An atlas of protein-protein interactions across mouse tissues.

Cellular processes arise from the dynamic organization of proteins in networks of physical interactions. Mapping the interactome has therefore been a central objective of high-throughput biology. However, the dynamics of protein interactions across physiological contexts remain poorly understood. Here, we develop a quantitative proteomic approach combining protein correlation profiling with stable isotope labeling of mammals (PCP-SILAM) to map the interactomes of seven mouse tissues. The resulting maps provide a proteome-scale survey of interactome rewiring across mammalian tissues, revealing more than 125,000 unique interactions at a quality comparable to the highest-quality human screens. We identify systematic suppression of cross-talk between the evolutionarily ancient housekeeping interactome and younger, tissue-specific modules. Rewired proteins are tightly regulated by multiple cellular mechanisms and are implicated in disease. Our study opens up new avenues to uncover regulatory mechanisms that shape in vivo interactome responses to physiological and pathophysiological stimuli in mammalian systems.

Proteomic Portraits Reveal Evolutionarily Conserved and Divergent Responses to Spinal Cord Injury.

Despite the emergence of promising therapeutic approaches in preclinical studies, the failure of large-scale clinical trials leaves clinicians without effective treatments for acute spinal cord injury (SCI). These trials are hindered by their reliance on detailed neurological examinations to establish outcomes, which inflate the time and resources required for completion. Moreover, therapeutic development takes place in animal models whose relevance to human injury remains unclear. Here, we address these challenges through targeted proteomic analyses of cerebrospinal fluid and serum samples from 111 patients with acute SCI and, in parallel, a large animal (porcine) model of SCI. We develop protein biomarkers of injury severity and recovery, including a prognostic model of neurological improvement at 6 months with an area under the receiver operating characteristic curve of 0.91, and validate these in an independent cohort. Through cross-species proteomic analyses, we dissect evolutionarily conserved and divergent aspects of the SCI response and establish the cerebrospinal fluid abundance of glial fibrillary acidic protein as a biochemical outcome measure in both humans and pigs. Our work opens up new avenues to catalyze translation by facilitating the evaluation of novel SCI therapies, while also providing a resource from which to direct future preclinical efforts.

Deep Profiling of the Cleavage Specificity and Human Substrates of Snake Venom Metalloprotease HF3 by Proteomic Identification of Cleavage Site Specificity (PICS) Using Proteome Derived Peptide Libraries and Terminal Amine Isotopic Labeling of Substrates (TAILS) N-Terminomics.

Snakebite is a major medical concern in many parts of the world with metalloproteases playing important roles in the pathological effects of Viperidae venoms, including local tissue damage, hemorrhage, and coagulopathy. Hemorrhagic Factor 3 (HF3), a metalloprotease from Bothrops jararaca venom, induces local hemorrhage and targets extracellular matrix (ECM) components, including collagens and proteoglycans, and plasma proteins. However, the full substrate repertoire of this metalloprotease is unknown. We report positional proteomic studies identifying >2000 N-termini, including neo-N-termini of HF3 cleavage sites in mouse embryonic fibroblast secretome proteins. Terminal amine isotopic labeling of substrates (TAILS) analysis identified a preference for Leu at the P1' position among candidate HF3 substrates including proteins of the ECM and focal adhesions and the cysteine protease inhibitor cystatin-C. Interestingly, 190 unique peptides matched to annotated cleavage sites in the TopFIND N-termini database, suggesting that these cleavages occurred at a site prone to cleavage or might have been generated by other proteases activated upon incubation with HF3, including caspases-3 and -7, cathepsins D and E, granzyme B, and MMPs 2 and 9. Using Proteomic identification of cleavage site specificity (PICS), a tryptic library derived from THP-1 monocytic cells was used as HF3 substrates for identifying protease cleavage sites and sequence preferences in peptides. A total of 799 unique cleavage sites were detected and, in accordance with TAILS analysis using native secreted protein substrates of MEF cells, revealed a clear preference for Leu at P1'. Taken together, these results greatly expand the known substrate degradome of HF3 and reveal potential new targets, which may serve as a basis to better elucidate the complex pathophysiology of snake envenomation.

