Schematic diagram of the experimental procedures and 1D SDS gel image after Coomassie staining. (A) GCF samples were collected from healthy individuals and chronic gingivitis, moderate periodontitis and severe periodontitis patients using a gel loading tip. The collected samples were diluted with 0.9% NaCl solution, and the GCF proteins were isolated. Analysis of the GCF proteome was performed using LC-MS/MS. The proteins selected as candidate biomarkers of periodontitis were verified using western blotting and ELISA. (B) Pooled samples from 2 sets were separated by 1D SDS-PAGE and stained with Coomassie Brilliant Blue for the visualization of proteins. The stained proteins were excised in 15 pieces for in-gel trypsin digestion. Peptides were isolated from four pooled samples, 2 normal and 2 chronic periodontitis, and analyzed in duplicate by LC-MS/MS (Additional files 1). All subjects were non-smokers and had no other disease indications or other conditions, e.g., diabetes, arteriosclerosis, hepatitis, AIDS, hypertension or pregnancy.