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Influenza A virus (IAV) coopts numerous host factors for efficient replication. The cysteine protease cathepsin W (CTSW) has been identified as one host factor required for IAV entry, specifically for the escape of IAVs from late endosomes. However, the substrate specificity of CTSW and the proviral mechanism are thus far unknown. Here, we show that intracellular but not secreted CTSW promotes viral entry. We reveal 79 potential direct and 31 potential indirect cellular target proteins of CTSW using the high-throughput proteomic approach terminal amine isotopic labeling of substrates (TAILS) and determine the cleavage motif shared by the substrates of CTSW. Subsequent integration with data from RNA interference (RNAi) screens for IAV host factors uncovers first insights into the proviral function of CTSW. Notably, CTSW-deficient mice display a 25% increase in survival and a delay in mortality compared to wild-type mice upon IAV infection. Altogether, these findings support the development of drugs targeting CTSW as novel host-directed antiviral therapies. IMPORTANCE Influenza viruses are respiratory pathogens and pose a constant threat to human health. Although antiviral drugs are available for influenza, the emergence and spread of drug-resistant viruses is cause for concern. Therefore, the development of new antivirals with lower chances of their target viruses acquiring resistance is urgently needed to reduce the high morbidity and mortality caused by influenza. Promising alternatives to drugs targeting viral proteins are those directed against host factors required for viral replication. The cysteine protease cathepsin W (CTSW) is an important host factor for IAV replication, and its proteolytic activity is required for fusion of viral and endosomal membranes. In this work, we identify a number of hitherto unknown CTSW substrates, providing new insights into virus-host interactions, and reveal that CTSW might also play a proviral role in an in vivo model. These results support the development of CTSW as a drug target for next-generation antivirals against influenza., Microbiology Spectrum, 10 (4), ISSN:2165-0497

We developed a simple and rapid method to enrich protein N-terminal peptides, in which the protease TrypN is first employed to generate protein N-terminal peptides without Lys or Arg and internal peptides with two positive charges at their N termini, and then, the N-terminal peptides with or without N-acetylation are separated from the internal peptides by strong cation exchange chromatography according to a retention model based on the charge/orientation of peptides. This approach was applied to 20 μg of human HEK293T cell lysate proteins to profile the N-terminal proteome. On average, 1550 acetylated and 200 unmodified protein N-terminal peptides were successfully identified in a single LC/MS/MS run with less than 3% contamination with internal peptides, even when we accepted only canonical protein N termini registered in the Swiss-Prot database. Because this method involves only two steps, protein digestion and chromatographic separation, without the need for tedious chemical reactions, it should be useful for comprehensive profiling of protein N termini, including proteoforms with neo-N termini., Graphical Abstract, Highlights • Isolation of protein N-terminal peptides without chemical reaction • Both N-acetylated and unmodified protein N termini can be isolated • Two-step isolation consisting of TrypN digestion and strong cation exchange separation • Less than 3% contamination with internal peptides, In Brief N-acetylated and unmodified protein N-terminal peptides were successfully isolated by strong cation exchange chromatographic separation of TrypN-digested peptides without chemical reaction. From 20 μg of human HEK293T cell lysate proteins, 1550 acetylated and 200 unmodified protein N-terminal peptides were identified in a single LC/MS/MS run with less than 3% contamination with internal peptides, even when we accepted only canonical protein N termini registered in the Swiss-Prot database.

Loss-of-function mutations in the secreted enzyme ADAMTS7 (a disintegrin and metalloproteinase with thrombospondin motifs 7) are associated with protection for coronary artery disease (CAD). ADAMTS7 catalytic inhibition has been proposed as a therapeutic strategy for treating CAD; however, the lack of an endogenous substrate has hindered the development of activity-based biomarkers. To identify ADAMTS7 extracellular substrates and their cleavage sites relevant to vascular disease, we used TAILS (terminal amine isotopic labeling of substrates), a method for identifying protease-generated neo-N termini. We compared the secreted proteome of vascular smooth muscle and endothelial cells expressing either full-length mouse ADAMTS7 WT, catalytic mutant ADAMTS7 E373Q or a control luciferase adenovirus. Significantly enriched N-terminal cleavage sites in ADAMTS7 WT samples were compared to the negative control conditions and filtered for stringency, resulting in catalogs of high confidence candidate ADAMTS7 cleavage sites from our three independent TAILS experiments. Within the overlap of these discovery sets, we identified 24 unique cleavage sites from 16 protein substrates, including cleavage sites in EFEMP1 (EGF-containing fibulin-like extracellular matrix protein 1/Fibulin-3). The ADAMTS7 TAILS preference for EMEMP1 cleavage at the amino acids 123.124 over the adjacent 124.125 site was validated using both endogenous EFEMP1 and purified EFEMP1 in a binary in vitro cleavage assay. Collectively our TAILS discovery experiments have uncovered hundreds of potential substrates and cleavage sites to explore disease related biological substrates and facilitate activity-based ADAMTS7 biomarker development. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/469331v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@1520ad4org.highwire.dtl.DTLVardef@144f148org.highwire.dtl.DTLVardef@13cdbc1org.highwire.dtl.DTLVardef@7f7b4f_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

