Table2_Acute exposure to ultraviolet radiation targets proteins involved in collagen fibrillogenesis.XLSX

Introduction: Exposure to chronic, low-dose UV irradiation (UVR) can lead to premature ageing of the skin. Understanding which proteins are affected by acute UVR and photo-dynamically produced reactive oxygen species (ROS) could help to inform strategies to delay photoageing. Conventional biochemical analyses can be used to characterize UVR/ROS-induced damage on a protein-by-protein basis and we have previously shown using SDS-PAGE that collagen I and plasma fibronectin are respectively resistant and susceptible to physiological doses of UVR. The aim of this study was to screen a complex proteome for UVR-affected proteins.
Methods: This study employed a sensitive mass spectrometry technique (peptide location fingerprinting: PLF) which can identify structure associated differences following trypsin digestion to characterize the impact of UVR exposure on purified collagen I and tissue fibronectin and to identify UVR-susceptible proteins in an ECM-enriched proteome.
Results: Using LC/MS-MS and PLF we show that purified mature type-I collagen is resistant to UVR, whereas purified tissue fibronectin is susceptible. UV irradiation of a human dermal fibroblast-deposited ECM-enriched proteome in vitro, followed by LC/MS-MS and PLF analysis revealed two protein cluster groups of UV susceptible proteins involved in i) matrix collagen fibril assembly and ii) protein translation and motor activity. Furthermore, PLF highlighted UV susceptible domains within targeted matrix proteins, suggesting that UV damage of matrix proteins is localized.
Discussion: Here we show that PLF can be used to identify protein targets of UVR and that collagen accessory proteins may be key targets in UVR exposed tissues.

引言：长期低剂量紫外线照射（UV irradiation, UVR）暴露可诱发皮肤过早老化。明确受急性UVR及光动力产生活性氧（reactive oxygen species, ROS）调控的蛋白靶点，可为延缓光老化的干预策略提供理论依据。传统生化分析可实现单蛋白水平的表征，以解析UVR/ROS诱导的蛋白损伤；本团队此前通过SDS-PAGE实验证实，胶原蛋白I型与血浆纤连蛋白分别对生理剂量的UVR具有抗性与易感性。本研究旨在从复杂蛋白质组中筛选受UVR影响的蛋白。
方法：本研究采用高灵敏度质谱技术——肽段定位指纹图谱（peptide location fingerprinting, PLF），该技术可识别胰蛋白酶消化后与蛋白结构相关的差异，以此表征UVR暴露对纯化胶原蛋白I型及组织纤连蛋白的影响，并从富集细胞外基质（extracellular matrix, ECM）的蛋白质组中筛选UVR易感蛋白。
结果：通过液相色谱-串联质谱（LC/MS-MS）与PLF分析，本研究证实纯化的成熟I型胶原蛋白对UVR具有抗性，而纯化的组织纤连蛋白则易受UVR损伤。对体外培养的人真皮成纤维细胞沉积的富集ECM蛋白质组进行UV照射，随后通过LC/MS-MS与PLF分析，发现两类UV易感蛋白簇：其一参与基质胶原纤维组装，其二参与蛋白质翻译与分子运动活性。此外，PLF技术还明确了靶向基质蛋白内部的UV易感结构域，提示基质蛋白的UV损伤具有局域特异性。
讨论：本研究证实，PLF技术可用于识别UVR的蛋白作用靶点，且胶原蛋白辅助蛋白可能是UVR暴露组织中的关键作用靶点。