Degradomics technologies in matrisome exploration.

Consisting of a defined set of extracellular proteins secreted from resident cells and with minor contributions from serum proteins, the extracellular matrix (ECM) is an essential component of all tissues. Maintaining tissue homeostasis, structural support and cellular control through cell-ECM communication, the ECM has come to be viewed as not just a passive structural entity but rather as a dynamic signaling conduit between cells and the extracellular compartment. Proteins and their cleavage products mediate this communication, and aberrant signaling, either directly or indirectly distorting the ECM, results in pathological conditions including cancer, inflammation, fibrosis, and neurodegenerative diseases.

Comparative Degradomics of Porcine and Human Wound Exudates Unravels Biomarker Candidates for Assessment of Wound Healing Progression in Trauma Patients.

Impaired cutaneous wound healing is a major complication in elderly people and patients suffering from diabetes, the rate of which is rising in industrialized countries. Heterogeneity of clinical manifestations hampers effective molecular diagnostics and decisions for appropriate therapeutic regimens. Using a customized positional quantitative proteomics workflow, we have established a time-resolved proteome and N-terminome resource from wound exudates in a clinically relevant pig wound model that we exploited as a robust template to interpret a heterogeneous dataset from patients undergoing the same wound treatment.

In vivo assessment of protease dynamics in cutaneous wound healing by degradomics analysis of porcine wound exudates.

Proteases control complex tissue responses by modulating inflammation, cell proliferation and migration, and matrix remodeling. All these processes are orchestrated in cutaneous wound healing to restore the skin's barrier function upon injury. Altered protease activity has been implicated in the pathogenesis of healing impairments, and proteases are important targets in diagnosis and therapy of this pathology.

Proteolytic post-translational modification of proteins: proteomic tools and methodology.

Proteolytic processing is a ubiquitous and irreversible post-translational modification involving limited and highly specific hydrolysis of peptide and isopeptide bonds of a protein by a protease. Cleavage generates shorter protein chains displaying neo-N and -C termini, often with new or modified biological activities. Within the past decade, degradomics and terminomics have emerged as significant proteomics subfields dedicated to characterizing proteolysis products as well as natural protein N and C termini.

Systems-level analysis of proteolytic events in increased vascular permeability and complement activation in skin inflammation.

During inflammation, vascular permeability is increased by various proteolytic events, such as the generation of bradykinin, that augment local tissue responses by enabling tissue penetration of serum proteins, including complement and acute-phase proteins. Proteases also govern inflammatory responses by processing extracellular matrix proteins and soluble bioactive mediators. We quantified changes in the proteome and the nature of protein amino termini (the N-terminome) and the altered abundance of murine proteases and inhibitors during skin inflammation.

Protein TAILS: when termini tell tales of proteolysis and function.

Among the hundreds of posttranslational modifications, limited proteolysis, also known as processing, is special: It is irreversible, near ubiquitous, and by trimming peptide chains from their ends or cutting proteins into two, proteolysis forms shorter chains displaying new termini. The unique chemistry and location of α-amino-termini and carboxyl-termini in a protein engender special chemical and physical properties to a protein. Hence, modification of protein termini is often associated with new biological activities of a protein.

Multiplex N-terminome analysis of MMP-2 and MMP-9 substrate degradomes by iTRAQ-TAILS quantitative proteomics.

Proteolysis is a major protein posttranslational modification that, by altering protein structure, affects protein function and, by truncating the protein sequence, alters peptide signatures of proteins analyzed by proteomics. To identify such modified and shortened protease-generated neo-N-termini on a proteome-wide basis, we developed a whole protein isobaric tag for relative and absolute quantitation (iTRAQ) labeling method that simultaneously labels and blocks all primary amines including protein N- termini and lysine side chains. Blocking lysines limits trypsin cleavage to arginine, which effectively elongates the proteolytically truncated peptides for improved MS/MS analysis and peptide identification.

A statistics-based platform for quantitative N-terminome analysis and identification of protease cleavage products.

Terminal amine isotopic labeling of substrates (TAILS), our recently introduced platform for quantitative N-terminome analysis, enables wide dynamic range identification of original mature protein N-termini and protease cleavage products. Modifying TAILS by use of isobaric tag for relative and absolute quantification (iTRAQ)-like labels for quantification together with a robust statistical classifier derived from experimental protease cleavage data, we report reliable and statistically valid identification of proteolytic events in complex biological systems in MS2 mode. The statistical classifier is supported by a novel parameter evaluating ion intensity-dependent quantification confidences of single peptide quantifications, the quantification confidence factor (QCF).

Metadegradomics: toward in vivo quantitative degradomics of proteolytic post-translational modifications of the cancer proteome.

Post-translational modifications enable extra layers of control of the proteome, and perhaps the most important is proteolysis, a major irreversible modification affecting every protein. The intersection of the protease web with a proteome sculpts that proteome, dynamically modifying its state and function. Protease expression is distorted in cancer, so perturbing signaling pathways and the secretome of the tumor and reactive stromal cells.

Protease proteomics: revealing protease in vivo functions using systems biology approaches.

Proteases irreversibly modify proteins by cleaving their amide bonds and are implicated in virtually every important biological process such as immunity, development and tissue repair. Accordingly, it is easy to see that deregulated proteolysis is a pathognomic feature of many diseases. Most of the current information available on proteases was acquired using in vitro methods, which reveals molecular structure, enzyme kinetics and active-site specificity.

Proteome-derived, database-searchable peptide libraries for identifying protease cleavage sites.

We introduce human proteome-derived, database-searchable peptide libraries for characterizing sequence-specific protein interactions. To identify endoprotease cleavage sites, we used peptides in such libraries with protected primary amines to simultaneously determine sequence preferences on the N-terminal (nonprime P) and C-terminal (prime P') sides of the scissile bond. Prime-side cleavage products were tagged with biotin, isolated and identified by tandem mass spectrometry, and the corresponding nonprime-side sequences were derived from human proteome databases using bioinformatics.

Identification of candidate angiogenic inhibitors processed by matrix metalloproteinase 2 (MMP-2) in cell-based proteomic screens: disruption of vascular endothelial growth factor (VEGF)/heparin affin regulatory peptide (pleiotrophin) and VEGF/Connective tissue growth factor angiogenic inhibitory complexes by MMP-2 proteolysis.

Matrix metalloproteinases (MMPs) exert both pro- and antiangiogenic functions by the release of cytokines or proteolytically generated angiogenic inhibitors from extracellular matrix and basement membrane remodeling. In the Mmp2-/- mouse neovascularization is greatly reduced, but the mechanistic aspects of this remain unclear. Using isotope-coded affinity tag labeling of proteins analyzed by multidimensional liquid chromatography and tandem mass spectrometry we explored proteome differences between Mmp2-/- cells and those rescued by MMP-2 transfection.

Proteomics discovery of metalloproteinase substrates in the cellular context by iTRAQ labeling reveals a diverse MMP-2 substrate degradome.

Elucidation of protease substrate degradomes is essential for understanding the function of proteolytic pathways in the protease web and how proteases regulate cell function. We identified matrix metalloproteinase-2 (MMP-2) cleaved proteins, solubilized pericellular matrix, and shed cellular ectodomains in the cellular context using a new multiplex proteomics approach. Tryptic peptides of intact and cleaved proteins, collected from conditioned culture medium of Mmp2(-/-) fibroblasts expressing low levels of transfected active human MMP-2 at different time points, were amine-labeled with iTRAQ mass tags.