Infective endocarditis is a life-threatening infection of heart valves and adjacent structures characterized by vegetations on valves and other endocardial surfaces, with tissue destruction and risk of embolization. We used high-resolution mass spectrometry to define the proteome of staphylococcal and non-staphylococcal vegetations and Terminal Amine Isotopic Labeling of Substrates (TAILS) to define their proteolytic landscapes. These approaches identified over 2000 human proteins in staphylococcal and non-staphylococcal vegetations. Individual vegetation proteomes demonstrated comparable profiles of quantitatively major constituents that overlapped with serum, platelet, and neutrophil proteomes. Staphylococcal vegetation proteomes resembled one another more than the proteomes of non-staphylococcal vegetations. TAILS demonstrated extensive proteolysis within vegetations, with numerous previously undescribed cleavages. Several proteases and pathogen-specific proteins, including virulence factors, were identified in most vegetations. Proteolytic peptides in fibronectin and complement C3 were identified as potential infective endocarditis biomarkers. Overlap of staphylococcal and non-staphylococcal vegetation proteomes suggests a convergent thrombotic and immune response to endocardial infection by diverse pathogens. However, the differences between staphylococcal and non-staphylococcal vegetations and internal variance within the non-staphylococcal group indicate that additional pathogen- or patient-specific effects exist. Pervasive proteolysis of vegetation components may arise from vegetation-intrinsic proteases and destabilize vegetations, contributing to embolism.

Protein N-termini and their modifications not only represent different protein isoforms but also relate to the functional annotation and proteolytic activities. Currently, negative selection methods, such as terminal amine isotopic labeling of substrates (TAILS), are the most popular strategy to analyze the protein N-terminome, in which dimethylation or acetylation modification is commonly used to block the free amines of proteome samples. However, after tryptic digestion, the generated long peptides, caused by the missing cleavage of blocked lysine, could hardly be identified by MS, which hindered the deep-coverage analysis of N-terminome. Herein, to solve this problem, we developed an approach, named terminal amine guanidination of substrates (TAGS). 1H-Pyrazole-1-carboxamidine was used to effectively guanidinate lysine e-amines and N-terminal α-amines, followed by tryptic digestion to generate N-terminal peptides without free amines and internal peptides with free amines. Then, the internal peptides with free amines were removed by hyperbranched polyglycerol-aldehyde polymers (HPG-ALDs) to achieve the negative enrichment of N-terminome. By TAGS, not only the cleavage rate of blocked lysine could be improved, but also the ionization efficiency of tryptic peptides was increased. In comparison, 1814 and 1620 protein N-termini were, respectively, identified by TAGS and TAILS in Saccharomyces cerevisiae (S. cerevisiae). Among them, 1012 N-termini were uniquely identified in TAGS. Furthermore, by the combination of TAGS and the stable isotope labeling with amino acids in cell culture (SILAC)/label-free quantitative method, we not only identified the known N-terminal cleavage fragment of gasdermin D but also identified some new cleavage sites during Val-boroPro-induced pyroptosis. All these results demonstrated that our developed approach, TAGS, might be of great promise for the comprehensive analysis of N-terminome and beneficial for promoting the identification of protein isoforms and studying in-depth the proteolytic activity of proteins.

Kallikrein-related peptidase 7 (KLK7) is a serine peptidase that is over expressed in ovarian cancer. In vitro functional analyses have suggested KLK7 to play a cancer progressive role, although monitoring of KLK7 expression has suggested a contradictory protective role for KLK7 in ovarian cancer patients. In order to help delineate its mechanism of action and thereby the functional roles, information on its substrate repertoire is crucial. Therefore, in this study a quantitative proteomics approach-PROtein TOpography and Migration Analysis Platform (PROTOMAP)-coupled with SILAC was used for in-depth analysis of putative KLK7 substrates from a representative ovarian cancer cell line, SKOV-3, secreted proteins. The Terminal Amine Isotopic Labeling of Substrates (TAILS) approach was used to determine the exact cleavage sites and to validate qPROTOMAP-identified putative substrates. By employing these two technically divergent approaches, exact cleavage sites on 16 novel putative substrates and two established substrates, matrix metalloprotease (MMP) 2 and insulin growth factor binding protein 3 (IGFBP3), were identified in the SKOV-3 secretome. Eight of these substrates were also identified on TAILS analysis of another ovarian cancer cell (OVMZ-6) secretome, with a further seven OVMZ-6 substrates common to the SKOV-3 qPROTOMAP profile. Identified substrates were significantly associated with the common processes of cell adhesion, extracellular matrix remodeling and cell migration according to the gene ontology (GO) biological process analysis. Biochemical validation supports a role for KLK7 in directly activating pro-MMP10, hydrolysis of IGFBP6 and cleavage of thrombospondin 1 with generation of a potentially bioactive N-terminal fragment. Overall, this study constitutes the most comprehensive analysis of the putative KLK7 degradome in any cancer to date, thereby opening new avenues for KLK7 research.

Toxoplasma gondii is one of the most successful human parasites. Central to its success is the arsenal of virulence proteins introduced into the infected host cell. Several of these virulence proteins require direct maturation by the aspartyl protease ASP5, and all require ASP5 for translocation into the host cell, yet the true number of ASP5 substrates and complete repertoire of effectors is currently unknown. Here we selectively enrich N-terminally derived peptides using Terminal Amine Isotopic Labeling of Substrates (TAILS) and use quantitative proteomics to reveal novel ASP5 substrates. We identify, using two different enrichment techniques, new ASP5 substrates and their specific cleavage sites. ASP5 substrates include two kinases and one phosphatase that reside at the host-parasite interface, which are important for infection., Toxoplasma gondii infects approximately 30% of the world’s population, causing disease primarily during pregnancy and in individuals with weakened immune systems. Toxoplasma secretes and exports effector proteins that modulate the host during infection, and several of these proteins are processed by the Golgi-associated aspartyl protease 5 (ASP5). Here, we identify ASP5 substrates by selectively enriching N-terminally derived peptides from wild-type and Δasp5 parasites. We reveal more than 2,000 unique Toxoplasma N-terminal peptides, mapping to both natural N termini and protease cleavage sites. Several of these peptides mapped directly downstream of the characterized ASP5 cleavage site, arginine-arginine-leucine (RRL). We validate candidates as true ASP5 substrates, revealing they are not processed in parasites lacking ASP5 or in wild-type parasites following mutation of the motif from RRL to ARL. All identified ASP5 substrates are dense granule proteins, and interestingly, none appear to be exported, thus differing from the analogous system in related Plasmodium spp. Instead we show that the majority of substrates reside within the parasitophorous vacuole (PV), and its membrane (the PVM), including two kinases and one phosphatase. We show that genetic deletion of WNG2 leads to attenuation in a mouse model, suggesting that this putative kinase is a new virulence factor in Toxoplasma. Collectively, these data constitute the first in-depth analyses of ASP5 substrates and shed new light on the role of ASP5 as a maturase of dense granule proteins during the Toxoplasma lytic cycle.

