Terminal Amine Isotopic Labeling Of Substrates Research Articles

Abstract 267: Identification of Proteolytic Substrates for ADAMTS7 Using Terminal Amine Isotope Labeling of Substrates

ADAMTS7 , a member of the disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family, has been recently identified as a novel genetic locus for coronary artery disease. Although a handful of ADAMTS7 substrates have been identified through traditional yeast two-hybrid screening and standard proteomic profiling, the molecular mechanisms of ADAMTS7 contribution to atherosclerosis have not been fully explored in the context of its proteolytic action. Here we utilized terminal amine isotope labeling of substrates (TAILS), a cutting-edge proteomic approach to enrich N-terminal peptides, enable systemic characterization of proteolytic events and substrate identification for ADAMTS7. Briefly, primary vascular smooth muscle cells (SMC) from ADAMTS7 WT and KO mice were treated with TNFα to stimulated endogenous ADAMTS7 expression, followed by TAILS using dimethyl labeling in cellular proteome and secretome. In 3 biological replicates, we identified 1,720 and 1,616 quantified N-terminal peptides in SMC proteome and secretome, which composed of 415 and 122 unique N-termini of mature protein as well as 1,185 and 1,161 cleaved neo-N-termini driven by proteolytic processing. As expected, most of acetylated peptides in SMC proteome and secretome are natural N-termini of mature proteins (82% and 77%, respectively) while free N-terminal peptides are remarkably enriched with cleaved neo-N termini (85% and 96%, respectively). Importantly, 106 and 86 neo-N-termini (corresponding to 65 and 54 unique proteins) were enriched in ADAMTS7 WT SMC proteome and secretome respectively (WT/KO isotope ratio >2), suggesting that these proteins underwent elevated proteolysis in ADAMTS WT SMCs and thus are proteolytic substrate candidates for ADAMTS7. Interestingly, gene pathway analysis indicates that these proteins are enriched with genes involved in proteinaceous extracellular matrix and extracellular region, such as vimentin, fibulin and MMPs. Biochemistry studies are ongoing to validate these ADAMTS7 substrate candidates. In summary, our study presents a systemic profiling on proteolytic events in SMC cellular proteome and secretome and identifies candidate ADAMTS7 substrates relevant to smooth muscle function and vascular biology.

Degradome of the Interverteral Disc

Introduction The intervertebral disc (IVD) is a complex structure consisting of the nucleus pulposus (NP), annulus fibrosus (AF) and cartilage endplates (EP), which work collectively for proper disc function. Both aging and degeneration alter the biochemical and mechanical function of the IVD – potentially leading to degeneration, which is still not fully understood. In human IVD, sparsely populated cells embedded within the matrix are responsible for the production of matrix, of which its upkeep is important for disc maintenance and function. However, the processes involved with disc maintenance in the non-degenerated condition, versus those that lead to IVD degeneration remain to be identified. Moreover, the presence of reported enzymes in the IVD are likely to contribute to matrix changes by cleaving proteins, thereby altering protein function, and potentially disc function. Studying the degradome of the IVD may identify fragmented proteins and enzymes that degrade these proteins and elucidate events that occur in IVD maintenance versus IVD degeneration, of which cannot be identified by conventional mass spectrometry techniques. Here, we examined the degradome of non-degenerated and degenerated IVD using Terminal Amine Isotopic Labeling of Substrates (TAILS) which facilitates the enrichment of N-termini peptides of degraded protein fragments. Material and Methods NP and AF isolated from three non-degenerated IVD and three degenerated IVD were snap frozen and pulverized in liquid nitrogen. Samples were extracted with 1% SDS with protease inhibitor. N-terminal peptides were enriched using the TAILS method. Briefly, each sample was individually labeled with a Tandem Mass Tag (TMT) and mixed together at equal ratios. The pooled sample was digested with trypsin, and the peptides containing free amines were depleted by polymerization. Samples were analyzed by mass spectrometry (Bruker Daltonics Impact II QTOF), and data analyzed using Scaffold Software. Results Sixty-one peptides (corresponding to 61 protein fragments) were identified in the NP samples. Degenerate NP had more cleaved matrix proteins, including fibronectin, COMP, and lubricin. In non-degenerated NP, the cleaved proteins included collagen II and lysozyme. Ninety-one peptides (corresponding to 91 protein fragments) were identified in the AF samples. Degenerated AF had more cleaved matrix proteins including COMP and fibronectin, in addition to CILP and HAPLN1. Non-degenerate AF had more cleaved collagen I, II, and VI. Conclusion In degenerate AF and NP, the presence of more cleaved matrix proteins, particularly those that are associated with stability, can potentially lead to alterations in the mechanical properties and function of the IVD. Conversely, the cleaved proteins in the non-degenerate tissue could indicate turnover of proteins associated with maintaining a non-degenerate disc environment (such as collagen II in NP, and collagens I and II in AF). Understanding the degenerative process and maintenance of the disc via analysis of the degradome can highlight events that are occurring in health and in degeneration, in addition to the identification of enzymes that cleave these proteins, of which are currently in progress. Acknowledgment This work was supported by the Research Grants Committee of Hong Kong–Theme-based Research Scheme (T-12–708/12N).

