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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

Using approaches of transcriptomics and proteomics we have shown that the phenotype of Bothrops jararaca venom undergoes a significant rearrangement upon neonate to adult transition. Most regulatory processes in biology are intrinsically related to modifications of protein structure, function, and abundance. However, it is unclear to which extent intrinsic proteolysis affects toxins and snake venom phenotypes upon ontogenesis. Here we assessed the natural N-terminome of Bothrops jararaca newborn and adult venoms and explored the degree of N-terminal protein truncation in ontogenetic-based proteome variation. To this end we applied the Terminal Amine Isotopic Labeling of Substrates (TAILS) technology to characterize venom collected in the presence of proteinase inhibitors. We identified natural N-terminal sequences in the newborn (71) and adult (84) venoms, from which only 37 were common to both. However, truncated toxins were found in higher number in the newborn (212) than in the adult (140) venom. Moreover, sequences N-terminally blocked by pyroglutamic acid were identified in the newborn (55) and adult (49) venoms. Most toxin classes identified by their natural N-terminal sequences showed a similar number of unique peptides in the newborn and adult venoms, however, those of serine proteinases and C-type lectins were more abundant in the adult venom. Truncated sequences from at least ten toxin classes were detected, however the catalytic and cysteine-rich domains of metalloproteinases were the most prone to proteolysis, mainly in the newborn venom. Our results underscore the pervasiveness of truncations in most toxin classes and highlight variable post-translational events in newborn and adult venoms.

Gluten, a group of proteins found in wheat, barley, and rye, is the trigger of celiac disease, an immune disorder that affects about 1% of people worldwide. The toxicity of partially hydrolyzed gluten (PHG) in fermented products is less well understood due to the significant analytical challenges in PHG characterization. In this project, an N-terminal labeling mass spectrometry method, terminal amine isotopic labeling of substrates (TAILS), was optimized for the in-depth analysis of PHG and validated using a test protease (trypsin) with known cleavage specificity. Gluten N-termini in test and control groups were labeled with heavy and light formaldehyde, respectively. Trypsin-generated neo N-termini were identified by exhibiting an MS1 Log2 H:L peak area ratio with a significant difference (p .01) from zero and native N-termini with no significant difference from zero (p .01). Using this strategy, all abundant, theoretical, test protease-generated peptides in exemplar alpha/beta gliadins and gamma gliadins were identified. SIGNIFICANCE: This study is the first study that modified and evaluated TAILS analysis for the analysis of partially hydrolyzed gluten proteins. The evaluation indicated that the TAILS analysis could be modified and expanded to the identification of multiple protease cleavage sites in gluten proteins and is worth further evaluation as a novel strategy for the analysis of natural hydrolysis of gluten in food processes. This strategy also may be further applied to characterize a broader range of partially hydrolyzed allergens in foods and provide reference for their safety assessment to both industry and regulatory authorities.

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.

Quantification by series of b, y fragment ion pairs generated from isobaric-labeled peptides in MS2 spectra has recently been considered an accurate strategy in quantitative proteomics. Here we developed a novel MS2 quantification approach named quantitation by isobaric terminal labeling (QITL) by coupling (18)O labeling with dimethylation. Trypsin-digested peptides were labeled with two (16)O or (18)O atoms at their C-termini in H(2)(16)O or H(2)(18)O. After blocking all ε-amino groups of lysines through guanidination, the N-termini of the peptides were accordingly labeled with formaldehyde-d(2) or formaldehyde. These indistinguishable, isobaric-labeled peptides in MS1 spectra produce b, y fragment ion pairs in the whole mass range of MS2 spectra that can be used for quantification. In this study, the feasibility of QITL was first demonstrated using standard proteins. An accurate and reproducible quantification over a wide dynamic range was achieved. Then, complex rat liver samples were used to verify the applicability of QITL for large-scale quantitative analysis. Finally, QITL was applied to profile the quantitative proteome of hepatocellular carcinoma (HCC) and adjacent non-tumor liver tissues. Given its simplicity, low-cost, and accuracy, QITL can be widely applied in biological samples (cell lines, tissues, and body fluids, etc.) for quantitative proteomic research.

In the quantitative proteomic studies, numerous in vitro and in vivo peptide labeling strategies have been successfully applied to measure differentially regulated protein and peptide abundance. These approaches have been proven to be versatile and repeatable in biological discoveries. (15)N metabolic labeling is one of these widely adopted and economical methods. However, due to the differential incorporation rates of (15)N or (14)N, the labeling results produce imperfectly matched isotopic envelopes between the heavy and light nitrogen-labeled peptides. In the present study, we have modified the solid Arabidopsis growth medium to standardize the (15)N supply, which led to a uniform incorporation of (15)N into the whole plant protein complement. The incorporation rate (97.43±0.11%) of (15)N into (15)N-coded peptides was determined by correlating the intensities of peptide ions with the labeling efficiencies according to Gaussian distribution. The resulting actual incorporation rate (97.44%) and natural abundance of (15)N/(14)N-coded peptides are used to re-calculate the intensities of isotopic envelopes of differentially labeled peptides, respectively. A modified (15)N/(14)N stable isotope labeling strategy, SILIA, is assessed and the results demonstrate that this approach is able to differentiate the fold change in protein abundance down to 10%. The machine dynamic range limitation and purification step will make the precursor ion ratio deriving from the actual ratio fold change. It is suggested that the differentially mixed (15)N-coded and (14)N-coded plant protein samples that are used to establish the protein abundance standard curve should be prepared following a similar protein isolation protocol used to isolate the proteins to be quantitated.

Proteolysis constitutes a major post-translational modification but specificity and substrate selectivity of numerous proteases have remained elusive. In this review, we highlight how advanced techniques in the areas of proteomics and activity-based probes can be used to investigate i) protease active site specificity; ii) protease in vivo substrates; iii) protease contribution to proteome homeostasis and composition; and iv) detection and localization of active proteases. Peptide libraries together with genetical or biochemical selection have traditionally been used for active site profiling of proteases. These are now complemented by proteome-derived peptide libraries that simultaneously determine prime and non-prime specificity and characterize subsite cooperativity. Cell-contextual discovery of protease substrates is rendered possible by techniques that isolate and quantitate protein termini. Here, a novel approach termed Terminal Amine Isotopic Labeling of Substrates (TAILS) provides an integrated platform for substrate discovery and appropriate statistical evaluation of terminal peptide identification and quantification. Proteolytically generated carboxy-termini can now also be analyzed on a proteome-wide level. Proteolytic regulation of proteome composition is monitored by quantitative proteomic approaches employing stable isotope coding or label free quantification. Activity-based probes specifically recognize active proteases. In proteomic screens, they can be used to detect and quantitate proteolytic activity while their application in cellular histology allows to locate proteolytic activity in situ. Activity-based probes - especially in conjunction with positron emission tomography - are also promising tools to monitor proteolytic activities on an organism-wide basis with a focus on in vivo tumor imaging. Together, this array of methodological possibilities enables unveiling physiological protease substrate repertoires and defining protease function in the cellular- and organism-wide context.

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.