Background: There is no plasma marker for detecting oral cancer, one of the most frequent cancers worldwide. We developed a bead-based affinity-fractionated proteomic method to discover a novel plasma marker for oral cancer. Methods: Affinity purification of heparinized plasma with magnetic beads and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis were used to screen potential oral cancer markers. We compiled MS protein profiles for 57 patients with oral cancer and compared them with profiles from 29 healthy controls. The spectra were analyzed statistically using flexAnalysis TM and ClinProt TM bioinformatic software. In each MS analysis, the peak intensities of interest were normalized with an internal standard (adrenocorticotropic hormone 18 -39). For identification, affinity bead-purified plasma protein was subjected to MALDI TOF/TOF analysis followed by Mascot identification of the peptide sequences and a search of the National Center for Biotechnology Information protein database. Results: To optimize MALDI-TOF analysis based on the best discriminator of the cancer and control spectra, copper-chelated beads were used for plasma protein profiling. The within-and between-run CVs for assays were <4% and 7%, respectively. Six markers that differentiated between cancer and control spectra were found,

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