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Bothrops alcatraz, a species endemic to Alcatrazes Islands, is regarded as critically endangered due to its small area of occurrence and the declining quality of its habitat. We recently reported the identification of N-glycans attached to toxins of Bothrops species, showing similar compositions in venoms of the B. jararaca complex (B. jararaca, B. insularis, and B. alcatraz). Here, we characterized B. alcatraz venom using electrophoretic, proteomic, and glycoproteomic approaches. Electrophoresis showed that B. alcatraz venom differs from B. jararaca and B. insularis; however, N-glycan removal revealed similarities between them, indicating that the occupation of N-glycosylation sites contributes to interspecies variability in the B. jararaca complex. Metalloproteinase was the major toxin class identified in the B. alcatraz venom proteome followed by serine proteinase and C-type lectin, and overall, the adult B. alcatraz venom resembles that of B. jararaca juvenile specimens. The comparative glycoproteomic analysis of B. alcatraz venom with B. jararaca and B. insularis indicated that there may be differences in the utilization of N-glycosylation motifs among their different toxin classes. Furthermore, we prospected for the first time the N-terminome of a snake venom using the terminal amine isotopic labeling of substrates (TAILS) approach and report the presence of ∼30% of N-termini corresponding to truncated toxin forms and ∼37% N-terminal sequences blocked by pyroglutamic acid in B. alcatraz venom. These findings underscore a low correlation between venom gland transcriptomes and proteomes and support the view that post-translational processes play a major role in shaping venom phenotypes.

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

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

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.

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.

With 2129 proteins still classified by the Human Proteome Organisation Human Proteome Project (HPP) as "missing" without compelling evidence of protein existence (PE) in humans, we hypothesized that in-depth proteomic characterization of tissues that are technically challenging to access and extract would yield evidence for tissue-specific missing proteins. Paradoxically, although the skeleton is the most massive tissue system in humans, as one of the poorest characterized by proteomics, bone falls under the HPP umbrella term as a "rare tissue". Therefore, we aimed to optimize mineralized tissue protein extraction methodology and workflows for proteomic and data analyses of small quantities of healthy young adult human alveolar bone. Osteoid was solubilized by GuHCl extraction, with hydroxyapatite-bound proteins then released by ethylenediaminetetraacetic acid demineralization. A subsequent GuHCl solubilization extraction was followed by solid-phase digestion of the remaining insoluble cross-linked protein using trypsin and then 6 M urea dissolution incorporating LysC digestion. Bone extracts were digested in parallel using trypsin, LysargiNase, AspN, or GluC prior to liquid chromatography-mass spectrometry analysis. Terminal Amine Isotopic Labeling of Substrates was used to purify semitryptic peptides, identifying natural and proteolytic-cleaved neo N-termini of bone proteins. Our strategy enabled complete solubilization of the organic bone matrix leading to extensive categorization of bone proteins in different bone matrix extracts, and hence matrix compartments, for the first time. Moreover, this led to the high confidence identification of pannexin-3, a "missing protein", found only in the insoluble collagenous matrix and revealed for the first time by trypsin solid-phase digestion. We also found a singleton proteotypic peptide of another missing protein, meiosis inhibitor protein 1. We also identified 17 proteins classified in neXtprot as PE1 based on evidence other than from MS, termed non-MS PE1 proteins, including ≥9-mer proteotypic peptides of four proteins.

Global characterization of protein N termini provides valuable information on proteome dynamics and diversity in health and disease. Driven by the progress in mass spectrometry-based proteomics, novel approaches for the dedicated investigation of protein N termini and protease substrates have been recently developed. Terminal amine isotopic labeling of substrates (TAILS) is a quantitative proteomics approach suitable for high-throughput and system-wide profiling of protein N termini in complex biological matrices. TAILS employs isotopic labeling of primary amines of intact proteins in combination with an amine-reactive high molecular weight polymer (HPG-ALD) for depletion of internal tryptic peptides and high enrichment of protein N termini by negative selection. Thereby, TAILS allows simultaneous identification of the natural N termini, protease-generated neo-N termini, and endogenously modified (e.g., acetylated) N termini. In this chapter, we provide a protocol for tandem mass tag (TMT)-TAILS analysis and further discuss specific considerations regarding N-terminome data interpretation using Proteome Discoverer™ software.

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.

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.

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.

