Quantifying Protein Temporal Changes with Mass Spectrometry in Plasma Samples from Breast Cancer Patients Undergoing Radiotherapy

Radiation therapy (RT) is a central component of cancer treatment, including breast cancer, and primarily damages DNA in cancer cells, causing cell death. However, secondary effects of RT that may influence tumor and normal tissue both within and beyond the site of irradiation are not well characterized, particularly at the level of the circulating blood proteome. In this study, isotopic acrylamide labeling of proteins and high-resolution LC-MS/MS were used to analyze plasma proteins from breast cancer patients before, during, and after RT. Clustering analysis was used to group proteins according to how their levels changed over time. A total of 31 proteins were identified whose levels were altered during RT but returned to baseline following the conclusion of treatment, representing key parts of the immune response to RT. Another set of 31 proteins related to the extracellular matrix and other functions was altered during RT and remained altered after RT. Specific time points were also examined, and acute changes were identified at the start of RT in the levels of 63 proteins involved in coagulation, cell-cell adhesion, and carbohydrate metabolism. After the end of RT, the circulating levels of 23 extracellular matrix proteins remained altered. These LC-MS/MS findings were validated orthogonally by ELISAs of three proteins in a larger group of breast cancer patients undergoing RT. Overall, in this study, temporal changes in the circulating blood proteome of breast cancer patients undergoing RT were characterized, and these altered protein levels were linked to potential alterations in biological pathways.