Matrix Metalloproteinase 10 Degradomics in Keratinocytes and Epidermal Tissue Identifies Bioactive Substrates With Pleiotropic Functions*

Matrix metalloproteinases (MMPs) are important players in skin homeostasis, wound repair, and in the pathogenesis of skin cancer. It is now well established that most of their functions are related to processing of bioactive proteins rather than components of the extracellular matrix (ECM). MMP10 is highly expressed in keratinocytes at the wound edge and at the invasive front of tumors, but hardly any non-ECM substrates have been identified and its function in tissue repair and carcinogenesis is unclear. To better understand the role of MMP10 in the epidermis, we employed multiplexed iTRAQ-based Terminal Amine Isotopic Labeling of Substrates (TAILS) and monitored MMP10-dependent proteolysis over time in secretomes from keratinocytes. Time-resolved abundance clustering of neo-N termini classified MMP10-dependent cleavage events by efficiency and refined the MMP10 cleavage site specificity by revealing a so far unknown preference for glutamate in the P1 position. Moreover, we identified and validated the integrin alpha 6 subunit, cysteine-rich angiogenic inducer 61 and dermokine as novel direct MMP10 substrates and provide evidence for MMP10-dependent but indirect processing of phosphatidylethanolamine-binding protein 1. Finally, we sampled the epidermal proteome and degradome in unprecedented depth and confirmed MMP10-dependent processing of dermokine in vivo by TAILS analysis of epidermis from transgenic mice that overexpress a constitutively active mutant of MMP10 in basal keratinocytes. The newly identified substrates are involved in cell adhesion, migration, proliferation, and/or differentiation, indicating a contribution of MMP10 to local modulation of these processes during wound healing and cancer development. Data are available via ProteomeXchange with identifier PXD002474.

Positional proteomics in the era of the human proteome project on the doorstep of precision medicine

Proteolytic processing is a pervasive and irreversible post-translational modification that expands the protein universe by generating new proteoforms (protein isoforms). Unlike signal peptide or prodomain removal, protease-generated proteoforms can rarely be predicted from gene sequences. Positional proteomic techniques that enrich for N- or C-terminal peptides from proteomes are indispensable for a comprehensive understanding of a protein's function in biological environments since protease cleavage frequently results in altered protein activity and localization. Proteases often process other proteases and protease inhibitors which perturbs proteolytic networks and potentiates the initial cleavage event to affect other molecular networks and cellular processes in physiological and pathological conditions. This review is aimed at researchers with a keen interest in state of the art systems level positional proteomic approaches that: (i) enable the study of complex protease-protease, protease-inhibitor and protease-substrate crosstalk and networks; (ii) allow the identification of proteolytic signatures as candidate disease biomarkers; and (iii) are expected to fill the Human Proteome Project missing proteins gap. We predict that these methodologies will be an integral part of emerging precision medicine initiatives that aim to customize healthcare, converting reactive medicine into a personalized and proactive approach, improving clinical care and maximizing patient health and wellbeing, while decreasing health costs by eliminating ineffective therapies, trial-and-error prescribing, and adverse drug effects. Such initiatives require quantitative and functional proteome profiling and dynamic disease biomarkers in addition to current pharmacogenomics approaches. With proteases at the pathogenic center of many diseases, high-throughput protein termini identification techniques such as TAILS (Terminal Amine Isotopic Labeling of Substrates) and COFRADIC (COmbined FRActional DIagonal Chromatography) will be fundamental for individual and comprehensive assessment of health and disease.

TAILS N-terminomic and proteomic datasets of healthy human dental pulp.