Interactome disassembly during apoptosis occurs independent of caspase cleavage.

Protein-protein interaction networks (interactomes) define the functionality of all biological systems. In apoptosis, proteolysis by caspases is thought to initiate disassembly of protein complexes and cell death. Here we used a quantitative proteomics approach, protein correlation profiling (PCP), to explore changes in cytoplasmic and mitochondrial interactomes in response to apoptosis initiation as a function of caspase activity. We measured the response to initiation of Fas-mediated apoptosis in 17,991 interactions among 2,779 proteins, comprising the largest dynamic interactome to date. The majority of interactions were unaffected early in apoptosis, but multiple complexes containing known caspase targets were disassembled. Nonetheless, proteome-wide analysis of proteolytic processing by terminal amine isotopic labeling of substrates (TAILS) revealed little correlation between proteolytic and interactome changes. Our findings show that, in apoptosis, significant interactome alterations occur before and independently of caspase activity. Thus, apoptosis initiation includes a tight program of interactome rearrangement, leading to disassembly of relatively few, select complexes. These early interactome alterations occur independently of cleavage of these protein by caspases.

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

Proteases, 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.

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.

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. Incorporating iTRAQ whole protein labeling with terminal amine isotopic labeling of substrates (iTRAQ-TAILS) to enrich the N-terminome by negative selection of the blocked mature original N-termini and neo-N-termini has many advantages. It enables simultaneous characterization of the natural N-termini of proteins, their N-terminal modifications, and proteolysis product and cleavage site identification. Furthermore, iTRAQ-TAILS also enables multiplex N-terminomics analysis of up to eight samples and allows for quantification in MS2 mode, thus preventing an increase in spectral complexity and extending proteome coverage by signal amplification of low abundance proteins. We compared the substrate degradomes of two closely related matrix metalloproteinases, MMP-2 (gelatinase A) and MMP-9 (gelatinase B), in fibroblast secreted proteins. Among 3,152 unique N-terminal peptides identified corresponding to 1,054 proteins, we detected 201 cleavage products for MMP-2 and unexpectedly only 19 for the homologous MMP-9 under identical conditions. Novel substrates identified and biochemically validated include insulin-like growth factor binding protein-4, complement C1r component A, galectin-1, dickkopf-related protein-3, and thrombospondin-2. Hence, N-terminomics analyses using iTRAQ-TAILS links gelatinases with new mechanisms of action in angiogenesis and reveals unpredicted restrictions in substrate repertoires for these two very similar proteases.

Proteomic Screens for Suppressors of Anoikis Identify IL1RAP as a Promising Surface Target in Ewing Sarcoma.

Cancer cells must overcome anoikis (detachment-induced death) to successfully metastasize. Using proteomic screens, we found that distinct oncoproteins upregulate IL1 receptor accessory protein (IL1RAP) to suppress anoikis. IL1RAP is directly induced by oncogenic fusions of Ewing sarcoma, a highly metastatic childhood sarcoma. IL1RAP inactivation triggers anoikis and impedes metastatic dissemination of Ewing sarcoma cells. Mechanistically, IL1RAP binds the cell-surface system Xc - transporter to enhance exogenous cystine uptake, thereby replenishing cysteine and the glutathione antioxidant. Under cystine depletion, IL1RAP induces cystathionine gamma lyase (CTH) to activate the transsulfuration pathway for de novo cysteine synthesis. Therefore, IL1RAP maintains cyst(e)ine and glutathione pools, which are vital for redox homeostasis and anoikis resistance. IL1RAP is minimally expressed in pediatric and adult normal tissues, and human anti-IL1RAP antibodies induce potent antibody-dependent cellular cytotoxicity of Ewing sarcoma cells. Therefore, we define IL1RAP as a new cell-surface target in Ewing sarcoma, which is potentially exploitable for immunotherapy. SIGNIFICANCE: Here, we identify cell-surface protein IL1RAP as a key driver of metastasis in Ewing sarcoma, a highly aggressive childhood sarcoma. Minimal expression in pediatric and adult normal tissues nominates IL1RAP as a promising target for immunotherapy.See related commentary by Yoon and DeNicola, p. 2679.This article is highlighted in the In This Issue feature, p. 2659.

