Comparative proteomics of cancer stem cells in osteosarcoma using ultra-high-performance liquid chromatography and Orbitrap Fusion mass spectrometer.

Osteosarcoma is the most common malignant tumour found in bones, and it has a poor prognosis. For improved therapy, it is significant to have a deep understanding of the proteomics changes in the cancer stem cells (CSCs) of osteosarcoma. Therefore, a comparative proteomics approach based on ultra-high-performance liquid chromatography coupled to an Orbitrap Fusion mass spectrometer (UHPLC-Orbitrap Fusion MS) was established to investigate the key molecular changes between CSCs and non-CSCs in human osteosarcoma HOS cells. A proteomic analysis was performed on these samples and a total of more than 6600 proteins were identified in each run. Moreover, most of the correlation coefficients between three biological repeats were more than 0.9 in each group. That finding demonstrated not only that the reproducibility of the test is good but also that the stability of this MS is excellent. A label-free quantitative method was applied to analyse differentially expressed proteins. Using the criteria of greater than 1.5-fold changes and a p value < 0.05, 124 proteins were identified as being significantly different between HOS-CSCs and non-CSCs. A pathway analysis of differentially expressed proteins by Ingenuity Pathway Analysis (IPA) revealed the potential molecular regulatory networks that may regulate CSCs. Selected differential α-actinin 4 (ACTN4) proteins were validated by Western blot assay. These findings enhance our understanding of the molecular changes in CSCs and may provide additional improvements in therapy for treating osteosarcoma. Moreover, the UHPLC-Orbitrap Fusion MS-based proteomics method is helpful in cancer research.