The Data described here provide the in depth proteomic assessment of the human dental pulp proteome and N-terminome (Eckhard et al., 2015) [1]. A total of 9 human dental pulps were processed and analyzed by the positional proteomics technique TAILS (Terminal Amine Isotopic Labeling of Substrates) N-terminomics. 38 liquid chromatography tandem mass spectrometry (LC-MS/MS) datasets were collected and analyzed using four database search engines in combination with statistical downstream evaluation, to yield the by far largest proteomic and N-terminomic dataset of any dental tissue to date. The raw mass spectrometry data and the corresponding metadata have been deposited in ProteomeXchange with the PXD identifier <PXD002264>; Supplementary Tables described in this article are available via Mendeley Data (10.17632/555j3kk4sw.1).

Neutrophil serine proteases exert proteolytic activity on endothelial cells

Neutrophil serine proteases (NSPs) are released from activated neutrophils during inflammation. Here we studied the transfer of the three major NSPs, namely proteinase 3, human neutrophil elastase, and cathepsin G, from neutrophils to endothelial cells and used an unbiased approach to identify novel endothelial NSP substrates. Enzymatically active NSPs were released from stimulated neutrophils and internalized by endothelial cells in a dose- and time-dependent manner as shown by immunoblotting, flow cytometry, and the Boc-Ala substrate assay. Using terminal-amine isotopic labeling of substrates in endothelial cells, we identified 121 peptides from 82 different proteins consisting of 36 substrates for proteinase 3, 30 for neutrophil elastase, and 28 for cathepsin G, respectively. We characterized the extended cleavage pattern and provide corresponding IceLogos. Gene ontology analysis showed significant cytoskeletal substrate enrichment and confirmed several cytoskeletal protein substrates by immunoblotting. Finally, ANCA-stimulated neutrophils released all three active NSPs into the supernatant. Supernatants increased endothelial albumin flux and disturbed the endothelial cell cytoskeletal architecture. Serine protease inhibition abrogated this effect. Longer exposure to NSPs reduced endothelial cell viability and increased apoptosis. Thus, we identified novel NSP substrates and suggest NSP inhibition as a therapeutic measure to inhibit neutrophil-mediated inflammatory vascular diseases.

The Human Dental Pulp Proteome and N-Terminome: Levering the Unexplored Potential of Semitryptic Peptides Enriched by TAILS to Identify Missing Proteins in the Human Proteome Project in Underexplored Tissues.

An underexplored yet widespread feature of the human proteome is the proteolytic proteoforms of proteins. We used terminal amine isotopic labeling of substrates (TAILS), a high-content N-terminal positional proteomics technique, for in-depth characterization of the human dental pulp proteome from its N-terminome and to provide data for the Chromosome-centric Human Proteome Project (C-HPP). Dental pulp is a unique connective tissue maintaining tooth sensation and structure by supporting a single cell layer of odontoblasts that synthesize mineralization-competent dentine extracellular matrix. Therefore, we posited pulp to be a rich source of unique tissue-specific proteins and hence an abundant source of "missing" proteins as defined by neXtProt. From the identified 4332 proteins (false discovery rate (FDR) ≤ 0.7%), 21 528 unique peptides (FDR ≤ 1.0%) and 9079 unique N-termini, we analyzed N-terminal methionine excision, co- and posttranslational Nα-acetylation, protein maturation, and proteolytic processing. Apart from 227 candidate alternative translation initiation sites, most identified N-termini (78%) represented proteolytic processing and mechanism-informative internal neo-N-termini, confirming a pervasive amount of proteolytic-processing generated proteoforms in vivo. Furthermore, we identified 17 missing protein candidates for the C-HPP, highlighting the importance of using (i) less studied human specimens and (ii) orthogonal proteomic approaches such as TAILS to map the human proteome. The mass spectrometry raw data and metadata have been deposited to ProteomeXchange with the PXD identifier <PXD002264>.

