Toward a standardized saliva proteome analysis methodology.

The present study aimed the evaluation of saliva sample pre-treatment, in particular the sample clearance usually performed by centrifugation, to the contribution of salivary proteome and peptidome. Using in-gel and off-gel approaches, a large content of salivary proteins was detected in the pellet fraction that is usually discarded. In addition, chaotropic/detergent treatment in combination with sonication, before the centrifugation step, resulted in salivary complex disruption and consequently in the extraction of high amounts of proteins. Based on this data, we suggest the use of urea/detergent with sonication as a standard saliva sample pre-treatment procedure. We also described a procedure to extract salivary peptides which can be performed even after saliva sample treatment with chaotropic/detergents. In overall, we reported for the first time the contribution of the pellet fraction to the whole saliva proteome. iTRAQ analysis highlighted a higher number of different peptides as well as distinct quantities of each protein class when after sample treatment with urea and sonication, acetone precipitation followed by solubilization with acetonitrile/HCl was performed.