Fibroblast activation protein-alpha (FAP) is a cell-surface transmembrane-anchored dimeric protease. This unique, constitutively active serine protease has both dipeptidyl aminopeptidase and endopeptidase activities and can hydrolyze the post-proline bond. FAP expression is very low in adult organs but is upregulated by activated fibroblasts in sites of tissue remodeling, including fibrosis, atherosclerosis, arthritis and tumors. To identify the endogenous substrates of FAP, we immortalized primary mouse embryonic fibroblasts (MEFs) from FAP gene knockout embryos and then stably transduced them to express either enzymatically active or inactive FAP. The MEF secretomes were then analyzed using degradomic and proteomic techniques. Terminal amine isotopic labeling of substrates (TAILS)-based degradomics identified cleavage sites in collagens, many other extracellular matrix (ECM) and associated proteins, and lysyl oxidase-like-1, CXCL-5, CSF-1, and C1qT6, that were confirmed in vitro. In addition, differential metabolic labeling coupled with quantitative proteomic analysis also implicated FAP in ECM-cell interactions, as well as with coagulation, metabolism and wound healing associated proteins. Plasma from FAP-deficient mice exhibited slower than wild-type clotting times. This study provides a significant expansion of the substrate repertoire of FAP and provides insight into the physiological and potential pathological roles of this enigmatic protease.

Enteroviruses encode proteinases that are essential for processing of the translated viral polyprotein. In addition, viral proteinases also target host proteins to manipulate cellular processes and evade innate antiviral responses to promote replication and infection. Although some host protein substrates of enterovirus proteinases have been identified, the full repertoire of targets remains unknown. We used a novel quantitative in vitro proteomics-based approach, termed t erminal a mine i sotopic l abeling of s ubstrates (TAILS), to identify with high confidence 72 and 34 new host protein targets of poliovirus and coxsackievirus B3 (CVB3) 3C proteinases (3C pro s) in HeLa cell and cardiomyocyte HL-1 cell lysates, respectively. We validated a subset of candidate substrates that are targets of poliovirus 3C pro in vitro including three common protein targets, phosphoribosylformylglycinamidine synthetase (PFAS), hnRNP K, and hnRNP M, of both proteinases. 3C pro -targeted substrates were also cleaved in virus-infected cells but not noncleavable mutant proteins designed from the TAILS-identified cleavage sites. Knockdown of TAILS-identified target proteins modulated infection both negatively and positively, suggesting that cleavage by 3C pro promotes infection. Indeed, expression of a cleavage-resistant mutant form of the endoplasmic reticulum (ER)-Golgi vesicle-tethering protein p115 decreased viral replication and yield. As the first comprehensive study to identify and validate functional enterovirus 3C pro substrates in vivo , we conclude that N-terminomics by TAILS is an effective strategy to identify host targets of viral proteinases in a nonbiased manner. IMPORTANCE Enteroviruses are positive-strand RNA viruses that encode proteases that cleave the viral polyprotein into the individual mature viral proteins. In addition, viral proteases target host proteins in order to modulate cellular pathways and block antiviral responses in order to facilitate virus infection. Although several host protein targets have been identified, the entire list of proteins that are targeted is not known. In this study, we used a novel unbiased proteomics approach to identify ∼100 novel host targets of the enterovirus 3C protease, thus providing further insights into the network of cellular pathways that are modulated to promote virus infection.

Proteases are in the center of many diseases and consequently proteases and their substrates are important drug targets as represented by an estimated 5-10% of all drugs under development. Mass spectrometry has been an indispensable tool for the discovery of novel protease substrates, particularly through the proteome-scale enrichment of so-called N-terminal peptides representing endogenous protein N-termini. Methods such as COmbined FRActional DIagonal Chromatography (COFRADIC) and later Terminal Amine Isotopic Labeling of Substrates (TAILS) have revealed numerous insights into protease substrates and consensus motifs. We present an alternative and simple protocol for N-terminal peptide enrichment, based on Charge-based FRActional DIagonal Chromatography (ChaFRADIC) and requiring only well-established protein chemistry and a pipette tip. Using iTRAQ-8plex we quantified on average 2073±52 unique N-terminal peptides from only 4.3 μg per sample/channel, allowing the identification of proteolytic targets and consensus motifs. This high sensitivity may even allow working with clinical samples such as needle biopsies in the future. We applied our method to study the dynamics of staurosporine-induced apoptosis. Our data demonstrate an orchestrated regulation of specific pathways after 1.5 h, 3 h and 6 h of treatment, with many important players of homeostasis targeted already after 1.5 h. We additionally observed an early multi-level modulation of the splicing machinery both by proteolysis and phosphorylation. This may reflect the known role of alternative splicing variants for a variety of apoptotic genes which seems to be a driving-force of staurosporine-induced apoptosis. ispartof: Molecular & Cellular Proteomics vol:17 issue:4 pages:826-834 ispartof: location:United States status: published