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. Indeed many cancer biomarkers are stable proteolytic fragments. It is crucial to determine which proteases contribute to the pathology versus their roles in homeostasis and in mitigating cancer. Thus the full substrate repertoire of a protease, termed the substrate degradome, must be deciphered to define protease function and to identify drug targets. Degradomics has been used to identify many substrates of matrix metalloproteinases that are important proteases in cancer. Here we review recent degradomics technologies that allow for the broadly applicable identification and quantification of proteases (the protease degradome) and their activity state, substrates, and interactors. Quantitative proteomics using stable isotope labeling, such as ICAT, isobaric tags for relative and absolute quantification (iTRAQ), and stable isotope labeling by amino acids in cell culture (SILAC), can reveal protease substrates by taking advantage of the natural compartmentalization of membrane proteins that are shed into the extracellular space. Identifying the actual cleavage sites in a complex proteome relies on positional proteomics and utilizes selection strategies to enrich for protease-generated neo-N termini of proteins. In so doing, important functional information is generated. Finally protease substrates and interactors can be identified by interactomics based on affinity purification of protease complexes using exosite scanning and inactive catalytic domain capture strategies followed by mass spectrometry analysis. At the global level, the N terminome analysis of whole communities of proteases in tissues and organs in vivo provides a full scale understanding of the protease web and the web-sculpted proteome, so defining metadegradomics.

Transcriptomic and proteomic host response to Aspergillus fumigatus conidia in an air-liquid interface model of human bronchial epithelium.

Aspergillus fumigatus (A. fumigatus) is a wide-spread fungus that is a potent allergen in hypersensitive individuals but also an opportunistic pathogen in immunocompromised patients. It reproduces asexually by releasing airborne conidiospores (conidia). Upon inhalation, fungal conidia are capable of reaching the airway epithelial cells (AECs) in bronchial and alveolar tissues. Previous studies have predominantly used submerged monolayer cultures for studying this host-pathogen interaction; however, these cultures do not recapitulate the mucocililary differentiation phenotype of the in vivo epithelium in the respiratory tract. Thus, the aim of this study was to use well-differentiated primary human bronchial epithelial cells (HBECs) grown at the air-liquid interface (ALI) to determine their transcriptomic and proteomic responses following interaction with A. fumigatus conidia. We visualized conidial interaction with HBECs using confocal laser scanning microscopy (CLSM), and applied NanoString nCounter and shotgun proteomics to assess gene expression changes in the human cells upon interaction with A. fumigatus conidia. Western blot analysis was used to assess the expression of top three differentially expressed proteins, CALR, SET and NUCB2. CLSM showed that, unlike submerged monolayer cultures, well-differentiated ALI cultures of primary HBECs were estimated to internalize less than 1% of bound conidia. Nevertheless, transcriptomic and proteomic analyses revealed numerous differentially expressed host genes; these were enriched for pathways including apoptosis/autophagy, translation, unfolded protein response and cell cycle (up-regulated); complement and coagulation pathways, iron homeostasis, nonsense mediated decay and rRNA binding (down-regulated). CALR and SET were confirmed to be up-regulated in ALI cultures of primary HBECs upon exposure to A. fumigatus via western blot analysis. Therefore, using transcriptomics and proteomics approaches, ALI models recapitulating the bronchial epithelial barrier in the conductive zone of the respiratory tract can provide novel insights to the molecular response of bronchial epithelial cells upon exposure to A. fumigatus conidia.

Melanocyte development in the mouse tail epidermis requires the Adamts9 metalloproteinase.

