Figure 1: The process for 2D-DIGE-based target identification, confirmation, and validation. Surgical samples are collected from patients with osteosarcoma. Collected samples contain proteins associated with clinical information. All protein samples are labeled with different fluorescent dyes (The internal control sample is a mixture of a small portion of all individual samples labeled by Cy3, and the individual samples are labeled by Cy 5). The protein expression profiles are obtained using 2D-DIGE with highly sensitive fluorescent dyes. The protein expression profiles are analyzed to identify candidate biomarkers through data-mining using the proteomic profiles and clinical data. The protein expression levels are then confirmed by a western blot analysis and/or immunohistochemistry. The biomarker candidates are validated using additional large-variation cohorts to develop them for clinical applications.