Abstract
In ageing tissues, long-lived extracellular matrix (ECM) proteins are susceptible to the accumulation of structural damage due to diverse mechanisms including glycation, oxidation and protease cleavage. Peptide location fingerprinting (PLF) is a new mass spectrometry (MS) analysis technique capable of identifying proteins exhibiting structural differences in complex proteomes. PLF applied to published young and aged intervertebral disc (IVD) MS datasets (posterior, lateral and anterior regions of the annulus fibrosus) identified 268 proteins with age-associated structural differences. For several ECM assemblies (collagens I, II and V and aggrecan), these differences were markedly conserved between degeneration-prone (posterior and lateral) and -resistant (anterior) regions. Significant differences in peptide yields, observed within collagen I α2, collagen II α1 and collagen V α1, were located within their triple-helical regions and/or cleaved C-terminal propeptides, indicating potential accumulation of damage and impaired maintenance. Several proteins (collagen V α1, collagen II α1 and aggrecan) also exhibited tissue region (lateral)-specific differences in structure between aged and young samples, suggesting that some ageing mechanisms may act locally within tissues. This study not only reveals possible age-associated differences in ECM protein structures which are tissue-region specific, but also highlights the ability of PLF as a proteomic tool to aid in biomarker discovery.
Highlights
Ageing and degeneration of intervertebral disc (IVD) are the result of genetics, environments and their interplays [5], this study focused predominantly on the identification of extracellular matrix (ECM)
This was successfully enabled through the application of Peptide location fingerprinting (PLF) to historic LC–mass spectrometry (MS)/MS datasets generated from the inner annulus fibrosus (AF) of posterior, lateral and anterior regions of young and aged human
Many of these affected proteins were region-specific, several were identified in all three regions tested
Summary
Since degeneration in the ageing disc is profoundly regional [8,9], and PLF can be applied post hoc to historical LC–MS/MS data (as shown for ageing tendon [25]), further analysis of the DIPPER dataset presents us with a timely opportunity to test: (i) the hypothesis that protein structures (as indicated by peptide yield pattern) are spatially and potentially age-dependent in the IVD and (ii) the ability of PLF to detect protein structural differences within localised tissue regions. IVD regions (posterior, left lateral and anterior; Figure 1a) This enabled the detection of structural differences in ECM proteins between aged and young IVDs, which could be further compared between degeneration-prone (posterior and lateral) and -resistant (anterior) regions of the IVD (Figure 1b), revealing potentially age-affected proteins with tissue region-specific peptide yield patterns. To visualise these differences along the protein structure, average PSM counts per segment in the young group were subtracted from those in the aged one and compared between posterior, left lateral and anterior IVD regions
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