Mass spectrometry-based stable isotope labeling has become a key technology for protein and small-molecule analyses. We developed a multiplexed quantification method for simultaneous proteomics and amine metabolomics analyses via nano reversed-phase liquid chromatography–tandem mass spectrometry (nanoRPLC–MS/MS), called mass defect-based N,N-dimethyl leucine (mdDiLeu) labeling. The duplex mdDiLeu reagents were custom-synthesized with a mass difference of 20.5 mDa, arising from the subtle variation in nuclear binding energy between the two DiLeu isotopologues. Optimal MS resolving powers were determined to be 240K for labeled peptides and 120K for labeled metabolites on the Orbitrap Fusion Lumos instrument. The mdDiLeu labeling does not suffer from precursor interference and dynamic range compression, providing excellent accuracy for MS1-centric quantification. Quantitative information is only revealed at high MS resolution without increasing spectrum complexity and overlapping isotope distribution. Chromatographic performance of polar metabolites was dramatically improved by mdDiLeu labeling with modified hydrophobicity, enhanced ionization efficiency, and picomole levels of detection limits. Paralleled proteomics and amine metabolomics analyses using mdDiLeu were systematically evaluated and then applied to pancreatic cancer cells.

Proteolysis is an irreversible post-translational modification that affects intra- and intercellular communication by modulating the activity of bioactive mediators. Key to understanding protease function is the system-wide identification of cleavage events and their dynamics in physiological contexts. Despite recent advances in mass spectrometry-based proteomics for high-throughput substrate screening, current approaches suffer from high false positive rates and only capture single states of protease activity. Here, we present a workflow based on multiplexed terminal amine isotopic labeling of substrates for time-resolved substrate degradomics in complex proteomes. This approach significantly enhances confidence in substrate identification and categorizes cleavage events by specificity and structural accessibility of the cleavage site. We demonstrate concomitant quantification of cleavage site spanning peptides and neo-N and/or neo-C termini to estimate relative ratios of noncleaved and cleaved forms of substrate proteins. By applying this strategy to dissect the matrix metalloproteinase 10 (MMP10) substrate degradome in fibroblast secretomes, we identified the extracellular matrix protein ADAMTS-like protein 1 (ADAMTSL1) as a direct MMP10 substrate and revealed MMP10-dependent ectodomain shedding of platelet-derived growth factor receptor alpha (PDGFRα) as well as sequential processing of type I collagen. The data have been deposited to the ProteomeXchange Consortium with identifier PXD000503.

Objectives Oral tongue squamous cell carcinoma (OTSCC)is an aggressive cancer with poor survival and increasing incidence. Matrix metalloproteinase-8 (MMP-8), unlike most MMPs, provides protective effects in various human cancers including OTSCC. Yet, the mechanism behind these effects remain unclear. Knowledge of the molecular basis of disease progression is crucial for developing novel treatments. Thus our study aimed to identify novel candidate substrates of MMP-8 to examine the mechanisms behind its tumor-suppressive actions in OTSCC. Findings We have previously generated stably MMP-8 overexpressing oral squamous cell carcinoma cell line (HSC-3 MMP-8+ cells) and showed that overexpression of MMP-8 significantly decreased the cell migration and invasion. However, the molecular mechanisms hampering the motility of these cells remained unrevealed. The current study aimed to unravel these mechanisms by subjecting the secretomes of MMP-8+ and control HSC-3 cells to Terminal Amine Isotopic Labeling of Substrates (TAILS) analysis to find novel candidate substrates for MMP-8. The analysis revealed cleaved proteins, including dysadherin, 60S ribosomal protein L13, kallikrein-5, lipolysis-stimulated lipoprotein receptor, matrix-remodeling-associated protein 7, POTE ankyrin domain family member E, stathmin 1 and tubulin alpha-1C chain, which were enriched in MMP-8+ secretomes. Dysadherin is known to promote metastasis of various cancers and decrease cell adhesion. MMP-8+ cells showed decreased levels of dysadherin, suggesting a cleavage by MMP-8 from the cell membrane. Moreover, the adhesion of MMP-8+ cells was enhanced which might affect the migration. Conclusions Several novel candidate substrates of MMP-8 were revealed by TAILS analysis. The potential substrates, including dysadherin, may play crucial role in the changed behavior of MMP-8+ cells and needs to be further explored for their potential role in OTSCC. The cleavage of tumor-promoting dysadherin from the cell membrane might be one of the mechanisms by which MMP-8 increases tumor cell adhesion and thereby suppresses migration.

Background: Biological roles for intracellular dipeptidyl peptidases 8 and 9 are unknown. Results: By degradomics, 29 new in vivo substrates were identified (nine validated) for DP8/DP9, including adenylate kinase 2 and calreticulin. Conclusion: These substrates indicate roles for DP8 and DP9 in metabolism and energy homeostasis. Significance: Being the first proteomics screen for DP8/DP9 substrates, unexpected new cellular roles were revealed., 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.

Stable isotope labeling is at present one of the most powerful methods in quantitative proteomics. Stable isotope labeling has been performed at both the protein as well as the peptide level using either metabolic or chemical labeling. Here, we present a straightforward and cost-effective triplex quantification method that is based on stable isotope dimethyl labeling at the peptide level. Herein, all proteolytic peptides are chemically labeled at their alpha- and epsilon-amino groups. We use three different isotopomers of formaldehyde to enable the parallel analysis of three different samples. These labels provide a minimum of 4 Da mass difference between peaks in the generated peptide triplets. The method was evaluated based on the quantitative analysis of a cell lysate, using a typical "shotgun" proteomics experiment. While peptide complexity was increased by introducing three labels, still more than 1300 proteins could be identified using 60 microg of starting material, whereby more than 600 proteins could be quantified using at least four peptides per protein. The triplex labeling was further utilized to distinguish specific from aspecific cAMP binding proteins in a chemical proteomics experiment using immobilized cAMP. Thereby, differences in abundance ratio of more than two orders of magnitude could be quantified.

