 11,301±2 and 22,362±3Da) eluting in the 32.5-35.0min range. Treatment in reducing conditions allowed establishing that they are S-derivatives of N-terminal acetylated cystatin B, namely its S-glutathionyl, S-cysteinyl and S-S dimer. The identification was confirmed by high resolution HPLC-ESI-MS-MS experiments on the intact naturally occurring proteins and their tryptic digests. S-unmodified cystatin B is rarely detectable in whole saliva of healthy adults (5 subjects out of 65) and its percentage does not overcome approximately 20% of total cystatin B (11±9%). In the majority of subjects (60 out of 65) the mean percentages of the S-modified derivatives were S-glutathionyl 53±13%, S-cysteinyl 15±5%, S-S 2-mer 32±13%. Variations of the percentages of these S-modified derivatives of cystatin B could be indicative of oral oxidative stress. As we are aware, this is the first time that S-glutathionylation and S-cysteinylation were described as extensive PTM of a salivary protein and the first time that these PTMs were detected in naturally occurring cystatin B.