The mouse tail has an important role in the study of melanogenesis, because mouse tail skin can be used to model human skin pigmentation. To better understand the development of melanocytes in the mouse tail, we cloned two dominant ENU-generated mutations of the Adamts9 gene, Und3 and Und4, which cause an unpigmented ring of epidermis in the middle of the tail, but do not alter pigmentation in the rest of the mouse. Adamts9 encodes a widely expressed zinc metalloprotease with thrombospondin type 1 repeats with few known substrates. Melanocytes are lost in the Adamts9 mutant tail epidermis at a relatively late stage of development, around E18.5. Studies of our Adamts9 conditional allele suggest that there is a melanocyte cell-autonomous requirement for Adamts9. In addition, we used a proteomics approach, TAILS N-terminomics, to identify new Adamts9 candidate substrates in the extracellular matrix of the skin. The tail phenotype of Adamts9 mutants is strikingly similar to the unpigmented trunk belt in Adamts20 mutants, which suggests a particular requirement for Adamts family activity at certain positions along the anterior-posterior axis.

Testosterone regulation of homocysteine metabolism modulates redox status in human prostate cancer cells.

Clearance of homocysteine via the transsulfuration pathway provides an endogenous route for cysteine synthesis and represents a quantitatively significant source of this amino acid needed for glutathione synthesis. Men have higher plasma levels of total homocysteine than do women, but the mechanism of this sex-dependent difference is not known. In this study, we investigated regulation by testosterone of cystathionine beta-synthase (CBS), which catalyzes the committing step in the transsulfuration pathway. We report that testosterone downregulates CBS expression via a posttranscriptional mechanism in the androgen-responsive prostate cancer cell line, LNCaP. This diminution in CBS levels is accompanied by a decrease in flux through the transsulfuration pathway and by a lower intracellular glutathione concentration. The lower antioxidant capacity in testosterone-treated prostate cancer cells increases their susceptibility to oxidative stress conditions. These results demonstrate regulation of the homocysteine-clearing enzyme, CBS, by testosterone and suggest the potential utility of targeting this enzyme as a chemotherapeutic strategy.

A Targeted Proteomics Analysis of Cerebrospinal Fluid after Acute Human Spinal Cord Injury.

Efforts to validate novel therapies in acute clinical trials for spinal cord injury (SCI) are impeded by the lack of objective quantitative measures that reflect injury severity and accurately predict neurological recovery. Therefore, a strong rationale exists for establishing neurochemical biomarkers that objectively quantify injury severity and predict outcome. Here, we conducted a targeted proteomics analysis of cerebrospinal fluid (CSF) samples derived from 29 acute SCI patients (American Spinal Injury Association Impairment Scale [AIS] A, B, or C) acquired at 24, 48, and 72 h post-injury. From a total of 165 proteins, we identified 27 potential biomarkers of injury severity (baseline AIS A, B, or C), with triosephosphate isomerase having the strongest relationship to AIS grade. The majority of affected proteins (24 of 27) were more abundant in samples from AIS A patients than in those from AIS C patients, suggesting that for the most part, these proteins are released into the CSF more readily with more severe trauma to the spinal cord. We then analyzed the relationship between CSF protein abundance and neurological recovery. For AIS grade improvement over 6 months, we identified 34 proteins that were associated with AIS grade conversion (p < 0.05); however, these associations were not statistically significant after adjusting for multiple comparisons. For total motor score (TMS) recovery over 6 months, after adjusting for baseline neurological injury level, we identified 46 proteins with a statistically significant association with TMS recovery. Twenty-two of these proteins were among the 27 proteins that were related to baseline AIS grade, consistent with the notion that protein markers that reflect a more severe injury also appropriately predict a poorer recovery of motor function. In summary, this study provides a description of the CSF proteome changes that occur after acute human SCI, and reveals a number of protein candidates for further validation as potential biomarkers of injury severity.

Analysis of pathological defects in methionine metabolism using a simple mathematical model.