Quantitation of protein abundance is a vital component in the proteomic analysis of biological systems, which can be achieved by differential stable isotopic labeling. To analyze tissue-derived samples, the isotopic labeling can be performed using chemical labeling of the peptides post-digestion. Standard chemical labeling procedures often require many manual sample handling steps, reducing the accuracy of measurements. Here, we describe a fully automated, online (in nanoLC columns), labeling procedure, which allows protein quantitation using differential isotopic dimethyl labeling of peptide N termini and lysine residues. We show that the method allows reliable quantitation over a wide dynamic range and can be used to quantify differential protein abundances in lysates and, more targeted, differences in composition between purified protein complexes. We apply the method to determine the differences in composition between bovine liver and spleen 20 S core proteasome complexes. We find that although all catalytically active immunoproteasome subunits were up-regulated in spleen (compared with liver), only one of the normal catalytic subunits was down-regulated, suggesting that the tissue-specific immunoproteasome assembly is more diverse than previously assumed.

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.

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.

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.

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.

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.

Determining which proteins are unique among one or several protein populations is an often-encountered task in proteomics. To this purpose, we present a new method based on trypsin-catalyzed incorporation of the stabile isotope (18)O in the C-termini of tryptic peptides, followed by LC-MALDI MS analysis. The analytical strategy was designed such that proteins unique to a given population out of several can be assigned in a single experiment by the isotopic signal intensity distributions of their tryptic peptides in the recorded mass spectra. The method is demonstrated for protein-protein interaction analysis, in which the differential isotope labeling was used to distinguish endogenous human brain proteins interacting with a recombinant bait protein from nonbiospecific background binders.

Current non-gel techniques for analyzing proteomes rely heavily on mass spectrometric analysis of enzymatically digested protein mixtures. Prior to analysis, a highly complex peptide mixture is either separated on a multidimensional chromatographic system or it is first reduced in complexity by isolating sets of representative peptides. Recently, we developed a peptide isolation procedure based on diagonal electrophoresis and diagonal chromatography. We call it combined fractional diagonal chromatography (COFRADIC). In previous experiments, we used COFRADIC to identify more than 800 Escherichia coli proteins by tandem mass spectrometric (MS/MS) analysis of isolated methionine-containing peptides. Here, we describe a diagonal method to isolate N-terminal peptides. This reduces the complexity of the peptide sample, because each protein has one N terminus and is thus represented by only one peptide. In this new procedure, free amino groups in proteins are first blocked by acetylation and then digested with trypsin. After reverse-phase (RP) chromatographic fractionation of the generated peptide mixture, internal peptides are blocked using 2,4,6-trinitrobenzenesulfonic acid (TNBS); they display a strong hydrophobic shift and therefore segregate from the unaltered N-terminal peptides during a second identical separation step. N-terminal peptides can thereby be specifically collected for further liquid chromatography (LC)-MS/MS analysis. Omitting the acetylation step results in the isolation of non-lysine-containing N-terminal peptides from in vivo blocked proteins.

Cysteine-selective proteomics approaches simplify complex protein mixtures and improve the chance of detecting low abundant proteins. It is possible that cysteinyl-peptide/protein enrichment methods could be coupled to isotopic labeling and isobaric tagging methods for quantitative proteomics analyses in as few as two or up to 10 samples, respectively. Here we present two novel cysteine-selective proteomics approaches: cysteine-selective dimethyl labeling (cysDML) and cysteine-selective combined precursor isotopic labeling and isobaric tagging (cPILOT). CysDML is a duplex precursor quantification technique that couples cysteinyl-peptide enrichment with on-resin stable-isotope dimethyl labeling. Cysteine-selective cPILOT is a novel 12-plex workflow based on cysteinyl-peptide enrichment, on-resin stable-isotope dimethyl labeling, and iodoTMT tagging on cysteine residues. To demonstrate the broad applicability of the approaches, we applied cysDML and cPILOT methods to liver tissues from an Alzheimer’s disease (AD) mouse model and wild-type (WT) controls. From the cysDML experiments, an average of 850 proteins were identified and 594 were quantified, whereas from the cPILOT experiment, 330 and 151 proteins were identified and quantified, respectively. Overall, 2259 unique total proteins were detected from both cysDML and cPILOT experiments. There is tremendous overlap in the proteins identified and quantified between both experiments, and many proteins have AD/WT fold-change values that are within ~20% error. A total of 65 statistically significant proteins are differentially expressed in the liver proteome of AD mice relative to WT. The performance of cysDML and cPILOT are demonstrated and advantages and limitations of using multiple duplex experiments versus a single 12-plex experiment are highlighted.

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between samples using mass spectrometry. ReDi labeling is performed using either regular (light) or deuterated (heavy) forms of formaldehyde and sodium cyanoborohydride to add two methyl groups to each free amine. Here we demonstrate a robust protocol for ReDi labeling and quantitative comparison of complex protein mixtures. Protein samples for comparison are digested into peptides, labeled to carry either light or heavy methyl tags, mixed, and co-analyzed by LC-MS/MS. Relative protein abundances are quantified by comparing the ion chromatogram peak areas of heavy and light labeled versions of the constituent peptide extracted from the full MS spectra. The method described here includes sample preparation by reversed-phase solid phase extraction, on-column ReDi labeling of peptides, peptide fractionation by basic pH reversed-phase (BPRP) chromatography, and StageTip peptide purification. We discuss advantages and limitations of ReDi labeling with respect to other methods for stable isotope incorporation. We highlight novel applications using ReDi labeling as a fast, inexpensive, and accurate method to compare protein abundances in nearly any type of sample.

