Peptide Location Fingerprinting Reveals Tissue Region-Specific Differences in Protein Structures in an Ageing Human Organ

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.

Keywords: ageing; extracellular matrix; intervertebral disc; mass spectrometry; peptide location fingerprinting; proteomics; spine.

Figure 1

Experimental design for the application of PLF to three distinct regions of young and aged IVDs. (a) Raw LC–MS/MS datasets (from two males, one young [16 years old] and one aged [59 years old]), corresponding to the posterior inner AF/NP, left lateral inner AF and anterior inner AF/NP regions of the L3/4, L4/5 and L5/S1 lower lumbar IVDs were downloaded from the PRIDE repository (PXD017740) [13]. (b) After MS/MS peptide identification, the MPLF webtool [25] was used to perform PLF on the identified peptides and compare peptide yields across protein structures between young and aged discs—three vs. three discs for anterior and left lateral datasets and three vs. two discs for posterior datasets (the young anterior L4/5 disc was omitted from the analysis due to the insufficient number of identified peptides for PLF analysis). As previously described [25], ((b), i) proteins were divided into 50-amino acid (aa)-sized segments; the collagen I α2 chain (COL1A2) is shown here as an example. ((b), ii) Peptides were mapped to their respective segments, summed, normalised between aged and young groups based on the median whole protein spectrum count and averaged for each group (average peptide spectrum match [PSM] count per segment). ((b), iii) Peptide yields in each segment were statistically compared between young and aged IVDs using an unpaired, Bonferroni-corrected, repeated-measures ANOVA (* p ≤ 0.05; *** p ≤ 0.001). 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.

Figure 2

ECM-associated proteins make up half of the proteins identified with significant, age-associated structural differences in all three IVD regions. PLF revealed 126 proteins in posterior (Table S4), 117 in left lateral (Table S5) and 165 in anterior (Table S6) IVD regions with significant differences in peptide yield between aged and young IVDs, across their structures. (a) Of these, 38 were shared between the three regions. (b) Classification of these shared proteins revealed ECM as the major class, which included several ECM-associated proteins, collagens, two elastic fibre-specific proteins and two proteoglycans. The affected proteins which were uniquely discovered by PLF and not by label-free quantification of abundance in the DIPPER study [13] (Figure S3) are highlighted in bold.

Figure 3

COL1A2 and PCOLCE protein structures exhibit differences in peptide yield patterns between young and aged proteins, which were conserved in posterior, left lateral and anterior IVD regions. PSMs were summed in each 50-aa protein segment, normalised and averaged for young and aged groups (bar graphs = average, normalised PSMs, error bars = standard deviation). Average PSMs per segment in young were subtracted by those in aged and divided by segment aa length (50) to reveal fluctuations in peptide yield along protein structures (line graph y axes = aged–young PSM counts/segment length, normalised between tissue regions in composite graphs; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001, Bonferroni-corrected, repeated-measures ANOVA; black * in composite line graph = significant in all three IVD regions; functional domains, sourced from UniProt, are indicated). ((a), i) Several COL1A2 segments exhibited significant differences in peptide yield between young and aged samples within the C-terminal half of the protein (blue line: posterior, green: left lateral, red: anterior). ((a), ii) Crucially, these differences in peptide yield along the COL1A2 structure are markedly consistent between all the three regions, with a similarly higher peptide yield displayed in segment 20 of aged compared to young and lower peptide yield in the last three segments, nearest to the C-terminus. ((b), i) The last 50-aa segment on the C-terminal end of PCOLCE also exhibited a significantly lower peptide yield in aged compared to young for the inner AFs of all three IVD regions. ((b), ii) Peptide yield difference patterns along the primary structure were also similar between all the three regions, with a consistently higher peptide yield in aged compared to young measured on the N-terminal side of the protein followed by a lower yield in the last two segments near the C-terminal end.

Figure 4

A total of 166 proteins were identified with significant, age-associated structural differences that were entirely IVD-region specific. The affected proteins which were uniquely discovered by PLF and not by label-free quantification of abundance in the DIPPER study (Figure S3) [13] are highlighted in bold.

Figure 5

COL5A1, COL2A1 and aggrecan all exhibit lateral, inner AF-specific differences in peptide yield not seen in the posterior or anterior IVD regions. PSMs were summed in each 50-aa protein segment, normalised and averaged for young and aged groups (bar graphs = average, normalised PSMs, error bars = standard deviation). Average PSMs per segment in the young group were subtracted by those in the aged one and divided by segment aa length (50) to reveal fluctuations in peptide yield along protein structures (line graph y axes = aged–young PSM counts/segment length, normalised between tissue regions in composite graphs; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001, Bonferroni-corrected, repeated-measures ANOVA; black * in composite line graph = significant in all three IVD regions; functional domains, sourced from UniProt, are indicated). ((a), i) Several segments on the C-terminal end of COL5A1 exhibited significant differences in peptide yields between young and aged samples for all three IVD regions. However, a left lateral-specific significant difference in yield was also observed in its second segment, on the N-terminal end of the protein. ((a), ii) Fluctuations in COL5A1 peptide yield on the C-terminal end follow a similar pattern in all three regions, with a consistent fall and rise of peptide yield observed in aged compared to young samples for the last four segments of its primary structure. However, a clear left lateral-specific increase in peptide yield can also be seen on the N-terminal end. ((b), i) COL2A1 also had several protein segments on the C-terminal side which exhibited significant differences in peptide yield. ((b), ii) These displayed a characteristic rise-and-fall peptide yield pattern in aged compared to young samples, which were markedly consistent between the three IVD regions. However, once again, two COL2A1 segments on the N-terminal side of the protein exhibited significantly higher yields for aged than for young samples that were unique to the left lateral region of the disc. ((c), i) Segments two and three on the N-terminal side of the aggrecan protein (ACAN) also exhibited significant differences in peptide yield between young and aged samples. ((c), ii) These differences were significantly higher in aged aggrecan within the posterior and anterior inner AF regions, compared to young aggrecan, but significantly lower in the left lateral region.