Derangements in methionine metabolism are a hallmark of cancers and homocystinuria, an inborn error of metabolism. In this study, the metabolic consequences of the pathological changes associated with the key pathway enzymes, methionine adenosyl transferase (MAT), glycine N-methyl transferase (GNMT) and cystathionine beta-synthase (CBS) as well as an activation of polyamine metabolism, were analyzed using a simple mathematical model describing methionine metabolism in liver. The model predicts that the mere loss of allosteric regulation of CBS by adenosylmethionine (AdoMet) leads to an increase in homocysteine concentration. This is consistent with the experimental data on the corresponding genetic defects, which specifically impair allosteric activation but not basal enzyme activity. Application of the characteristics of transformed hepatocytes to our model, i.e., substitution of the MAT I/III isozyme by MAT II, loss of GNMT activity and activation of polyamine biosynthesis, leads to the prediction of a significantly different dependence of methionine metabolism on methionine concentrations. The theoretical predictions were found to be in good agreement with experimental data obtained with the human hepatoma cell line, HepG2.

TAILS N-Terminomics and Proteomics Show Protein Degradation Dominates over Proteolytic Processing by Cathepsins in Pancreatic Tumors.

Deregulated cathepsin proteolysis occurs across numerous cancers, but in vivo substrates mediating tumorigenesis remain ill-defined. Applying 8-plex iTRAQ terminal amine isotopic labeling of substrates (TAILS), a systems-level N-terminome degradomics approach, we identified cathepsin B, H, L, S, and Z in vivo substrates and cleavage sites with the use of six different cathepsin knockout genotypes in the Rip1-Tag2 mouse model of pancreatic neuroendocrine tumorigenesis. Among 1,935 proteins and 1,114 N termini identified by TAILS, stable proteolytic products were identified in wild-type tumors compared with one or more different cathepsin knockouts (17%-44% of 139 cleavages). This suggests a lack of compensation at the substrate level by other cathepsins. The majority of neo-N termini (56%-83%) for all cathepsins was consistent with protein degradation. We validated substrates, including the glycolytic enzyme pyruvate kinase M2 associated with the Warburg effect, the ER chaperone GRP78, and the oncoprotein prothymosin-alpha. Thus, the identification of cathepsin substrates in tumorigenesis improves the understanding of cathepsin functions in normal physiology and cancer.

Active site specificity profiling of the matrix metalloproteinase family: Proteomic identification of 4300 cleavage sites by nine MMPs explored with structural and synthetic peptide cleavage analyses.

Secreted and membrane tethered matrix metalloproteinases (MMPs) are key homeostatic proteases regulating the extracellular signaling and structural matrix environment of cells and tissues. For drug targeting of proteases, selectivity for individual molecules is highly desired and can be met by high yield active site specificity profiling. Using the high throughput Proteomic Identification of protease Cleavage Sites (PICS) method to simultaneously profile both the prime and non-prime sides of the cleavage sites of nine human MMPs, we identified more than 4300 cleavages from P6 to P6' in biologically diverse human peptide libraries. MMP specificity and kinetic efficiency were mainly guided by aliphatic and aromatic residues in P1' (with a ~32-93% preference for leucine depending on the MMP), and basic and small residues in P2' and P3', respectively. A wide differential preference for the hallmark P3 proline was found between MMPs ranging from 15 to 46%, yet when combined in the same peptide with the universally preferred P1' leucine, an unexpected negative cooperativity emerged. This was not observed in previous studies, probably due to the paucity of approaches that profile both the prime and non-prime sides together, and the masking of subsite cooperativity effects by global heat maps and iceLogos. These caveats make it critical to check for these biologically highly important effects by fixing all 20 amino acids one-by-one in the respective subsites and thorough assessing of the inferred specificity logo changes. Indeed an analysis of bona fide MEROPS physiological substrate cleavage data revealed that of the 37 natural substrates with either a P3-Pro or a P1'-Leu only 5 shared both features, confirming the PICS data. Upon probing with several new quenched-fluorescent peptides, rationally designed on our specificity data, the negative cooperativity was explained by reduced non-prime side flexibility constraining accommodation of the rigidifying P3 proline with leucine locked in S1'. Similar negative cooperativity between P3 proline and the novel preference for asparagine in P1 cements our conclusion that non-prime side flexibility greatly impacts MMP binding affinity and cleavage efficiency. Thus, unexpected sequence cooperativity consequences were revealed by PICS that uniquely encompasses both the non-prime and prime sides flanking the proteomic-pinpointed scissile bond.

