Identification of the proteolytic signature in CVB3-infected cells.

Coxsackievirus B3 (CVB3) encodes proteinases that are essential for processing of the translated viral polyprotein. Viral proteinases also target host proteins to manipulate cellular processes and evade innate antiviral responses to promote replication and infection. While some host protein substrates of the CVB3 3C and 2A cysteine proteinases have been identified, the full repertoire of targets is not known. Here, we utilize an unbiased quantitative proteomics-based approach termed terminal amine isotopic labeling of substrates (TAILS) to conduct a global analysis of CVB3 protease-generated N-terminal peptides in both human HeLa and mouse cardiomyocyte (HL-1) cell lines infected with CVB3. We identified >800 proteins that are cleaved in CVB3-infected HeLa and HL-1 cells including the viral polyprotein, known substrates of viral 3C proteinase such as PABP, DDX58, and HNRNPs M, K, and D and novel cellular proteins. Network and GO-term analysis showed an enrichment in biological processes including immune response and activation, RNA processing, and lipid metabolism. We validated a subset of candidate substrates that are cleaved under CVB3 infection and some are direct targets of 3C proteinase in vitro. Moreover, depletion of a subset of TAILS-identified target proteins decreased viral yield. Characterization of two target proteins showed that expression of 3Cpro-targeted cleaved fragments of emerin and aminoacyl-tRNA synthetase complex-interacting multifunctional protein 2 modulated autophagy and the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway, respectively. The comprehensive identification of host proteins targeted during virus infection provides insights into the cellular pathways manipulated to facilitate infection. IMPORTANCE: RNA viruses encode proteases that are responsible for processing viral proteins into their mature form. Viral proteases also target and cleave host cellular proteins; however, the full catalog of these target proteins is incomplete. We use a technique called terminal amine isotopic labeling of substrates (TAILS), an N-terminomics to identify host proteins that are cleaved under virus infection. We identify hundreds of cellular proteins that are cleaved under infection, some of which are targeted directly by viral protease. Revealing these target proteins provides insights into the host cellular pathways and antiviral signaling factors that are modulated to promote virus infection and potentially leading to virus-induced pathogenesis.

Substrate O-glycosylation actively regulates extracellular proteolysis.

Extracellular proteolysis critically regulates cellular and tissue responses and is often dysregulated in human diseases. The crosstalk between proteolytic processing and other major post-translational modifications (PTMs) is emerging as an important regulatory mechanism to modulate protease activity and maintain cellular and tissue homeostasis. Here, we focus on matrix metalloproteinase (MMP)-mediated cleavages and N-acetylgalactosamine (GalNAc)-type of O-glycosylation, two major PTMs of proteins in the extracellular space. We investigated the influence of truncated O-glycan trees, also referred to as Tn antigen, following the inactivation of C1GALT1-specific chaperone 1 (COSMC) on the general and MMP9-specific proteolytic processing in MDA-MB-231 breast cancer cells. Quantitative assessment of the proteome and N-terminome using terminal amine isotopic labelling of substrates (TAILS) technology revealed enhanced proteolysis by MMP9 within the extracellular proteomes of MDA-MB-231 cells expressing Tn antigen. In addition, we detected substantial modifications in the proteome and discovered novel ectodomain shedding events regulated by the truncation of O-glycans. These results highlight the critical role of mature O-glycosylation in fine-tuning proteolytic processing and proteome homeostasis by modulating protein susceptibility to proteolytic degradation. These data suggest a complex interplay between proteolysis and O-GalNAc glycosylation, possibly affecting cancer phenotypes.

CLIPPER 2.0: Peptide-Level Annotation and Data Analysis for Positional Proteomics.

Positional proteomics methodologies have transformed protease research, and have brought mass spectrometry (MS)-based degradomics studies to the forefront of protease characterization and system-wide interrogation of protease signaling. Considerable advancements in both sensitivity and throughput of liquid chromatography (LC)-MS/MS instrumentation enable the generation of enormous positional proteomics datasets of natural and protein termini and neo-termini of cleaved protease substrates. However, concomitant progress has not been observed to the same extent in data analysis and post-processing steps, arguably constituting the largest bottleneck in positional proteomics workflows. Here, we present a computational tool, CLIPPER 2.0, that builds on prior algorithms developed for MS-based protein termini analysis, facilitating peptide-level annotation and data analysis. CLIPPER 2.0 can be used with several sample preparation workflows and proteomics search algorithms and enables fast and automated database information retrieval, statistical and network analysis, as well as visualization of terminomic datasets. We demonstrate the applicability of our tool by analyzing GluC and MMP9 cleavages in HeLa lysates. CLIPPER 2.0 is available at https://github.com/UadKLab/CLIPPER-2.0.

