Peptide location fingerprinting reveals modification-associated biomarkers of ageing in human tissue proteomes

Abstract

Although dysfunctional protein homeostasis (proteostasis) is a key factor in many age-related diseases, the untargeted identification of structural modifications in proteins remains challenging. Peptide location fingerprinting is a proteomic analysis technique capable of identifying structural modification-associated differences in mass spectrometry (MS) datasets of complex biological samples. A new webtool (Manchester Peptide Location Fingerprinter), applied to photoaged and intrinsically aged skin proteomes, can relatively quantify peptides (spectral counting) and map statistically significant differences to regions within protein structures. New photoageing biomarkers were identified in multiple proteins including matrix components (collagens and proteoglycans), oxidation and protease modulators (peroxiredoxins and SERPINs) and cytoskeletal proteins (keratins). Crucially, for many extracellular biomarkers, structural modification-associated differences were not correlated with relative abundance (by ion intensity). By applying peptide location fingerprinting to published MS datasets, (identifying biomarkers including collagen V and versican in ageing tendon) we demonstrate the potential of the MPLF webtool to discover novel biomarkers.

Predicting Proteolysis in Complex Proteomes Using Deep Learning

“…In order to compare the predictive capabilities of PROSPER and DeepCleave against a complex proteome, we decellularized a post-confluent culture of HDFs and digested the remaining ECM with MMP9. Over 60 proteins were identified by LC–MS/MS in the baseline (non-MMP9 digested) matrix, many of which (COL6A1, COL6A2, COL6A3, EMILIN1, COL1A1, COL1A2) are found in the human dermis [ 44 , 45 ]. Putative MMP9 cleavage sites were identified in 40 out of 60 proteins, (including COL6A1, EMILIN1, FBLN2, COL1A2) resulting in 390 putative MMP9 cleavage sites ( Figure 1 d).…”

“…This is consistent with the ability of defensins to act as antioxidant proteins [ 63 ]. Crucially, MPSC not only predicts that known photoaging candidate biomarkers (LOX, TIMP3) will be susceptible to UVR/ROS degradation [ 44 , 64 , 65 ] but also highlights the potential susceptibility (TSHB, CTSB, CTSZ) and resistance (DCD, EMCN, CALM5 and APOC1) of new skin aging biomarker candidates ( Figure 3 c). In complex tissues, degradative mechanisms do not act in isolation [ 36 , 37 ], and we, and others, have shown that UVR exposure can enhance protease-mediated-proteolysis [ 7 , 66 ].…”