Saliva analysis by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF/MS): from sample collection to data analysis

Background: Saliva is one of the most promising and easy-to-collect source of potential biomarkers of oral and systemic disease. We standardized a protocol suitable for pre-analytical treatment and for the analysis of whole normal saliva by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF/MS). Methods: We evaluated the impact of storage time, freeze/thaw cycles, denaturing agents, glycoproteins depletion, centrifugation, type of matrix and ProteinChip((R)) used on the quality of the SELDI protein profile. Moreover, we explored the inter-individual and between-sex differences and the changes in the sample composition over the day. Results: Saliva was qualitatively stable, in the absence of protease inhibitors, for up to 3 h from the collection at room temperature, although the intensity of a number of peaks slightly decreased between 0 and 3 h and the addition of protease inhibitors did not completely revert this trend. The saliva proteome changed during the day and showed relevant between-sex differences. The protein profile remained stable for up to five freeze/thaw cycles. The addition of denaturing solutions and the depletion of glycoproteins improved the quality of the spectra without affecting their reproducibility. Conclusions: We defined a protocol that improved the quality and the reproducibility of SELDI-TOF/MS analysis, thus potentially supporting the search for putative biomarkers of disease.