Combined genetic-pharmacologic inactivation of tightly linked ADAMTS proteases in temporally specific windows uncovers distinct roles for versican proteolysis and glypican-6 in cardiac development.

Extracellular matrix remodeling mechanisms are understudied in cardiac development and congenital heart defects. We show that matrix-degrading metalloproteases ADAMTS1 and ADAMTS5, are extensively co-expressed during mouse cardiac development. The mouse mutants of each gene have mild cardiac anomalies, however, their combined genetic inactivation to elicit cooperative roles is precluded by tight gene linkage. Therefore, we coupled Adamts1 inactivation with pharmacologic ADAMTS5 blockade to uncover stage-specific cooperative roles and investigated their potential substrates in mouse cardiac development. ADAMTS5 blockade was achieved in Adamts1 null mouse embryos using an activity-blocking monoclonal antibody during distinct developmental windows spanning myocardial compaction or cardiac septation and outflow tract rotation. Synchrotron imaging, RNA in situ hybridization, immunofluorescence microscopy and electron microscopy were used to determine the impact on cardiac development and compared to Gpc6 and ADAMTS-cleavage resistant versican mutants. Mass spectrometry-based N-terminomics was used to seek relevant substrates. Combined inactivation of ADAMTS1 and ADAMTS5 prior to 12.5 days of gestation led to dramatic accumulation of versican-rich cardiac jelly and inhibited formation of compact and trabecular myocardium, which was also observed in mice with ADAMTS cleavage-resistant versican. Combined inactivation after 12.5 days impaired outflow tract development and ventricular septal closure, generating a tetralogy of Fallot-like defect. N-terminomics of combined ADAMTS knockout and control hearts identified a cleaved glypican-6 peptide only in the controls. ADAMTS1 and ADAMTS5 expression in cells was associated with specific glypican-6 cleavages. Paradoxically, combined ADAMTS1 and ADAMTS5 inactivation reduced cardiac glypican-6 and outflow tract Gpc6 transcription. Notably, Gpc6-/- hearts demonstrated similar rotational defects as combined ADAMTS inactivated hearts and both had reduced hedgehog signaling. Thus, versican proteolysis in cardiac jelly at the canonical Glu441-Ala442 site is cooperatively mediated by ADAMTS1 and ADAMTS5 and required for proper ventricular cardiomyogenesis, whereas, reduced glypican-6 after combined ADAMTS inactivation impairs hedgehog signaling, leading to outflow tract malrotation.

mTORC1-CTLH E3 ligase regulates the degradation of HMG-CoA synthase 1 through the Pro/N-degron pathway.

Mammalian target of rapamycin (mTOR) senses changes in nutrient status and stimulates the autophagic process to recycle amino acids. However, the impact of nutrient stress on protein degradation beyond autophagic turnover is incompletely understood. We report that several metabolic enzymes are proteasomal targets regulated by mTOR activity based on comparative proteome degradation analysis. In particular, 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) synthase 1 (HMGCS1), the initial enzyme in the mevalonate pathway, exhibits the most significant half-life adaptation. Degradation of HMGCS1 is regulated by the C-terminal to LisH (CTLH) E3 ligase through the Pro/N-degron motif. HMGCS1 is ubiquitylated on two C-terminal lysines during mTORC1 inhibition, and efficient degradation of HMGCS1 in cells requires a muskelin adaptor. Importantly, modulating HMGCS1 abundance has a dose-dependent impact on cell proliferation, which is restored by adding a mevalonate intermediate. Overall, our unbiased degradomics study provides new insights into mTORC1 function in cellular metabolism: mTORC1 regulates the stability of limiting metabolic enzymes through the ubiquitin system.

Extracellular proteolysis in cancer: Proteases, substrates, and mechanisms in tumor progression and metastasis.

