title: Whole-saliva proteolysis and its impact on salivary diagnostics.
abstract: There is growing interest in the use of human whole saliva for diagnostics and disease monitoring as an alternative to blood samples. In contrast to blood, whole saliva is a non-sterile body fluid. Proper hand-ling and storage are required to preserve the integrity of potential biomarkers. We investigated salivary autoproteolytic degradation using a variety of approaches. We determined inhibition of protease activities by monitoring the endogenous proteome. In addition, the stability of highly protease-susceptible proteins-histatin 5, statherin, and PRP1-was assessed. Experimental variables included (a) protease inhibitors, (b) salivary pH, (c) incubation temperatures, and (d) sample heating. A cocktail containing AEBSF, aprotinin, pancreatic trypsin inhibitor, leupeptin, antipain, and EDTA could not prevent histatin 5, statherin, or PRP1 degradation in whole saliva. Among the other treatments evaluated, short-term storage of freshly collected samples on ice was effective without interfering with the chemistry of the proteome. In conclusion, whole saliva contains a unique mixture of enzymes as evidenced from their resilience to protease inhibition. Analytical evidence on protein stability is needed to ensure the validity of salivary biomarker study outcomes. Analysis of the data presented will provide help and guidance for the use of saliva samples for diagnostic purposes.
authors.affiliation: Boston University Henry M. Goldman School of Dental Medicine, Dept. of Periodontology and Oral Biology, 700 Albany Street, CABR W201, Boston, MA 02118, USA.
authors.forename: K
authors.initials: K
authors.lastname: Thomadaki
authors.forename: E J
authors.initials: EJ
authors.lastname: Helmerhorst
authors.forename: N
authors.initials: N
authors.lastname: Tian
authors.forename: X
authors.initials: X
authors.lastname: Sun
authors.forename: W L
authors.initials: WL
authors.lastname: Siqueira
authors.forename: D R
authors.initials: DR
authors.lastname: Walt
authors.forename: F G
authors.initials: FG
authors.lastname: Oppenheim
mesh_terms: D015415:Biomarkers / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D054808:Histatins / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D010447:Peptide Hydrolases / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D055550:Protein Stability; D020543:Proteome / Q000737:chemistry; D012463:Saliva / Q000201:enzymology*; D055273:Salivary Proline-Rich Proteins / Q000378:metabolism; D012471:Salivary Proteins and Peptides / Q000737:chemistry* / Q000378:metabolism*; D013048:Specimen Handling; D013696:Temperature
chemical_list: D015415:Biomarkers; C068075:HTN3 protein, human; D054808:Histatins; D011480:Protease Inhibitors; D020543:Proteome; C018149:STATH protein, human; D055273:Salivary Proline-Rich Proteins; D012471:Salivary Proteins and Peptides; D010447:Peptide Hydrolases