The comprehensive profiling of the repertoire of secreted proteins from cancer cells samples provides information on the signaling events in oncogenesis as well as on the cross-talk between normal and tumoral cells. Moreover, the analysis of post-translational modifications in secreted proteins may unravel biological circuits regulated by irreversible modifications such as proteolytic processing. In this context, we used Terminal Amine Isotopic Labeling of Substrates (TAILS) to perform a system-wide investigation on the N-terminome of the secretomes derived from a paired set of mouse cell lines: Melan-a (a normal melanocyte) and Tm1 (its transformed phenotype). Evaluation of the amino acid identities at the scissile bond in internal peptides revealed significant differences, suggesting distinct proteolytic processes acting in the normal and tumoral secretomes. The mapping and annotation of cleavage sites in the tumoral secretome suggested functional roles of active proteases in central biological processes related to oncogenesis, such as the processing of growth factors, cleavage of extracellular matrix proteins and the shedding of ectopic domains from the cell surface, some of which may represent novel processed forms of the corresponding proteins. In the context of the tumor microenvironment, these results suggest important biological roles of proteolytic processing in murine melanoma secreted proteins.

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

Determination of drug targets and development of novel therapeutics for the treatment of different cancers are actively ongoing areas of research. Proteases being the second largest group of enzymes in humans present themselves as attractive targets for blocking and activation to treat malignancies. However, determination of the protease cleavage substrates is often missed by utilizing conventional modern proteomic approaches. The relatively low abundance of proteolytically processed, and mostly semi-tryptic, peptides compared to tryptic peptides generated in shotgun proteomics compounded with their poorer identification rates makes the identification of such critical peptides challenging and so are mostly overlooked. Our laboratory introduced Terminal Amine Isotopic Labeling of Substrates (TAILS) to identify N-terminal peptides from cleavage events. In this chapter we present a protocol from our complementary method carboxy-TAILS (C-TAILS) to identify C-terminal peptides in metabolically labeled cancer cell lines.

The proteome and N-terminome of the human odontoblast cell layer were identified for the first time by shotgun proteomic and terminal amine isotopic labeling of substrates (TAILS) N-terminomic analyses, respectively, and compared with that of human dental pulp stroma from 26 third molar teeth. After reverse-phase liquid chromatography-tandem mass spectrometry,170,000 spectra from the shotgun and TAILS analyses were matched by 4 search engines to 4,888 and 12,063 peptides in the odontoblast cell layer and pulp stroma, respectively. Within these peptide groups, 1,543 and 5,841 protein N-termini, as well as 895 and 2,423 unique proteins, were identified with a false discovery rate of ≤1%. Thus, the human dental pulp proteome was expanded by 974 proteins not previously identified among the 4,123 proteins in our 2015 dental pulp study. Further, 222 proteins of the odontoblast cell layer were not found in the pulp stroma, suggesting many of these proteins are synthesized only by odontoblasts. When comparing the proteomes of older and younger donors, differences were more apparent in the odontoblast cell layer than in the dental pulp stroma. In the odontoblast cell layer proteome, we found proteomic evidence for dentin sialophosphoprotein, which is cleaved into dentin sialoprotein and dentin phosphoprotein. By exploring the proteome of the odontoblast cell layer and expanding the known dental pulp proteome, we found distinct proteome differences compared with each other and with dentin. Moreover, between 61% and 66% of proteins also occurred as proteoforms commencing with a neo-N-terminus not annotated in UniProt. Hence, TAILS increased proteome coverage and revealed considerable proteolytic processing, by identifying stable proteoforms in these dynamic dental tissues. All mass spectrometry raw data have been deposited to ProteomeXchange with the identifierPXD006557, with the accompanying metadata at Mendeley Data ( https://data.mendeley.com/datasets/b57zfh6wmy/1 ).

Although extracellular proteases are confronted with substrate proteins expressed by multiple cell types in vivo, in most protease substrate discovery approaches, the test protease is exposed to a test proteome (secretome) derived only from a single cell type. This limits the potential substrate space and prohibits the formation of protein complexes constituted of components derived from multiple cellular origins. Mixing of secretomes collected from multiple cell types addresses this issue, but information on the cellular origin of a substrate protein is lost. Here, we describe a protocol and the corresponding data analysis workflow for a multidimensional substrate discovery approach termed SILAC -iTRAQ -TAILS that is based on hyperplexed terminal amine isotopic labeling of substrates (TAILS ), allowing identification of substrates and concomitant assignment to cellular origins in mixed secretomes within the same experiment.

