Figure 3: Our strategy for conducting proteomic studies using bone and soft tissue sarcomas is herein described. To develop biomarkers (blue arrows), we usually employ a three-step process: (i) 2D-DIGE-based target identification, (ii) confirmation, and (iii) validation. For the functional analyses (yellow arrows), we employ protein-based analyses (proteomic technologies) and DNA- and RNA-based analyses. In this article, we described the protein-based analyses used for the functional studies of the identified biomarkers (proteins). To develop biomarkers (blue arrows), surgical samples are collected from patients with bone and soft tissue tumors. We organize both the clinical samples and information to establish efficient strategies. Protein expression profiles are generated using 2D-DIGE and analyzed using data mining to identify biomarker candidates. The protein expression levels of the candidates are confirmed using Western blotting analyses, and/or immunohistochemistry. The diagnostic value of the biomarker candidates is verified using additional large variation cohorts. Finally, the validated biomarkers are subjected to novel clinical applications. In the functional analyses (yellow arrows), we focus on both the interaction proteins and regulated proteins associated with the biomarker proteins as proteomic approaches. The novel findings generated by the functional analyses are verified in validation studies, and/or are used in subsequent studies. Finally, we hope that the novel findings will provide beneficial effects to patients.