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. Global assessment of proteolysis at critical turning points after injury will define crucial events in acute healing that might be disturbed in healing disorders. As optimal biospecimens, wound exudates contain an ideal proteome to detect extracellular proteolytic events, are noninvasively accessible, and can be collected at multiple time points along the healing process from the same wound in the clinics. In this study, we applied multiplexed Terminal Amine Isotopic Labeling of Substrates (TAILS) to globally assess proteolysis in early phases of cutaneous wound healing. By quantitative analysis of proteins and protein N termini in wound fluids from a clinically relevant pig wound model, we identified more than 650 proteins and discerned major healing phases through distinctive abundance clustering of markers of inflammation, granulation tissue formation, and re-epithelialization. TAILS revealed a high degree of proteolysis at all time points after injury by detecting almost 1300 N-terminal peptides in ∼450 proteins. Quantitative positional proteomics mapped pivotal interdependent processing events in the blood coagulation and complement cascades, temporally discerned clotting and fibrinolysis during the healing process, and detected processing of complement C3 at distinct time points after wounding and by different proteases. Exploiting data on primary cleavage specificities, we related candidate proteases to cleavage events and revealed processing of the integrin adapter protein kindlin-3 by caspase-3, generating new hypotheses for protease-substrate relations in the healing skin wound in vivo. The data have been deposited to the ProteomeXchange Consortium with identifier PXD001198.

TAILS N-terminomics of human platelets reveals pervasive metalloproteinase-dependent proteolytic processing in storage

AbstractProteases, and specifically metalloproteinases, have been linked to the loss of platelet function during storage before transfusion, but the underlying mechanisms remain unknown. We used a dedicated N-terminomics technique, iTRAQ terminal amine isotopic labeling of substrates (TAILS), to characterize the human platelet N-terminome, proteome, and posttranslational modifications throughout platelet storage over 9 days under blood-banking conditions. From the identified 2938 proteins and 7503 unique peptides, we characterized N-terminal methionine excision, co- and posttranslational Nα acetylation, protein maturation, and proteolytic processing of proteins in human platelets. We also identified for the first time 10 proteins previously classified by the Human Proteome Organization as “missing” in the human proteome. Most N termini (77%) were internal neo-N termini (105 were novel potential alternative translation start sites, and 2180 represented stable proteolytic products), thus highlighting a prominent yet previously uncharacterized role of proteolytic processing during platelet storage. Protease inhibitor studies revealed metalloproteinases as being primarily responsible for proteolytic processing (as opposed to degradation) during storage. System-wide identification of metalloproteinase and other proteinase substrates and their respective cleavage sites suggests novel mechanisms of the effect of proteases on protein activity and platelet function during storage. All data sets and metadata are available through ProteomeXchange with the data set identifier PXD000906.

Macrophage Matrix Metalloproteinase-12 Dampens Inflammation and Neutrophil Influx in Arthritis

Resolution of inflammation reduces pathological tissue destruction and restores tissue homeostasis.Here, we used a proteomic protease substrate discoveryapproach, terminal amine isotopic labeling of substrates (TAILS), to analyze the role of the macrophage-specific matrix metalloproteinase-12 (MMP12) in inflammation. In murine peritonitis, MMP12 inactivates antithrombin and activates prothrombin, prolonging the activated partial thromboplastin time. Furthermore, MMP12 inactivates complement C3 to reduce complement activation and inactivates the chemoattractant anaphylatoxins C3a and C5a, whereas iC3b and C3b opsonin cleavage increases phagocytosis. Loss of these anti-inflammatory activities incollagen-induced arthritis in Mmp12(-/-) mice leadsto unresolved synovitis and extensive articularinflammation. Deep articular cartilage loss is associated with massive neutrophil infiltration and abnormal DNA neutrophil extracellular traps (NETs). The NETs are rich in fibrin and extracellular actin, which TAILS identified as MMP12 substrates. Thus, macrophage MMP12 in arthritis has multiple protective roles in countering neutrophil infiltration, clearing NETs, and dampening inflammatory pathways to prepare for the resolution of inflammation.

Identifying Natural Substrates for Dipeptidyl Peptidases 8 and 9 Using Terminal Amine Isotopic Labeling of Substrates (TAILS) Reveals in Vivo Roles in Cellular Homeostasis and Energy Metabolism