The protease web is a synonym for highly regulated molecular networks comprising enzymes, substrates, inhibitors, and other regulatory proteins. Latest high-throughput methods provided huge data sets, revealing an amazing complexity of proteolytic systems important for health and disease. Based on our previous studies, we discuss major problems and questions that have to be solved to gain precise insight into the regulation of the protease web and its impact on pathophysiological conditions. The goal is a combination of different proteomic approaches that help to investigate specific protease function at a glance. Exemplarily, the characterization of the metalloproteases meprin α and meprin β by proteomic identification of cleavage sites and terminal amine isotopic labeling of substrates demonstrates the power of MS-based techniques. Meprins are rather orphan proteases and could not be assigned to precise biological functions until recently. Proteomics helped to identify meprin α and meprin β being important for collagen assembly and deposition in skin, which makes them potential therapeutic targets in fibrotic conditions. Additionally, identification of the cleavage site specificity provides the basis for the development of activity-based probes and small compound inhibitors, important for the regulation of meprin activity and subsequent treatment of associated diseases.

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.

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.

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.

Quantitative proteomics using isobaric labeling typically involves sample digestion, peptide-level labeling and 2D LC-MS/MS. Proteomic analysis of complex samples can potentially be performed more comprehensively with GeLC-MS/MS. However, combining this approach with peptide-level labeling of multiple in-gel digests from entirely sectioned gel lanes can introduce many points of variation and adversely affect the final quantitative accuracy. Alternatively, samples labeled with isobaric tags at the protein level can be combined and analyzed by GeLC-MS/MS as a single gel lane. A caveat to this strategy is that only lysine residues are labeled, which might limit protein digestion and quantitation of peptides. Here we have compared a protein-level labeling GeLC-MS/MS strategy with a peptide-level labeling 2D LC-MS/MS approach, using mouse hippocampus synaptosomes and isobaric tandem mass tags. Protein-level labeling enabled the identification of 3 times more proteins (697 versus 241) than did peptide-level labeling, and importantly for quantitation, twice as many proteins with labeled peptides (480 versus 232) were identified. Preliminary in silico analysis also suggested the alternative use of Asp-N to trypsin to circumvent the interference of lysine labeling on protein digestion. Use of Asp-N resulted in the effective analysis of fewer peptides than with trypsin for the protein-level approach (1677 versus 3131), but yielded a similar quantitative proteomic coverage in terms of both peptides (1150 versus 1181) and proteins (448 versus 480). Taken together, these experiments demonstrate that protein-level labeling combined with GeLC-MS/MS is an effective strategy for the multiplexed quantitation of synaptosomal preparations, and may also be applicable to samples of a similar proteomic complexity and dynamic range of protein abundance.

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.

Metabolomic profiling involves relative quantification of metabolites in comparative samples and identification of the significant metabolites that differentiate different groups (e.g., diseased vs. controls). Chemical isotope labeling (CIL) liquid chromatography-mass spectrometry (LC-MS) is an enabling technique that can provide improved metabolome coverage and metabolite quantification. However, chemical identification of labeled metabolites can still be a challenge. In this work, a new set of isotopic labeling reagents offering versatile properties to enhance both detection and identification are described. They were prepared by a glycine molecule (or its isotopic counterpart) and an aromatic acid with varying structures through a simple three-step synthesis route. In addition to relatively low costs of synthesizing the reagents, this reaction route allows adjusting reagent property in accordance with the desired application objective. To date, two isotopic reagents, 4-dimethylaminobenzoylamido acetic acid N-hydroxylsuccinimide ester (DBAA-NHS) and 4-methoxybenzoylamido acetic acid N-hydroxylsuccinimide ester (MBAA-NHS), for labeling the amine-containing metabolites (i.e., amine submetabolome) have been synthesized. The labeling conditions and the related LC-MS method have been optimized. We demonstrate that DBAA labeling can increase the metabolite detectability because of the presence of an electrospray ionization (ESI)-active dimethylaminobenzoyl group. On the other hand, MBAA labeled metabolites can be fragmented in MS/MS and pseudo MS(3) experiments to provide structural information on metabolites of interest. Thus, these reagents can be tailored to quantitative profiling of the amine submetabolome as well as metabolite identification in metabolomics applications.