This unit describes the stepwise procedure for differential oxygen isotope labeling of peptides and mass spectrometric quantification of relative protein levels in comparative proteomic experiments. The [¹⁸O] labeling of peptides happens at the peptide C-terminus and is achieved via the enzymatic oxygen exchange of tryptic peptides via catalysis of immobilized trypsin. Experimental considerations in effective incorporation and stabilization of the oxygen labels are discussed. Methods for mass spectrometric quantification of peptides with differential [¹⁶O] and [¹⁸O] isotopes are presented.

A new method for proteolytic stable isotope labeling is introduced to provide quantitative and concurrent comparisons between individual proteins from two entire proteome pools or their subfractions. Two 18O atoms are incorporated universally into the carboxyl termini of all tryptic peptides during the proteolytic cleavage of all proteins in the first pool. Proteins in the second pool are cleaved analogously with the carboxyl termini of the resulting peptides containing two 16O atoms (i.e., no labeling). The two peptide mixtures are pooled for fractionation and separation, and the masses and isotope ratios of each peptide pair (differing by 4 Da) are measured by high-resolution mass spectrometry. Short sequences and/or accurate mass measurements combined with proteomics software tools allow the peptides to be related to the precursor proteins from which they are derived. Relative signal intensities of paired peptides quantify the expression levels of their precursor proteins from proteome pools to be compared, using an equation described in the paper. Observation of individual (unpaired) peptides is mainly interpreted as differential modification or sequence variation for the protein from the respective proteome pool. The method is evaluated here in a comparison of virion proteins for two serotypes (Ad5 and Ad2) of adenovirus, taking advantage of information already available about protein sequences and concentrations. In general, proteolytic 18O labeling enables a shotgun approach for proteomic studies with quantitation capability and is proposed as a useful tool for comparative proteomic studies of very complex protein mixtures.

We describe an approach for the accurate quantification and concurrent sequence identification of the individual proteins within complex mixtures. The method is based on a class of new chemical reagents termed isotope-coded affinity tags (ICATs) and tandem mass spectrometry. Using this strategy, we com- pared protein expression in the yeast Saccharomyces cerevisiae, using either ethanol or galactose as a carbon source. The measured differences in protein expression correlated with known yeast metabolic function under glucose-repressed conditions. The method is redundant if multiple cysteinyl residues are present, and the relative quantification is highly accurate because it is based on stable isotope dilution techniques. The ICAT approach should provide a widely applicable means to compare quantitatively glob- al protein expression in cells and tissues.

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.

While peptide-level labeling using isobaric tag reagents has been widely applied for quantitative proteomics experiments, there are comparatively few reports of protein-level labeling. Intact protein labeling could be broadly applied to quantification experiments utilizing protein-level separations or enrichment schemes. Here, protein-level isobaric labeling was explored as an alternative strategy to peptide-level labeling for serum glycoprotein quantification. Labeling and digestion conditions were optimized by comparing different organic solvents and enzymes. Digestions with Asp-N and trypsin were found highly complementary; combining the results enabled quantification of 30% more proteins than either enzyme alone. Three commercial reagents were compared for protein-level labeling. Protein identification rates were highest with iTRAQ 4-plex when compared to TMT 6-plex and iTRAQ 8-plex using higher-energy collisional dissociation on an Orbitrap Elite mass spectrometer. The compatibility of isobaric protein-level labeling with lectin-based glycoprotein enrichment was also investigated. More than 74% of lectin-bound labeled proteins were known glycoproteins, which was similar to results from unlabeled and peptide-level labeled serum samples. Finally, protein-level and peptide-level labeling strategies were compared for serum glycoprotein quantification. Isobaric protein-level labeling gave comparable identification levels and quantitative precision to peptide-level labeling.

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., Cellular and Molecular Life Sciences, 70 (2), ISSN:1420-682X, ISSN:1420-9071

Many proteomic experiments require selective labeling of either N- or C-termini of proteins and recovery of terminal peptides. Although N-termini can be selectively labeled, selective labeling of protein C-termini has not been possible due to the difficulty in discriminating between the carboxyl group on the C-terminus versus that on aspartate and glutamate residues. Here we describe the first simple proteomic approach for positive selection of protein C-termini, Profiling Protein C-Termini by Enzymatic Labeling (ProC-TEL). ProC-TEL uses carboxypeptidase Y and other readily available reagents to selectively add an affinity tag to protein C-termini and to capture C-terminal peptides from complex cell lysates for mass spectrometry (MS) identification. Using ProC-TEL, we identify novel C-terminal processing and internal proteolytic cleavage events. These results indicate that ProC-TEL provides a straightforward approach for profiling C-terminal peptides and identifying protein processing in complex biological samples.

Identification of physiologically relevant substrates is still the most challenging part in protease research for understanding the biological activity of these enzymes. The zinc-dependent metalloprotease meprin β is known to be expressed in many tissues with functions in health and disease. Here, we demonstrate unique interactions between meprin β and the amyloid precursor protein (APP). Although APP is intensively studied as a ubiquitously expressed cell surface protein, which is involved in Alzheimer disease, its precise physiological role and relevance remain elusive. Based on a novel proteomics technique termed terminal amine isotopic labeling of substrates (TAILS), APP was identified as a substrate for meprin β. Processing of APP by meprin β was subsequently validated using in vitro and in vivo approaches. N-terminal APP fragments of about 11 and 20 kDa were found in human and mouse brain lysates but not in meprin β(-/-) mouse brain lysates. Although these APP fragments were in the range of those responsible for caspase-induced neurodegeneration, we did not detect cytotoxicity to primary neurons treated by these fragments. Our data demonstrate that meprin β is a physiologically relevant enzyme in APP processing.

