Proteomic Pretreatment Strategies for Increased Diagnostic Value of Oral Fluids

INTODUCTION: The usage of oral fluids (OF) as a diagnostic tool has many advantages. However, two major barriers are i. Many informative analytes are generally present in lower amounts in OF than in serum and ii. The protein profile of OF is dominated by a subset of abundant proteins mainly salivary alpha amylases (sAA), albumins (Alb) and immunoglobulins (IgGs) that mask potential detection of low abundance biomarkers. AIM: To analyze human OF proteins composition following specific-depletion of high and medium abundance proteins. MATERIALS and METHODS: Pulled OF samples were obtained from 5 healthy volunteers and thereafter were subjected to sAA removing device. The filtrate was then transferred into Alb and IgG immuno-depletion column (Proteoprep, Sigma) followed by a multi immuno-depletion column (Proteoprep20S, Sigma), to remove 18 medium abundance proteins. Samples from none treated, triple-depleted and multi-depleted OF samples were analyzed by SDS-PAGE 2-DE and protein identification by ion-trap mass-spectrometry. RESULTS: The triple-depletion technique unmasked 36 spots, and increased the intensity of 58 spots. The multi depletion process revealed 45 new or increased intensity spots. CONCLUSION: Triple-depletion treatment improves the visualization of low abundance OF proteins and help to reveal new or low intensity spots in the 2-DE. The multi-depletion treatment intensifies the improvement process and provides high visibility of the low abundance OF proteins.