Active site specificity profiling datasets of matrix metalloproteinases (MMPs) 1, 2, 3, 7, 8, 9, 12, 13 and 14.

The data described provide a comprehensive resource for the family-wide active site specificity portrayal of the human matrix metalloproteinase family. We used the high-throughput proteomic technique PICS (Proteomic Identification of protease Cleavage Sites) to comprehensively assay 9 different MMPs. We identified more than 4300 peptide cleavage sites, spanning both the prime and non-prime sides of the scissile peptide bond allowing detailed subsite cooperativity analysis. The proteomic cleavage data were expanded by kinetic analysis using a set of 6 quenched-fluorescent peptide substrates designed using these results. These datasets represent one of the largest specificity profiling efforts with subsequent structural follow up for any protease family and put the spotlight on the specificity similarities and differences of the MMP family. A detailed analysis of this data may be found in Eckhard et al. (2015) [1]. The raw mass spectrometry data and the corresponding metadata have been deposited in PRIDE/ProteomeXchange with the accession number PXD002265.

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. Through analysis of the N-terminome by iTRAQ-TAILS, we identified cotranslational and posttranslational αN-acetylation motifs, quantitative increases in protein abundance, and qualitative changes in the proteolytic signature during inflammation. Of the proteins identified in normal skin, about half were cleaved, and phorbol ester-induced inflammation increased the proportion of cleaved proteins, including chemokines and complement proteins, that were processed at previously uncharacterized sites. In response to phorbol ester-induced inflammation, mice deficient in matrix metalloproteinase 2 (MMP2) showed reduced accumulation of serum proteins in the skin and exhibited different proteolytic networks from those of wild-type mice. We found that the complement 1 (C1) inhibitor attenuated the increase in serum protein accumulation in inflamed skin. Cleavage and inactivation of the C1 inhibitor by MMP2 increased complement activation and bradykinin generation in wild-type mice, leading to increased vessel permeability during inflammation, which was diminished in Mmp2(-/-) mice. Thus, our systems-level analysis of proteolysis dissected cleavage events associated with skin inflammation and demonstrated that loss of a single protease could perturb the proteolytic signaling network and enhance inflammation.

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.

Isotopic labeling of terminal amines in complex samples identifies protein N-termini and protease cleavage products.

Effective proteome-wide strategies that distinguish the N-termini of proteins from the N-termini of their protease cleavage products would accelerate identification of the substrates of proteases with broad or unknown specificity. Our approach, named terminal amine isotopic labeling of substrates (TAILS), addresses this challenge by using dendritic polyglycerol aldehyde polymers that remove tryptic and C-terminal peptides. We analyze unbound naturally acetylated, cyclized or labeled N-termini from proteins and their protease cleavage products by tandem mass spectrometry, and use peptide isotope quantification to discriminate between the substrates of the protease of interest and the products of background proteolysis. We identify 731 acetylated and 132 cyclized N-termini, and 288 matrix metalloproteinase (MMP)-2 cleavage sites in mouse fibroblast secretomes. We further demonstrate the potential of our strategy to link proteases with defined biological pathways in complex samples by analyzing mouse inflammatory bronchoalveolar fluid and showing that expression of the poorly defined breast cancer protease MMP-11 in MCF-7 human breast cancer cells cleaves both endoplasmin and the immunomodulator and apoptosis inducer galectin-1.