Proteomics analysis of cells in whole saliva from oral cancer patients via value-added three-dimensional peptide fractionation and tandem mass spectrometry

Whole human saliva possesses tremendous potential in clinical diagnostics, particularly for conditions within the oral cavity such as oral cancer. Although many have studied the soluble fraction of whole saliva, few have taken advantage of the diagnostic potential of the cells present in saliva, and none have taken advantage of proteomics capabilities for their study. We report on a novel proteomics method with which we characterized for the first time cells contained in whole saliva from patients diagnosed with oral squamous cell carcinoma. Our method uses three dimensions of peptide fractionation, combining the following steps: preparative IEF using free flow electrophoresis, strong cation exchange step gradient chromatography, and microcapillary reverse-phase liquid chromatography. We determined that the whole saliva samples contained enough cells, mostly exfoliated epithelial cells, providing adequate amounts of total protein for proteomics analysis. From a mixture of four oral cancer patient samples, the analysis resulted in a catalogue of over 1000 human proteins, each identified from at least two peptides, including numerous proteins with a role in oral squamous cell carcinoma signaling and tumorigenesis pathways. Additionally proteins from over 30 different bacteria were identified, some of which putatively contribute to cancer development. The combination of preparative IEF followed by strong cation exchange chromatography effectively fractionated the complex peptide mixtures despite the closely related physiochemical peptide properties of these separations (pl and solution phase charge, respectively). Furthermore compared with our two-step method combining preparative IIEF and reverse-phase liquid chromatography, our three-step method identified significantly more cellular proteins while retaining higher confidence protein identification enabled by peptide pl information gained through IEF. Thus, for detecting salivary markers of oral cancer and possibly other conditions of the oral cavity, the results confirm both the potential of analyzing the cells in whole saliva and doing so with our proteomics method.