Dipeptidyl peptidases (DP) 8 and 9 are homologous, cytoplasmic N-terminal post-proline-cleaving enzymes that are anti-targets for the development of DP4 (DPPIV/CD26) inhibitors for treating type II diabetes. To date, DP8 and DP9 have been implicated in immune responses and cancer biology, but their pathophysiological functions and substrate repertoire remain unknown. This study utilizes terminal amine isotopic labeling of substrates (TAILS), an N-terminal positional proteomic approach, for the discovery of in vivo DP8 and DP9 substrates. In vivo roles for DP8 and DP9 in cellular metabolism and homeostasis were revealed via the identification of more than 29 candidate natural substrates and pathways affected by DP8/DP9 overexpression. Cleavage of 14 substrates was investigated in vitro; 9/14 substrates for both DP8 and DP9 were confirmed by MALDI-TOF MS, including two of high confidence, calreticulin and adenylate kinase 2. Adenylate kinase 2 plays key roles in cellular energy and nucleotide homeostasis. These results demonstrate remarkable in vivo substrate overlap between DP8/DP9, suggesting compensatory roles for these enzymes. This work provides the first global investigation into DP8 and DP9 substrates, providing a number of leads for future investigations into the biological roles and significance of DP8 and DP9 in human health and disease.

Deletion of Cysteine Cathepsins B or L Yields Differential Impacts on Murine Skin Proteome and Degradome

Numerous studies highlight the fact that concerted proteolysis is essential for skin morphology and function. The cysteine protease cathepsin L (Ctsl) has been implicated in epidermal proliferation and desquamation, as well as in hair cycle regulation. In stark contrast, mice deficient in cathepsin B (Ctsb) do not display an overt skin phenotype. To understand the systematic consequences of deleting Ctsb or Ctsl, we determined the protein abundances of >1300 proteins and proteolytic cleavage events in skin samples of wild-type, Ctsb(-/-), and Ctsl(-/-) mice via mass-spectrometry-based proteomics. Both protease deficiencies revealed distinct quantitative changes in proteome composition. Ctsl(-/-) skin revealed increased levels of the cysteine protease inhibitors cystatin B and cystatin M/E, increased cathepsin D, and an accumulation of the extracellular glycoprotein periostin. Immunohistochemistry located periostin predominantly in the hypodermal connective tissue of Ctsl(-/-) skin. The proteomic identification of proteolytic cleavage sites within skin proteins revealed numerous processing sites that are underrepresented in Ctsl(-/-) or Ctsb(-/-) samples. Notably, few of the affected cleavage sites shared the canonical Ctsl or Ctsb specificity, providing further evidence of a complex proteolytic network in the skin. Novel processing sites in proteins such as dermokine and Notch-1 were detected. Simultaneous analysis of acetylated protein N termini showed prototypical mammalian N-alpha acetylation. These results illustrate an influence of both Ctsb and Ctsl on the murine skin proteome and degradome, with the phenotypic consequences of the absence of either protease differing considerably.

CLIPPER: an add-on to the Trans-Proteomic Pipeline for the automated analysis of TAILS N-terminomics data

Data analysis in proteomics is complex and with the extra challenges involved in the interpretation of data from N-terminomics experiments, this can be daunting.Therefore, we have devised a rational pipeline of steps to approach N-terminomics data analysis in a statistically based and valid manner. We have automated these steps in CLIPPER, an add-on to the Trans-Proteomic Pipeline(TPP). Applying CLIPPER to the analysis of N- terminomics data generated by terminal amine isotopic labeling of substrates (TAILS) enables high confidence peptide to protein assignment, protein N-terminal characterization and annotation, and for protease analysis readily allows protease substrate discovery with high confidence.

The substrate degradome of meprin metalloproteases reveals an unexpected proteolytic link between meprin β and ADAM10

The in vivo roles of meprin metalloproteases in pathophysiological conditions remain elusive. Substrates define protease roles. Therefore, to identify natural substrates for human meprin α and β we employed TAILS (terminal amine isotopic labeling of substrates), a proteomics approach that enriches for N-terminal peptides of proteins and cleavage fragments. Of the 151 new extracellular substrates we identified, it was notable that ADAM10 (a disintegrin and metalloprotease domain-containing protein 10)—the constitutive α-secretase—is activated by meprin β through cleavage of the propeptide. To validate this cleavage event, we expressed recombinant proADAM10 and after preincubation with meprin β, this resulted in significantly elevated ADAM10 activity. Cellular expression in murine primary fibroblasts confirmed activation. Other novel substrates including extracellular matrix proteins, growth factors and inhibitors were validated by western analyses and enzyme activity assays with Edman sequencing confirming the exact cleavage sites identified by TAILS. Cleavages in vivo were confirmed by comparing wild-type and meprin−/− mice. Our finding of cystatin C, elafin and fetuin-A as substrates and natural inhibitors for meprins reveal new mechanisms in the regulation of protease activity important for understanding pathophysiological processes.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-012-1106-2) contains supplementary material, which is available to authorized users.