Stable isotope labeling based on relative peptide/protein abundance measurements is commonly applied for quantitative proteomics. Recently, trypsin-catalyzed oxygen-18 labeling has grown in popularity due to its simplicity, cost-effectiveness, and its ability to universally label peptides with high sample recovery. In (18)O labeling, both C-terminal carboxyl group atoms of tryptic peptides can be enzymatically exchanged with (18)O, thus providing the labeled peptide with a 4 Da mass shift from the (16)O-labeled sample. Peptide (18)O labeling is ideally suited for generating a labeled "universal" reference sample used for obtaining accurate and reproducible quantitative measurements across large number of samples in quantitative discovery proteomics.

Accurate quantification of protein expression in biological systems is an increasingly important part of proteomics research. Incorporation of differential stable isotopes in samples for relative protein quantification has been widely used. Stable isotope incorporation at the peptide level using dimethyl labeling is a reliable, cost-effective and undemanding procedure that can be easily automated and applied in high-throughput proteomics experiments. Although alternative multiplex quantitative proteomics approaches introduce isotope labels at the organism level ('stable isotope labeling by amino acids in cell culture' (SILAC)) or enable the simultaneous analysis of eight samples (isobaric tagging for relative and absolute quantification (iTRAQ)), stable isotope dimethyl labeling is advantageous in that it uses inexpensive reagents and is applicable to virtually any sample. We describe in-solution, online and on-column protocols for stable isotope dimethyl labeling of sample amounts ranging from sub-micrograms to milligrams. The labeling steps take approximately 60–90 min, whereas the full protocol including digestion and (two-dimensional) liquid chromatography-mass spectrometry takes approximately 1.5–3 days to complete.

SummaryThe nearly 600 proteases in the human genome regulate a diversity of biological processes, including programmed cell death. Comprehensive characterization of protease signaling in complex biological samples is limited by available proteomic methods. We have developed a general approach for global identification of proteolytic cleavage sites using an engineered enzyme to selectively biotinylate free protein N termini for positive enrichment of corresponding N-terminal peptides. Using this method to study apoptosis, we have sequenced 333 caspase-like cleavage sites distributed among 292 protein substrates. These sites are generally not predicted by in vitro caspase substrate specificity but can be used to predict other physiological caspase cleavage sites. Structural bioinformatic studies show that caspase cleavage sites often appear in surface-accessible loops and even occasionally in helical regions. Strikingly, we also find that a disproportionate number of caspase substrates physically interact, suggesting that these dimeric proteases target protein complexes and networks to elicit apoptosis.

Labeling of proteins and peptides with stable heavy isotopes (deuterium, carbon-13, nitrogen-15, and oxygen-18) is widely used in quantitative proteomics. These are either incorporated metabolically in cells and small organisms, or postmetabolically in proteins and peptides by chemical or enzymatic reactions. Only upon measurement with mass spectrometers holding sufficient resolution, light, and heavy labeled peptide ions or reporter peptide fragment ions segregate and their intensity values are subsequently used for quantification. Targeted use of these labels or mass tags further leads to specific monitoring of diverse aspects of dynamic proteomes. In this review article, commonly used isotope labeling strategies are described, both for quantitative differential protein profiling and for targeted analysis of protein modifications.

Although stable isotopic labeling has found widespread use in the proteomics field, its application to carbohydrate quantification has been limited. Herein we report the design, synthesis, and application of a novel series of compounds that allow for the incorporation of isotopic variation within glycan structures. The novel feature of the compounds is the ability to incorporate the isotopes in a controlled manner, allowing for the generation of four tags that vary only in their isotopic content. This allows for the direct comparisons of three samples or triplicate measurements with an internal standard within one mass spectral analysis. Quantitation of partially depolymerized glycosaminoglycan mixtures, as well as N-linked glycans released from fetuin, is used to demonstrate the utility of the tetraplex tagging strategy.

Elucidation of protease substrate degradomes is essential for understanding the function of proteolytic pathways in the protease web and how proteases regulate cell function. We identified matrix metalloproteinase-2 (MMP-2) cleaved proteins, solubilized pericellular matrix, and shed cellular ectodomains in the cellular context using a new multiplex proteomics approach. Tryptic peptides of intact and cleaved proteins, collected from conditioned culture medium of Mmp2(-/-) fibroblasts expressing low levels of transfected active human MMP-2 at different time points, were amine-labeled with iTRAQ mass tags. Peptide identification and relative quantitation between active and inactive protease transfectants were achieved following tag fragmentation during tandem MS. Known substrates of MMP-2 were identified thereby validating this technique with many novel MMP-2 substrates including the CX(3)CL1 chemokine fractalkine, osteopontin, galectin-1, and HSP90alpha also being identified and biochemically confirmed. In comparison with ICAT-labeling and quantitation, 8-9-fold more proteins and substrates were identified by iTRAQ. "Peptide mapping," the location of multiple peptides identified within a particular protein by iTRAQ in combination with their relative abundance ratios, enabled the domain shed and general location of the cleavage site to be identified in the native cellular substrate. Hence this advance in degradomics cell-based screens for native protein substrates casts new light on the roles for proteases in cell function.

Labeling with 18O is currently one of the most commonly used methods for incorporating a stable isotopic label into samples for comparative proteomic studies. In this approach, isotopic labeling involves the enzymatic digestion, typically performed with trypsin, of a protein population in 18O-water, which incorporates the stable isotope into the C termini of the newly formed peptides. Although trypsin is often used to facilitate isotopic incorporation after digestion, it is typically overlooked that this same mechanism can lead to isotopic loss even under conditions such as low pH where it is assumed that trypsin is inactive. To examine the role that trypsin plays in isotopic loss, several experiments were performed on the rate of delabeling under conditions relevant to multidimensional proteomic experiments. Results from these studies demonstrate that enzyme-facilitated exchange of 18O in the peptide with 16O in the aqueous solvent was the major process by which the label is removed from the peptides, even under conditions of low pH and temperature where trypsin is thought to be inactive. This study brings the rapid, tryptic-facilitated exchange to the attention of laboratories using this scheme to prevent inaccuracies in quantitative labeling due to loss of the isotopic label.

