Extracellular matrix (ECM) in the intervertebral disc (IVD), lung and artery are thought to undergo the age-dependant accumulation of damage by chronic exposure to mechanisms such as reactive oxygen species, proteases and glycation. It is unknown whether this damage accumulation is species-dependant (via differing lifespans and hence cumulative exposures) or whether it can influence the progression of age-related diseases such as atherosclerosis. Peptide location fingerprinting (PLF) is a new proteomic analysis method, capable of the non-targeted identification of structure-associated changes within proteins. Here we applied PLF to publicly available ageing human IVD (outer annulus fibrosus), ageing mouse lung and human arterial atherosclerosis datasets and bioinformatically identified novel target proteins alongside common age-associated differences within protein structures which were conserved between three ECM-rich organs, two species, three IVD tissue regions, sexes and in an age-related disease. We identify peptide yield differences across protein structures which coincide with biological regions, potentially reflecting the functional consequences of ageing or atherosclerosis for macromolecular assemblies (collagen VI), enzyme/inhibitor activity (alpha-2 macroglobulin), activation states (complement C3) and interaction states (laminins, perlecan, fibronectin, filamin-A, collagen XIV and apolipoprotein-B). Furthermore, we show that alpha-2 macroglobulin and collagen XIV exhibit possible shared structural consequences in IVD ageing and arterial atherosclerosis, providing novel links between an age-related disease and intrinsic ageing. Crucially, we also demonstrate that fibronectin, laminin beta chains and filamin-A all exhibit conserved age-associated structural differences between mouse lung and human IVD, providing evidence that ECM, and their associating proteins, may be subjected to potentially similar mechanisms or consequences of ageing across both species, irrespective of differences in lifespan and tissue function.

椎间盘 (IVD)、肺和动脉中的细胞外基质 (ECM) 被认为会因长期暴露于活性氧、蛋白酶和糖基化等机制而经历与年龄相关的损伤累积。目前尚不清楚这种损伤累积是否取决于物种（通过不同的寿命和因此累积的暴露），或者它是否会影响与年龄相关的疾病（如动脉粥样硬化）的进展。肽定位指纹图谱 (PLF) 是一种新的蛋白质组学分析方法，能够非靶向识别蛋白质内部结构相关的变化。在这里，我们将 PLF 应用于公开可用的老化人类 IVD（外环纤维），老化的小鼠肺和人类动脉粥样硬化数据集和生物信息学鉴定的新靶蛋白以及蛋白质结构中常见的与年龄相关的差异，这些差异在三个富含 ECM 的器官、两个物种、三个 IVD 组织区域、性别和与年龄相关的疾病中是保守的。我们确定了与生物区域一致的蛋白质结构之间的肽产量差异，可能反映了老化或动脉粥样硬化对大分子组装（胶原蛋白 VI）、酶/抑制剂活性（α-2 巨球蛋白）、激活状态（补体 C3）和相互作用的功能性影响状态（层粘连蛋白、perlecan、纤连蛋白、filamin-A、胶原蛋白 XIV 和载脂蛋白-B）。此外，我们表明 alpha-2 巨球蛋白和胶原蛋白 XIV 在 IVD 老化和动脉粥样硬化中表现出可能的共同结构后果，提供了与年龄相关的疾病和内在老化之间的新联系。至关重要的是，我们还证明纤连蛋白、层粘连蛋白 β 链和细丝蛋白 A 在小鼠肺和人类 IVD 之间都表现出与年龄相关的保守结构差异，提供证据表明 ECM 及其相关蛋白可能受到潜在的类似机制或后果无论寿命和组织功能的差异如何，这两个物种都会老化。