Qualitative improvement and quantitative assessment of N-terminomics

Proteolysis represents one of the most tightly controlled physiological processes, as proteases create events that will typically commit pathways in an irreversible manner. Despite their implication in nearly all biological systems, our understanding of the role of proteases in disease pathology is often limited. Several approaches to studying proteolytic activity as it relates to biology, pathophysiology, and drug therapy have been published, including the recently described terminal amine isotopic labeling of substrates (TAILS) strategy by Kleifeld and colleagues. Here, we investigate TAILS as a methodology based on targeted enrichment and mass spectrometric detection of endogenous N-terminal peptides from clinically relevant biological samples and its potential to provide quantitative information on proteolysis and elucidation of the protease cleavage sites. While optimizing the most current protocol, by switching to a streamlined one-tube format and simplifying the reagents' removal steps, we demonstrate the advantages over previously published methods and provide solutions to some of the technical challenges presented in the Kleifeld publication. We also identify some of the current and unresolved limitations. We use human plasma as a model system to provide data, which illustrates some of the key analytical parameters of the modified TAILS procedure, including specificity, sensitivity, quantitative precision, and accuracy.

Biochemical Characterization and N-terminomics Analysis of Leukolysin, the Membrane-type 6 Matrix Metalloprotease (MMP25): CHEMOKINE AND VIMENTIN CLEAVAGES ENHANCE CELL MIGRATION AND MACROPHAGE PHAGOCYTIC ACTIVITIES

The neutrophil-specific protease membrane-type 6 matrix metalloproteinase (MT6-MMP)/MMP-25/leukolysin is implicated in multiple sclerosis and cancer yet remains poorly characterized. To characterize the biological roles of MT6-MMP, it is critical to identify its substrates for which only seven are currently known. Here, we biochemically characterized MT6-MMP, profiled its tissue inhibitor of metalloproteinase inhibitory spectrum, performed degradomics analyses, and screened 26 chemokines for cleavage using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. MT6-MMP processes seven each of the CXC and CC chemokine subfamilies. Notably, cleavage of the neutrophil chemoattractant CXCL5 activates the chemokine, thereby increasing its agonist activity, indicating a feed-forward mechanism for neutrophil recruitment. Likewise, cleavage also activated CCL15 and CCL23 to increase monocyte recruitment. Utilizing the proteomics approach proteomic identification of cleavage site specificity (PICS), we identified 286 peptidic cleavage sites spanning from P6 to P6' from which an unusual glutamate preference in P1 was identified. The degradomics screen terminal amine isotopic labeling of substrates (TAILS), which enriches for neo-N-terminal peptides of cleaved substrates, was used to identify 58 new native substrates in fibroblast secretomes after incubation with MT6-MMP. Vimentin, cystatin C, galectin-1, IGFBP-7, and secreted protein, acidic and rich in cysteine (SPARC) were among those substrates we biochemically confirmed. An extracellular "moonlighting" form of vimentin is a chemoattractant for THP-1 cells, but MT6-MMP cleavage abolished monocyte recruitment. Unexpectedly, the MT6-MMP-cleaved vimentin potently stimulated phagocytosis, which was not a property of the full-length protein. Hence, MT6-MMP regulates neutrophil and monocyte chemotaxis and by generating "eat-me" signals upon vimentin cleavage potentially increases phagocytic removal of neutrophils to resolve inflammation.

Contribution of cathepsin L to secretome composition and cleavage pattern of mouse embryonic fibroblasts

The endolysosomal cysteine endoprotease cathepsin L is secreted from cells in a variety of pathological conditions such as cancer and arthritis. We compared the secretome composition and extracellular proteolytic cleavage events in cell supernatants of cathepsin L-deficient and wild-type mouse embryonic fibroblasts (MEFs). Quantitative proteomic comparison of cell conditioned media indicated that cathepsin L deficiency affects, albeit in a limited manner, the abundances of extracellular matrix (ECM) components, signaling proteins, and further proteases as well as endogenous protease inhibitors. Immunodetection corroborated that cathepsin L deficiency results in decreased abundance of the ECM protein periostin and elevated abundance of matrix metalloprotease (MMP)-2. While mRNA levels of MMP-2 were not affected by cathepsin L ablation, periostin mRNA levels were reduced, potentially indicating a downstream effect. To characterize cathepsin L contribution to extracellular proteolysis, we performed terminal amine isotopic labeling of substrates (TAILS), an N-terminomic technique for the identification and quantification of native and proteolytically generated protein N-termini. TAILS identified >1500 protein N-termini. Cathepsin L deficiency predominantly reduced the magnitude of collagenous cleavage sites C-terminal to a proline residue. This contradicts cathepsin L active site specificity and indicates altered activity of further proteases as a result of cathepsin L ablation.

