BackgroundIn recent years, differential analysis of proteinsfrom human saliva, i.e., proteomic analysis, has receivedmuch attention mainly due to its unstressful sampling andits great potential for biomarker research. It is widelyconsidered that saliva is a highly stable medium for proteinsthanks to a large amount of antiprotease agents, even atambient and physiological temperatures.ObjectiveTo find the best protocol for the handling ofsamples, we have investigated the stability of saliva proteinsstored at different temperatures (from−80 to 20°C) by one-and two-dimensional electrophoresis.ResultsAt 20°C, no major changes were observed onprotein one-dimensional profiles following 1 day of storage;however, between 7 days and 30 days, the native alpha-amylase band decreased slightly to give several bands withmolecular weight between 35 and 25 kDa. The samephenomenon appeared after 30 days of storage at 4°C.Two-dimensional analysis of salivary maps revealed degra-dation from day 7 of several protein groups for samplesstored at 20°C.ConclusionAll these findings have to be carefully consid-ered when saliva is collected for clinical proteomicanalysis. We can conclude that, to maintain the optimumstability of saliva proteins, saliva samples should becollected on ice followed by the addition of protease in-hibitor cocktail, centrifuged to remove insoluble material,and stored at−20 or−80°C