A vast ensemble of extracellular proteins influences the development and progression of cancer, shaped and reshaped by a complex network of extracellular proteases. These proteases, belonging to the distinct classes of metalloproteases, serine proteases, cysteine proteases, and aspartic proteases, play a critical role in cancer. They often become dysregulated in cancer, with increases in pathological protease activity frequently driven by the loss of normal latency controls, diminished regulation by endogenous protease inhibitors, and changes in localization. Dysregulated proteases accelerate tumor progression and metastasis by degrading protein barriers within the extracellular matrix (ECM), stimulating tumor growth, reactivating dormant tumor cells, facilitating tumor cell escape from immune surveillance, and shifting stromal cells toward cancer-promoting behaviors through the precise proteolysis of specific substrates to alter their functions. These crucial substrates include ECM proteins and proteoglycans, soluble proteins secreted by tumor and stromal cells, and extracellular domains of cell surface proteins, including membrane receptors and adhesion proteins. The complexity of the extracellular protease web presents a significant challenge to untangle. Nevertheless, technological strides in proteomics, chemical biology, and the development of new probes and reagents are enabling progress and advancing our understanding of the pivotal importance of extracellular proteolysis in cancer.

N-Terminomic Identification of Intracellular MMP-2 Substrates in Cardiac Tissue.

Proteases are enzymes that induce irreversible post-translational modifications by hydrolyzing amide bonds in proteins. One of these proteases is matrix metalloproteinase-2 (MMP-2), which has been shown to modulate extracellular matrix remodeling and intracellular proteolysis during myocardial injury. However, the substrates of MMP-2 in heart tissue are limited, and lesser known are the cleavage sites. Here, we used degradomics to investigate the substrates of intracellular MMP-2 in rat ventricular extracts. First, we designed a novel, constitutively active MMP-2 fusion protein (MMP-2-Fc) that we expressed and purified from mammalian cells. Using this protease, we proteolyzed ventricular extracts and used subtiligase-mediated N-terminomic labeling which identified 95 putative MMP-2-Fc proteolytic cleavage sites using mass spectrometry. The intracellular MMP-2 cleavage sites identified in heart tissue extracts were enriched for proteins primarily involved in metabolism, as well as the breakdown of fatty acids and amino acids. We further characterized the cleavage of three of these MMP-2-Fc substrates based on the gene ontology analysis. We first characterized the cleavage of sarco/endoplasmic reticulum calcium ATPase (SERCA2a), a known MMP-2 substrate in myocardial injury. We then characterized the cleavage of malate dehydrogenase (MDHM) and phosphoglycerate kinase 1 (PGK1), representing new cardiac tissue substrates. Our findings provide insights into the intracellular substrates of MMP-2 in cardiac cells, suggesting that MMP-2 activation plays a role in cardiac metabolism.

Ion Mobility-Based Enrichment-Free N-Terminomics Analysis Reveals Novel Legumain Substrates in Murine Spleen.

Aberrant levels of the asparaginyl endopeptidase legumain have been linked to inflammation, neurodegeneration, and cancer, yet our understanding of this protease is incomplete. Systematic attempts to identify legumain substrates have been previously confined to in vitro studies, which fail to mirror physiological conditions and obscure biologically relevant cleavage events. Using high-field asymmetric waveform ion mobility spectrometry (FAIMS), we developed a streamlined approach for proteome and N-terminome analyses without the need for N-termini enrichment. Compared to unfractionated proteomic analysis, we demonstrate FAIMS fractionation improves N-termini identification by >2.5 fold, resulting in the identification of >2882 unique N-termini from limited sample amounts. In murine spleens, this approach identifies 6366 proteins and 2528 unique N-termini, with 235 cleavage events enriched in WT compared to legumain-deficient spleens. Among these, 119 neo-N-termini arose from asparaginyl endopeptidase activities, representing novel putative physiological legumain substrates. The direct cleavage of selected substrates by legumain was confirmed using in vitro assays, providing support for the existence of physiologically relevant extra-lysosomal legumain activity. Combined, these data shed critical light on the functions of legumain and demonstrate the utility of FAIMS as an accessible method to improve depth and quality of N-terminomics studies.

Data-Independent Acquisition Proteomics and N-Terminomics Methods Reveal Alterations in Mitochondrial Function and Metabolism in Ischemic-Reperfused Hearts.

