The number of hits in each cellular compartment in samples digested by either trypsin (blue) or elastase (orange).

<p>Identified proteins (Progenesis, 2 or more peptides) were categorized using PANTHER classification system. Although tryspin digestion yielded more hits, there was a similar distribution of proteins in the compartments.</p

The interactions of all proteins which were significantly reduced in torn samples compared to control, created using the STRING database.

<p>The different line colours represent the different types of associations between the differentially modulated proteins, which include known interactions such as those identified in the curated database (light blue), and experimentally determined (purple). Other interactions identified were via text mining (green), co-expression (black) and protein homology (grey). Proteins are: COL1A1, Collagen 1(I); COL1A2, Collagen 2(I); COL6A1, Collagen 1(VI); COL6A2, Collagen 2(VI); FBLN1, Fibrillin-1; MFAP5, microfibrillar associated protein 5; COMP, Cartilage oligomeric protein; THSD4, Thrombospondin-4; CILP1, Cartilage intermediate layer protein 1; CILP2, Cartilage intermediate layer protein 2; TGFB1, Latent-transforming growth factor beta-binding protein 2; FBN1, Fibulin-1; TNXB, Tenascin-X; MYOC, Myocilin.</p

The relative abundance of collagens and their subunits in supraspinatus tendon from control and pathologic female and male donors.

<p>Dot plots show biological replicates, means and standard error of the mean. (A) COL1A1, (B) COL1A2, (C) COL3A1, (D), COL5A1, (E) COL5A2, (F) COL6A1, (G) COL6A2, (H) COL6A3 and (I) COL14A1. (J) Floating bars showing minimum and maximum relative abundance of the subunits of collagen I with a line at the mean and (K) the relative abundance of the subunits of collagen VI. (L) The ratio of collagen III to I, average of ratios in individual runs within each group (duplicates per sample, n = 18 per group). *P<0.05, **P<0.01 significantly different from control.</p

Previously published studies confirming the disruption of COL1, COL6, FBN1, COMP and LTBP2 in torn and/or aged tendons.

<p>Previously published studies confirming the disruption of COL1, COL6, FBN1, COMP and LTBP2 in torn and/or aged tendons.</p

A quantitative label-free analysis of the extracellular proteome of human supraspinatus tendon reveals damage to the pericellular and elastic fibre niches in torn and aged tissue

<div><p>Tears of the human supraspinatus tendon are common and often cause painful and debilitating loss of function. Progressive failure of the tendon leading to structural abnormality and tearing is accompanied by numerous cellular and extra-cellular matrix (ECM) changes in the tendon tissue. This proteomics study aimed to compare torn and aged rotator cuff tissue to young and healthy tissue, and provide the first ECM inventory of human supraspinatus tendon generated using label-free quantitative LC-MS/MS. Employing two digestion protocols (trypsin and elastase), we analysed grain-sized tendon supraspinatus biopsies from older patients with torn tendons and from healthy, young controls. Our findings confirm measurable degradation of collagen fibrils and associated proteins in old and torn tendons, suggesting a significant loss of tissue organisation. A particularly marked reduction of cartilage oligomeric matrix protein (COMP) raises the possibility of using changes in levels of this glycoprotein as a marker of abnormal tissue, as previously suggested in horse models. Surprisingly, and despite using an elastase digestion for validation, elastin was not detected, suggesting that it is not highly abundant in human supraspinatus tendon as previously thought. Finally, we identified marked changes to the elastic fibre, fibrillin-rich niche and the pericellular matrix. Further investigation of these regions may yield other potential biomarkers and help to explain detrimental cellular processes associated with tendon ageing and tendinopathy.</p></div

The number of proteins in each cellular compartment of the proteome extracted from supraspinatus tendon samples using tryptic digestion.

<p>126 identified proteins (Progenesis, at least 2 identified peptides) were categorized using PANTHER classification system.</p

The relative abundance of differentially expressed elastic fibre components (A-D) and members of the thrombospondin family associated proteins (E-G).

<p>Dot plots show biological replicates, means and standard error of the mean. (A) fibrillin-1 (FBN1), (B) microfibrillar associated protein 5 (MFAP5), (C) Fibulin-1 (FBLN1), (D) Latent-transforming growth factor beta-binding protein 2 (LTBP2), (E) Thrombospondin-1, (F) Thrombospondin 4, and (G) cartilage oligomeric protein (COMP). (H) Western blot of COMP (around 100kDa), confirming higher prevalence in control compared to female and male torn and aged tendon samples. *P<0.05, **P<0.01 significantly different from control.</p

The ratios of COL3A1 to COL1A1, and of the subunits of Collagen I and Collagen VI in the three groups, Mean(SEM), *P<0.05 **P<0.01 significantly different from control.

<p>The ratios of COL3A1 to COL1A1, and of the subunits of Collagen I and Collagen VI in the three groups, Mean(SEM), *P<0.05 **P<0.01 significantly different from control.</p

The mean relative abundance of differentially expressed extracellular proteins in the elastase digested samples, mean (SEM), (*P<0.05 **P<0.01 significantly different from control).

<p>The mean relative abundance of differentially expressed extracellular proteins in the elastase digested samples, mean (SEM), (*P<0.05 **P<0.01 significantly different from control).</p

The ANOVA P value and mean fold change of differentially expressed extracellular proteins in the torn tissue of female and male patients compared to control in the trypsin digested samples.

<p>(*P <0.05, **P <0.01, significantly different from control in post-test).</p