Figure 1.

Shotgun LC-MS/MS workflow and analysis

Proteins extracted from eight saliva samples from two HVs, three post-transplanted patients without oral GVHD, and three post-transplanted patients with oral GVHD were proteolytically digested and labeled with isobaric iTRAQ tags. Peptides are then pooled at equal concentrations, fractioned using HPLC, and analyzed by mass spectrometer. Database search and bioinformatics procedures were used for protein identification, quantification, and selection of putative candidate biomarkers. Validation was performed by either WB analysis or immunofluorescence of two different cohorts of patients and five additional HVs.