Myocardial ischemia-reperfusion (IR) (stunning) injury triggers changes in the proteome and degradome of the heart. Here, we utilize quantitative proteomics and comprehensive degradomics to investigate the molecular mechanisms of IR injury in isolated rat hearts. The control group underwent aerobic perfusion, while the IR injury group underwent 20 min of ischemia and 30 min of reperfusion to induce a stunning injury. As MMP-2 activation has been shown to contribute to myocardial injury, hearts also underwent IR injury with ARP-100, an MMP-2-preferring inhibitor, to dissect the contribution of MMP-2 to IR injury. Using data-independent acquisition (DIA) and mass spectroscopy, we quantified 4468 proteins in ventricular extracts, whereby 447 proteins showed significant alterations among the three groups. We then used subtiligase-mediated N-terminomic labeling to identify more than a hundred specific cleavage sites. Among these protease substrates, 15 were identified following IR injury. We identified alterations in numerous proteins involved in mitochondrial function and metabolism following IR injury. Our findings provide valuable insights into the biochemical mechanisms of myocardial IR injury, suggesting alterations in reactive oxygen/nitrogen species handling and generation, fatty acid metabolism, mitochondrial function and metabolism, and cardiomyocyte contraction.

Redefining metalloproteases specificity through network proteolysis.

Proteolytic processes on cell surfaces and extracellular matrix (ECM) sustain cell behavior and tissue integrity in health and disease. Matrix metalloproteases (MMPs) and a disintegrin and metalloproteases (ADAMs) remodel cell microenvironments through irreversible proteolysis of ECM proteins and cell surface bioactive molecules. Pan-MMP inhibitors in inflammation and cancer clinical trials have encountered challenges due to promiscuous activities of MMPs. Systems biology advances revealed that MMPs initiate multifactorial proteolytic cascades, creating new substrates, activating or suppressing other MMPs, and generating signaling molecules. This review highlights the intricate network that underscores the role of MMPs beyond individual substrate-enzyme activities. Gaining insight into MMP function and tissue specificity is crucial for developing effective drug discovery strategies and novel therapeutics. This requires considering the dynamic cellular processes and consequences of network proteolysis.

Positional proteomics: is the technology ready to study clinical cohorts?

INTRODUCTION: Positional proteomics provides proteome-wide information on protein termini and their modifications, uniquely enabling unambiguous identification of site-specific, limited proteolysis. Such proteolytic cleavage irreversibly modifies protein sequences resulting in new proteoforms with distinct protease-generated neo-N and C-termini and altered localization and activity. Misregulated proteolysis is implicated in a wide variety of human diseases. Protein termini, therefore, constitute a huge, largely unexplored source of specific analytes that provides a deep view into the functional proteome and a treasure trove for biomarkers. AREAS COVERED: We briefly review principal approaches to define protein termini and discuss recent advances in method development. We further highlight the potential of positional proteomics to identify and trace specific proteoforms, with a focus on proteolytic processes altered in disease. Lastly, we discuss current challenges and potential for applying positional proteomics in biomarker and pre-clinical research. EXPERT OPINION: Recent developments in positional proteomics have provided significant advances in sensitivity and throughput. In-depth analysis of proteolytic processes in clinical cohorts thus appears feasible in the near future. We argue that this will provide insights into the functional state of the proteome and offer new opportunities to utilize proteolytic processes altered or targeted in disease as specific diagnostic, prognostic and companion biomarkers.

Sensitive and High-Throughput Exploration of Protein N-Termini by TMT-TAILS N-Terminomics.

Terminal amine isotopic labeling of substrates (TAILS) is a sensitive and robust quantitative mass spectrometry (MS)-based proteomics method used for the characterization of physiological or proteolytically processed protein N-termini, as well as other N-terminal posttranslational modifications (PTMs). TAILS is a well-established, high-throughput, negative enrichment workflow that enables system-wide exploration of N-terminomes independent of sample complexity. TAILS makes use of amine reactivity of free N-termini and a highly efficient aldehyde-functionalized polymer to deplete internal peptides generated after proteolytic digestion during sample preparation. Thereby, it enriches for natural N-termini, allowing for unbiased and complete investigation of differential proteolysis, protease substrate discovery, and analysis of N-terminal PTMs. In this chapter, we provide a state-of-the-art protocol, with detailed steps in all parts of the TAILS sample preparation, MS analysis, and post-processing of acquired data.

Innovative workflow for the identification of cathepsin K cleavage sites in type I collagen.

