X-Ray Exposure Induces Structural Changes in Human Breast Proteins

During radiotherapy, X-rays can deliver significant doses of ionising radiation to both cancerous and surrounding healthy tissues, often leading to undesirable side effects that compromise patient outcomes. While the cellular effects of such therapeutic x-ray exposures are well studied, the impact on extracellular matrix (ECM) proteins, which are critical to tissue integrity, remains poorly understood. This study characterises the response of breast proteins, including the major ECM components collagen I and fibronectin, to therapeutic X-rays doses of 50Gy (as employed in breast radiotherapy) and 100Gy, using a combination of gel electrophoresis, biochemical assays, and mass spectrometry-based peptide location fingerprinting (PLF) analysis. In purified protein solutions, X-ray exposure led to fragmentation of constituent α chains of collagen I, and in fibronectin localised structural modifications (as detected by LC-MS/MS peptide location fingerprinting [PLF]) which increased its binding affinity for collagen I. In complex environments, such as newly synthesised fibroblast-derived ECM (fECM) and mature ex vivo breast tissue, therapeutic x-rays induced proteolytic changes in, not just collagen I and fibronectin, but also key basement membrane proteins, including collagen IV, laminin, nidogen-1, and perlecan. Intracellular proteins associated with gene expression (RPS3, MeCP2), cytoskeleton (moesin, plectin), and the endoplasmic reticulum (calnexin) were also found to be structurally compromised. These structural changes may impair the ECM integrity and alter cell-ECM interactions, with potential implications for tissue stiffening, fibrosis, and impaired wound healing in irradiated tissues.