Oral cancer is one of the most frequent cancers worldwide. Tobacco, alcohol and betel quid chewing are the factors for oral cancer, being associated with the increase in the rate of oral cancer incidence in Taiwan. Early diagnosis of oral cancer can provide adequate treatment and increase survival. The goal of the study is to identify oral cancer-specific salivary biomarkers such as peptides and proteins using the proteomic approaches. We collected salivary samples from oral cancer-free individuals and oral cancer patients attended in the China Medical University hospital during 2008 and 2009. Firstly, commercial C8-functionalized magnetic beads were used for salivary peptide separations. C8- affinity peptides were incubated with the matrix 2,5-dihydroxyacetophenone (2,5-DHB) or a-cyano-4-hydroxycinnamic acid (HCCA), and then analyzed using Matrix-Assisted Laser Desoption/Ionization Time-of-flight Mass Spectrometry (MALDI-TOF MS). ClinProtTM Bruker Daltonic analyses revealed that unique mass peak of 4438 Da peptide in 2,5-DHB was in 10% of the saliva from oral cancer-free individuals and 41.18％of the saliva from oral cancer patients with the T2 stage. In addition, unique mass peak of 2918 Da peptide in HCCA was in 0% of the saliva from oral cancer-free individuals and 80％of the saliva from oral cancer patients with the T4 stage. Moreover, MALDI-TOF/TOF MS/MS revealed the amino acid sequence CNSWEVNLQSISEFIINNRNYSTK for 2918 Da peptide, being one fragment of the zinc finger protein 510 (ZNF510). ELISA and Western blotting assays confirmed the amount of 2918 Da peptide in saliva using rabbit polyclonal sera against the 2918 Da peptide. In addition, transferring was identified as the up-regulated protein in the saliva from oral cancer patients using two-dimension gel electrophoresis (2DE) and MALDI-TOF MS. ELISA and Western blotting assays showed a significantly higher protein level in the saliva from oral cancer patients than the saliva from oral cancer-free individuals. The study identified 2918 Da peptide and transferring as oral cancer-specific salivary biomarkers using the proteomic approaches, being helpful for developing the diagnostic kids for oral cancer.
