Salivary biomarkers in Multiple Sclerosis and Autoimmune Hepatitis explored by an integrate top-down and bottom-up platform

Multiple Sclerosis and Autoimmune Hepatitis are serious diseases whose diagnosis is extremely difficult. To date, no causes have been found in the manifestation of both pathologies, but several factors that lead to their progression such as infectious agents, environment and ethnicity and genetic predisposition. In this study, the salivary proteome and peptidome of affected individuals from these pathologies has been explored by mass spectrometry, through a top-down and bottom-up platform integrated and compared with groups of healthy controls, with the aim to assess whether qualitative and quantitative changes in proteins and salivary peptides could be associated with immune defenses distinctive imbalance of any illness and in order to have suggestions of specific potential salivary biomarkers. The comparative analysis of salivary proteome in Multiple Sclerosis patients with respect to controls allowed the identification and the structural characterization of new proteoforms of salivary cystatins never detected before in saliva. Moreover, this study highlighted quantitative alterations at the level of different peptides and proteins of specific glands secretion as well as some proteins non-specifically detectable in oral cavity. The proteoforms detected and characterized in saliva for the first time during this study were cystatin A Thr96→Met and its acetylated derivative; cystatin B N-terminally acetylated and CMC at Cys3; N-terminally truncated cystatin D with the N-terminal Q converted to pyro-E and lacking the first 5 amino acid residues (pGlu-cystatin D Cys26→R Des1-5); N-terminally truncated forms of cystatin SN and SN P11→L lacking the first 4 amino acids (cystatin SN Des1-4 and cystatin SN P11→L Des1-4) and the first 7 amino acids (cystatin SN Des1-7 and cystatin SN P11→L Des1-7); N-terminally truncated cystatin SA lacking the first 7 amino acids (cystatin SA Des1-7); oxidized derivatives of cystatins SN and S1 at W23 and W107. The quantitative analysis on Multiple Sclerosis subject performed on 102 salivary peptides/proteins, showed a high number of statistically variated proteins belonging to cystatins family. Among these, cystatin A Thr96→Met, cystatin SN Des1-4 and SN and P11→L; oxidized derivatives of cystatins SN and S1 were also found with altered level in Multiple Sclerosis with respect to controls group. Moreover, a higher number of protein statistically variated were found among those not specifically secreted from salivary glands such as S100A7, S100A8-SNO, antileukoproteinase and ASVD. This preliminary comparative analysis of salivary proteome in Autoimmune Hepatitis patients with respect to controls allowed to determine that the levels of proteins and peptides secreted by salivary glands, such as S-type cystatins, histatins and statherins and their naturally occurring proteoforms deriving from post-translational modifications were higher in autoimmune subjects respect to control. Further studies will have to be carried out to better explain the high overexpression of proteins involved in the protection of the oral cavity in subjects affected by autoimmune hepatitis. A possible cause could be the simultaneous presence of autoimmune diseases involving the oral cavity in half of patients with Autoimmune Hepatitis recruited for this study. The concomitance of these pathologies could either have damage the oral mucosa more or modified the natural bacterial flora present in the oral cavity or both, generating an over-expression by the protein classes involved in the protection of the oral cavity and explaining its high presence.