Since the late 1990s, cathepsin K cleavage sites in type I collagen have been extensively studied due to its ability to release bone resorption biomarkers such as CTX and NTX. However, gel-based methods and N-sequencing used in these studies lack sensitivity, especially for small to medium peptides. In this work, we propose a degradomics mass spectrometry-based workflow that combines protein digestion, Nano-LC-UDMSE, and several software tools to identify cathepsin K cleavage sites. This workflow not only identified previously known cleavage sites, but also discovered new ones. Multiple cleavage hotspots were found and described in type I α1 and type I α2 collagen, many of which coincided with pyridinoline crosslinks, known to stabilize the triple helix. Our results allowed us to establish a chronology of digestion and conclude that cathepsin K preferentially cleaves the extremities of type I collagen before the helical part. We also found that cathepsin K preferentially cleaves amino acid residues with long and hydrophobic lateral chains at the beginning of digestion, whereas no preferred amino acid residues were identified later in the digestion. In conclusion, our workflow successfully identified new cleavage sites and can be easily applied to other proteins or proteases.

Degradomic Identification of Membrane Type 1-Matrix Metalloproteinase as an ADAMTS9 and ADAMTS20 Substrate.

The secreted metalloproteases ADAMTS9 and ADAMTS20 are implicated in extracellular matrix proteolysis and primary cilium biogenesis. Here, we show that clonal gene-edited RPE-1 cells in which ADAMTS9 was inactivated, and which constitutively lack ADAMTS20 expression, have morphologic characteristics distinct from parental RPE-1 cells. To investigate underlying proteolytic mechanisms, a quantitative terminomics method, terminal amine isotopic labeling of substrates was used to compare the parental and gene-edited RPE-1 cells and their medium to identify ADAMTS9 substrates. Among differentially abundant neo-amino (N) terminal peptides arising from secreted and transmembrane proteins, a peptide with lower abundance in the medium of gene-edited cells suggested cleavage at the Tyr314-Gly315 bond in the ectodomain of the transmembrane metalloprotease membrane type 1-matrix metalloproteinase (MT1-MMP), whose mRNA was also reduced in gene-edited cells. This cleavage, occurring in the MT1-MMP hinge, that is, between the catalytic and hemopexin domains, was orthogonally validated both by lack of an MT1-MMP catalytic domain fragment in the medium of gene-edited cells and restoration of its release from the cell surface by reexpression of ADAMTS9 and ADAMTS20 and was dependent on hinge O-glycosylation. A C-terminally semitryptic MT1-MMP peptide with greater abundance in WT RPE-1 medium identified a second ADAMTS9 cleavage site in the MT1-MMP hemopexin domain. Consistent with greater retention of MT1-MMP on the surface of gene-edited cells, pro-MMP2 activation, which requires cell surface MT1-MMP, was increased. MT1-MMP knockdown in gene-edited ADAMTS9/20-deficient cells restored focal adhesions but not ciliogenesis. The findings expand the web of interacting proteases at the cell surface, suggest a role for ADAMTS9 and ADAMTS20 in regulating cell surface activity of MT1-MMP, and indicate that MT1-MMP shedding does not underlie their observed requirement in ciliogenesis.

In-Depth Characterization of Apoptosis N-Terminome Reveals a Link Between Caspase-3 Cleavage and Posttranslational N-Terminal Acetylation.

The N termini of proteins contain information about their biochemical properties and functions. These N termini can be processed by proteases and can undergo other co- or posttranslational modifications. We have developed LATE (LysN Amino Terminal Enrichment), a method that uses selective chemical derivatization of α-amines to isolate the N-terminal peptides, in order to improve N-terminome identification in conjunction with other enrichment strategies. We applied LATE alongside another N-terminomic method to study caspase-3-mediated proteolysis both in vitro and during apoptosis in cells. This has enabled us to identify many unreported caspase-3 cleavages, some of which cannot be identified by other methods. Moreover, we have found direct evidence that neo-N-termini generated by caspase-3 cleavage can be further modified by Nt-acetylation. Some of these neo-Nt-acetylation events occur in the early phase of the apoptotic process and may have a role in translation inhibition. This has provided a comprehensive overview of the caspase-3 degradome and has uncovered previously unrecognized cross talk between posttranslational Nt-acetylation and caspase proteolytic pathways.

Identification and quantification of degradome components in human synovial fluid reveals an increased proteolytic activity in knee osteoarthritis patients vs controls.