We describe here a multiplexed protein quantitation strategy that provides relative and absolute measurements of proteins in complex mixtures. At the core of this methodology is a multiplexed set of isobaric reagents that yield amine-derivatized peptides. The derivatized peptides are indistinguishable in MS, but exhibit intense low-mass MS/MS signature ions that support quantitation. In this study, we have examined the global protein expression of a wild-type yeast strain and the isogenic upf1Delta and xrn1Delta mutant strains that are defective in the nonsense-mediated mRNA decay and the general 5' to 3' decay pathways, respectively. We also demonstrate the use of 4-fold multiplexing to enable relative protein measurements simultaneously with determination of absolute levels of a target protein using synthetic isobaric peptide standards. We find that inactivation of Upf1p and Xrn1p causes common as well as unique effects on protein expression.

Phenotype in multicellular organisms is the consequence of dynamic metabolic events that occur in a spatially dependent fashion. This spatial and temporal complexity presents challenges for investigating metabolism; creating a need for improved methods that effectively probe biochemical events such as amino acid biosynthesis. Isotopic labeling can provide a temporal-spatial recording of metabolic events through, for example, the description of enriched amino acids in the protein pool. Proteins are therefore an important readout of metabolism and can be assessed with modern mass spectrometers. We compared the measurement of isotopic labeling in MS2 spectra obtained from tandem mass spectrometry under either higher energy collision dissociation (HCD) or collision induced dissociation (CID) at varied energy levels. Developing soybean embryos cultured with or without 13C-labeled substrates, and Escherichia coli MG1655 enriched by feeding 7% uniformly labeled glucose served as a source of biological material for protein evaluation. CID with low energies resulted in a disproportionate amount of heavier isotopologues remaining in the precursor isotopic distribution. HCD resulted in fewer quantifiable products; however deviation from predicted distributions were small relative to the CID-based comparisons. Fragment ions have the potential to provide information on the labeling of amino acids in peptides, but our results indicate that without further development the use of this readout in quantitative methods such as metabolic flux analysis is limited.

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.

Determining the relative abundances of proteins in biological systems is an important aspect of proteomics. Quantitation provides the possibility to unravel the often subtle molecular differences that regulate biological processes in cells and organisms. A common method to analyze differences in protein expression in complex samples is differential stable isotopic labeling combined with 2D-LC-MS separation. In such experiments, proteins or peptides from different samples are labeled with different stable isotopes and their relative amounts are determined from the peptide ion intensities using mass spectrometry. When human tissue samples are investigated, chemical stable isotope labeling strategies instead of metabolic labeling strategies are required. However, biological variation in protein expression between individuals is a key concern. Here we describe a method that allows for fully automated quantitative proteome analysis; involving desalting, triplex stable isotopic dimethyl labeling and multi-dimensional strong cation exchange/reversed phase separation of peptides prior to mass spectrometric analysis that can be applied to complex samples such as human tissue lysates. We highlight the usability of the method by characterizing the extent of biological variation between the proteomes of primary human leukocytes from three healthy donors. Using our method we were able to quantify 967 proteins with a minimum of 2 peptides, revealing very limited biological variation between the donors. The discovery is noteworthy considering the presence of significant endogenous protease activity, originating primarily from the enzyme neutrophil elastase. This dataset represents the largest quantitative dataset for human leukocytesproteins, which was made possible by the use of an automated labeling strategy.

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 ; Supplementary Tables described in this article are available via Mendeley Data (10.17632/555j3kk4sw.1).

The protease ADAMTS7 functions in the extracellular matrix (ECM) of the cardiovascular system. However, its physiological substrate specificity and mechanism of regulation remain to be explored. To address this, we conducted an unbiased substrate analysis using terminal amine isotopic labeling of substrates (TAILS). The analysis identified candidate substrates of ADAMTS7 in the human fibroblast secretome, including proteins with a wide range of functions, such as collagenous and noncollagenous extracellular matrix proteins, growth factors, proteases, and cell-surface receptors. It also suggested that autolysis occurs at Glu-729–Val-730 and Glu-732–Ala-733 in the ADAMTS7 Spacer domain, which was corroborated by N-terminal sequencing and Western blotting. Importantly, TAILS also identified proteolysis of the latent TGF-β–binding proteins 3 and 4 (LTBP3/4) at a Glu-Val and Glu-Ala site, respectively. Using purified enzyme and substrate, we confirmed ADAMTS7-catalyzed proteolysis of recombinant LTBP4. Moreover, we identified multiple additional scissile bonds in an N-terminal linker region of LTBP4 that connects fibulin-5/tropoelastin and fibrillin-1–binding regions, which have an important role in elastogenesis. ADAMTS7-mediated cleavage of LTBP4 was efficiently inhibited by the metalloprotease inhibitor TIMP-4, but not by TIMP-1 and less efficiently by TIMP-2 and TIMP-3. As TIMP-4 expression is prevalent in cardiovascular tissues, we propose that TIMP-4 represents the primary endogenous ADAMTS7 inhibitor. In summary, our findings reveal LTBP4 as an ADAMTS7 substrate, whose cleavage may potentially impact elastogenesis in the cardiovascular system. We also identify TIMP-4 as a likely physiological ADAMTS7 inhibitor.