Identifying and quantifying proteolytic events and the natural N terminome by terminal amine isotopic labeling of substrates

Analysis of the sequence and nature of protein N termini has many applications. Defining the termini of proteins for proteome annotation in the Human Proteome Project is of increasing importance. Terminomics analysis of protease cleavage sites in degradomics for substrate discovery is a key new application. Here we describe the step-by-step procedures for performing terminal amine isotopic labeling of substrates (TAILS), a 2- to 3-d (depending on method of labeling) high-throughput method to identify and distinguish protease-generated neo-N termini from mature protein N termini with all natural modifications with high confidence. TAILS uses negative selection to enrich for all N-terminal peptides and uses primary amine labeling-based quantification as the discriminating factor. Labeling is versatile and suited to many applications, including biochemical and cell culture analyses in vitro; in vivo analyses using tissue samples from animal and human sources can also be readily performed. At the protein level, N-terminal and lysine amines are blocked by dimethylation (formaldehyde/sodium cyanoborohydride) and isotopically labeled by incorporating heavy and light dimethylation reagents or stable isotope labeling with amino acids in cell culture labels. Alternatively, easy multiplex sample analysis can be achieved using amine blocking and labeling with isobaric tags for relative and absolute quantification, also known as iTRAQ. After tryptic digestion, N-terminal peptide separation is achieved using a high-molecular-weight dendritic polyglycerol aldehyde polymer that binds internal tryptic and C-terminal peptides that now have N-terminal alpha amines. The unbound naturally blocked (acetylation, cyclization, methylation and so on) or labeled mature N-terminal and neo-N-terminal peptides are recovered by ultrafiltration and analyzed by tandem mass spectrometry (MS/MS). Hierarchical substrate winnowing discriminates substrates from the background proteolysis products and non-cleaved proteins by peptide isotope quantification and bioinformatics search criteria.

Proteomic Analyses Reveal an Acidic Prime Side Specificity for the Astacin Metalloprotease Family Reflected by Physiological Substrates

Astacins are secreted and membrane-bound metalloproteases with clear associations to many important pathological and physiological processes. Yet with only a few substrates described their biological roles are enigmatic. Moreover, the lack of knowledge of astacin cleavage site specificities hampers assay and drug development. Using PICS (proteomic identification of protease cleavage site specificity) and TAILS (terminal amine isotopic labeling of substrates) degradomics approaches >3000 cleavage sites were proteomically identified for five different astacins. Such broad coverage enables family-wide determination of specificities N- and C-terminal to the scissile peptide bond. Remarkably, meprin α, meprin β, and LAST_MAM proteases exhibit a strong preference for aspartate in the peptide (P)1′ position because of a conserved positively charged residue in the active cleft subsite (S)1′. This unparalleled specificity has not been found for other families of extracellular proteases. Interestingly, cleavage specificity is also strongly influenced by proline in P2′ or P3′ leading to a rare example of subsite cooperativity. This specificity characterizes the astacins as unique contributors to extracellular proteolysis that is corroborated by known cleavage sites in procollagen I+III, VEGF (vascular endothelial growth factor)-A, IL (interleukin)-1β, and pro-kallikrein 7. Indeed, cleavage sites in VEGF-A and pro-kallikrein 7 identified by terminal amine isotopic labeling of substrates matched those reported by Edman degradation. Moreover, the novel substrate FGF-19 was validated biochemically and shown to exhibit altered biological activity after meprin processing.

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). Furthermore, the isoform assignment score (IAS) is introduced, a new scoring system for the evaluation of single peptide-to-protein assignments based on high confidence protein identifications in the same sample prior to negative selection enrichment of N-terminal peptides. By these approaches, we identified and validated, in addition to known substrates, low abundance novel bioactive MMP-2 targets including the plasminogen receptor S100A10 (p11) and the proinflammatory cytokine proEMAP/p43 that were previously undescribed.