Synovial fluid (SF) may contain cleavage products of proteolytic activities. Our aim was to characterize the degradome through analysis of proteolytic activity and differential abundance of these components in a peptidomic analysis of SF in knee osteoarthritis (OA) patients versus controls (n = 23). SF samples from end-stage knee osteoarthritis patients undergoing total knee replacement surgery and controls, that is, deceased donors without known knee disease were previously run using liquid chromatography mass spectrometry (LC-MS). This data was used to perform new database searches generating results for non-tryptic and semi-tryptic peptides for studies of degradomics in OA. We used linear mixed models to estimate differences in peptide-level expression between the two groups. Known proteolytic events (from the MEROPS peptidase database) were mapped to the dataset, allowing the identification of potential proteases and which substrates they cleave. We also developed a peptide-centric R tool, proteasy, which facilitates analyses that involve retrieval and mapping of proteolytic events. We identified 429 differentially abundant peptides. We found that the increased abundance of cleaved APOA1 peptides is likely a consequence of enzymatic degradation by metalloproteinases and chymase. We identified metalloproteinase, chymase, and cathepsins as the main proteolytic actors. The analysis indicated increased activity of these proteases irrespective of their abundance.

Identifying E3 Ligase Substrates With Quantitative Degradation Proteomics.

Controlled protein degradation by the ubiquitin-proteasome pathway is critical for almost all cellular processes. E3 ubiquitin ligases are responsible for targeting proteins for ubiquitylation and subsequent proteasomal degradation with spatial and temporal precision. While studies have revealed various E3-substrate pairs involved in distinct biological processes, the complete substrate profiles of individual E3 ligases are largely unknown. Here we report a new approach to identify substrates of an E3 ligase for proteasomal degradation using unnatural amino acid incorporation pulse-chase proteomics (degradomics). Applying this approach, we determine the steady-state substrates of the C-terminal to LisH (CTLH) E3 ligase, a multi-component complex with poorly defined substrates. By comparing the proteome degradation profiles of active and inactive CTLH-expressing cells, we successfully identify previously known and new potential substrates of CTLH ligase. Altogether, degradomics can comprehensively identify degradation substrates of an E3 ligase, which can be adapted for other E3 ligases in various cellular contexts.

Comparative Degradome Analysis of the Bovine Piroplasmid Pathogens Babesia bovis and Theileria annulata.

Babesia bovis and Theileria annulata are tick-borne hemoprotozoans that impact bovine health and are responsible for considerable fatalities in tropical and subtropical regions around the world. Both pathogens infect the same vertebrate host, are closely related, and contain similar-sized genomes; however, they differ in invertebrate host specificity, absence vs. presence of a schizont stage, erythrocyte invasion mechanism, and transovarial vs. transstadial transmission. Phylogenetic analysis and bidirectional best hit (BBH) identified a similar number of aspartic, metallo, and threonine proteinases and nonproteinase homologs. In contrast, a considerably increased number of S54 serine rhomboid proteinases and S9 nonproteinase homologs were identified in B. bovis, whereas C1A cysteine proteinases and A1 aspartic nonproteinase homologs were found to be expanded in T. annulata. Furthermore, a single proteinase of families S8 (subtilisin-like protein) and C12 (ubiquitin carboxyl-terminal hydrolase), as well as four nonproteinase homologs, one with dual domains M23-M23 and three with S9-S9, were exclusively present in B. bovis. Finally, a pronounced difference in species-specific ancillary domains was observed between both species. We hypothesize that the observed degradome differences represent functional correlates of the dissimilar life history features of B. bovis and T. annulata. The presented improved classification of piroplasmid proteinases will facilitate an informed choice for future in-depth functional studies.

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. Characterization of ECM components, the matrisome, the extracellular environment and their changes in disease is therefore of importance to understand and mitigate by developing novel therapeutics. Liquid chromatography-mass spectrometry (LC-MS) proteomics has been integral to protein and proteome research for decades and long superseded the obsolescent gel-based approaches. A continuous effort has ensured progress with increased sensitivity and throughput as more advanced equipment has been developed hand in hand with specialized enrichment, detection, and identification methods. Part of this effort lies in the field of degradomics, a branch of proteomics focused on discovering novel protease substrates by identification of protease-generated neo-N termini, the N-terminome, and characterizing the responsible protease networks. Various methods to do so have been developed, some specialized for specific tissue types, others for particular proteases, throughput, or ease of use. This review aims to provide an overview of the state-of-the-art proteomics techniques that have successfully been recently utilized to characterize proteolytic cleavages in the ECM and thereby guided new research and understanding of the ECM and matrisome biology.