Stable-isotope labeling coupled with liquid-phase separation and MS analysis is a powerful technique for comparative proteomics. We developed a dimethyl labeling strategy (Anal. Chem. 2003, 75, 6843-6852 and J. Proteome Res. 2005, 4, 101-108) to label peptide N-terminus and epsilon-amino groups of Lys with water-soluble formaldehyde via reductive methylation, and an isotopic pair of formaldehyde is used for binary labeling on two sets of samples. In this study, this approach is extended to a four sample labeling by combining the binary isotopic reagents of formaldehyde (d0, d2) and the binary isotopic reducing reagents, sodium cyanoborohydride (d0, d3). To ensure sufficient mass difference, this multiplexed labeling is coupled with endoproteinase Lys-C instead of trypsin for digestion, resulting in at least two labeling sites with a mass difference of 4 Da for each pair of peptide digest. Moreover, multiplex dimethyl labeling was proved to have no significant isotopic effect during RP LC elution. This method was further applied for monitoring Lys-C digestion using hemoglobin as a model. Dimethyl labeled digests derived from seven time points (1-30 h) were grouped into two sets of sample mixtures, separated by nano-LC to reduce the complexity, and then analyzed by ESI-MS/MS. The temporal study reveals that Lys-C digestion was completed in 10-15 h for all detected peptides. The multiplex dimethyl method has not only provided a simultaneous detection mean for four sample sets but has also conserved all the advantages associated with the original binary method.

Secreted proteases act on interstitial tissue secretomes released from multiple cell types. Thus, substrate proteins might be part of higher molecular complexes constituted by many proteins with diverse and potentially unknown cellular origin. In cell culture, these may be reconstituted by mixing native secretomes from different cell types prior to incubation with a test protease. Although current degradomics techniques could identify novel substrate proteins in these complexes, all information on the cellular origin is lost. To address this limitation, we combined iTRAQ-based terminal amine isotopic labeling of substrates (iTRAQ-TAILS) with SILAC to assign proteins to a specific cell type by MS1- and their cleavage by MS2-based quantification in the same experiment. We demonstrate the power of our newly established workflow by monitoring matrix metalloproteinase (MMP) 10 dependent cleavages in mixtures from light-labeled keratinocyte and heavy-labeled fibroblast secretomes. This analysis correctly assigned extracellular matrix components, such as laminins and collagens, to their respective cellular origins and revealed their processing in an MMP10-dependent manner. Hence, our newly devised degradomics workflow facilitates deeper insight into protease activity in complex intercellular compartments such as the epidermal-dermal interface by integrating multiple modes of quantification with positional proteomics. All MS data have been deposited in the ProteomeXchange with identifier PXD001643 (http://proteomecentral.proteomexchange.org/dataset/PXD001643).

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.

While phosphorylation and O-GlcNAc (cytoplasmic and nuclear glycosylation) are linked to normal and pathological changes in cell states, these post-translational modifications have been difficult to analyze in proteomic studies. We describe advances in beta-elimination / Michael addition-based approaches which allow for mass spectrometry-based identification and comparative quantification of O-phosphate or O-GlcNAc-modified peptides, as well as cysteine-containing peptides for expression analysis. The method (BEMAD) involves differential isotopic labeling through Michael addition with normal dithiothreitol (DTT) (d0) or deuterated DTT (d6), and enrichment of these peptides by thiol chromatography. BEMAD was comparable to isotope-coded affinity tags (ICAT; a commercially available differential isotopic quantification technique) in protein expression analysis, but also provided the identity and relative amounts of both O-phosphorylation and O-GlcNAc modification sites. Specificity of O-phosphate vs. O-GlcNAc mapping is achieved through coupling enzymatic dephosphorylation or O-GlcNAc hydrolysis with differential isotopic labeling. Blocking of cysteine labeling by prior oxidation of a cytosolic lysate from mouse brain allowed specific targeting of serine / threonine post-translational modifications as demonstrated through identification of 21 phosphorylation sites (5 previously reported) in a single mass spectrometry analysis. These results demonstate BEMAD is suitable for large-scale quantitative analysis of both protein expression and serine / threonine post-translational modifications.

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.

Here I describe the history of the Stable Isotope Labeling by Amino Acids in Cell culture (SILAC) technology. Although published in 2002, it had already been developed and used in my laboratory for a number of years. From the beginning, it was applied to challenging problems in cell signaling that were considered out of reach for proteomics at the time. It was also used to pioneer proteomic interactomics, time series and dynamic posttranslational modification studies. While initially developed for metabolically accessible systems, such as cell lines, it was subsequently extended to whole animal labeling as well as to clinical applications-in the form or spike-in or super-SILAC. New formats and applications for SILAC labeling continue to be developed, for instance for protein-turnover studies.

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.

Bottom-up proteomics is the analysis of peptides derived from single proteins or protein mixtures, and because each protein generates tens of peptides, there is scope for controlled reduction in complexity. We report here a new strategy for selective isolation of the N-terminal peptides of a protein mixture, yielding positionally defined peptides. The method is tolerant of several fragmentation methods, and the databases that must be searched are substantially less complex.

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

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

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.