Anna Prudova

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

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

Multiplex N-terminome Analysis of MMP-2 and MMP-9 Substrate Degradomes by iTRAQ-TAILS Quantitative Proteomics

Proteolysis is a major protein posttranslational modification that, by altering protein structure, affects protein function and, by truncating the protein sequence, alters peptide signatures of proteins analyzed by proteomics. To identify such modified and shortened protease-generated neo-N-termini on a proteome-wide basis, we developed a whole protein isobaric tag for relative and absolute quantitation (iTRAQ) labeling method that simultaneously labels and blocks all primary amines including protein N- termini and lysine side chains. Blocking lysines limits trypsin cleavage to arginine, which effectively elongates the proteolytically truncated peptides for improved MS/MS analysis and peptide identification. Incorporating iTRAQ whole protein labeling with terminal amine isotopic labeling of substrates (iTRAQ-TAILS) to enrich the N-terminome by negative selection of the blocked mature original N-termini and neo-N-termini has many advantages. It enables simultaneous characterization of the natural N-termini of proteins, their N-terminal modifications, and proteolysis product and cleavage site identification. Furthermore, iTRAQ-TAILS also enables multiplex N-terminomics analysis of up to eight samples and allows for quantification in MS2 mode, thus preventing an increase in spectral complexity and extending proteome coverage by signal amplification of low abundance proteins. We compared the substrate degradomes of two closely related matrix metalloproteinases, MMP-2 (gelatinase A) and MMP-9 (gelatinase B), in fibroblast secreted proteins. Among 3,152 unique N-terminal peptides identified corresponding to 1,054 proteins, we detected 201 cleavage products for MMP-2 and unexpectedly only 19 for the homologous MMP-9 under identical conditions. Novel substrates identified and biochemically validated include insulin-like growth factor binding protein-4, complement C1r component A, galectin-1, dickkopf-related protein-3, and thrombospondin-2. Hence, N-terminomics analyses using iTRAQ-TAILS links gelatinases with new mechanisms of action in angiogenesis and reveals unpredicted restrictions in substrate repertoires for these two very similar proteases.

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

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

Metadegradomics Toward in Vivo Quantitative Degradomics of Proteolytic Post-translational Modifications of the Cancer Proteome

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

Systems-level analysis of proteolytic events in increased vascular permeability and complement activation in skin inflammation.

Quantitative proteomic analysis of inflamed mouse skin reveals mediators of the inflammatory response in vivo. Taking a Snapshot of Inflammation Studies of inflammatory mediators, such as proteases and their inhibitors, often involve in vitro analyses or focus on a single target in vivo. However, given the complex interplay among all of the players in the inflammatory response, a system-wide analysis would provide a better understanding of how each factor works in its natural environment. With their approach of quantitatively labeling the N termini of proteins (both uncleaved and after protease-mediated cleavage) from inflamed mouse skin tissue, auf dem Keller et al. provide a snapshot of the inflammatory response in vivo. Their analysis identified previously uncharacterized cleavage events within the complement cascade and highlighted a role for matrix metalloproteinase 2, which contributed to increased blood vessel permeability during inflammation. Together, these data provide a system-wide proteolytic fingerprint of skin inflammation and generate functional hypotheses that warrant further investigation. During inflammation, vascular permeability is increased by various proteolytic events, such as the generation of bradykinin, that augment local tissue responses by enabling tissue penetration of serum proteins, including complement and acute-phase proteins. Proteases also govern inflammatory responses by processing extracellular matrix proteins and soluble bioactive mediators. We quantified changes in the proteome and the nature of protein amino termini (the N-terminome) and the altered abundance of murine proteases and inhibitors during skin inflammation. Through analysis of the N-terminome by iTRAQ-TAILS, we identified cotranslational and posttranslational αN-acetylation motifs, quantitative increases in protein abundance, and qualitative changes in the proteolytic signature during inflammation. Of the proteins identified in normal skin, about half were cleaved, and phorbol ester–induced inflammation increased the proportion of cleaved proteins, including chemokines and complement proteins, that were processed at previously uncharacterized sites. In response to phorbol ester–induced inflammation, mice deficient in matrix metalloproteinase 2 (MMP2) showed reduced accumulation of serum proteins in the skin and exhibited different proteolytic networks from those of wild-type mice. We found that the complement 1 (C1) inhibitor attenuated the increase in serum protein accumulation in inflamed skin. Cleavage and inactivation of the C1 inhibitor by MMP2 increased complement activation and bradykinin generation in wild-type mice, leading to increased vessel permeability during inflammation, which was diminished in Mmp2−/− mice. Thus, our systems-level analysis of proteolysis dissected cleavage events associated with skin inflammation and demonstrated that loss of a single protease could perturb the proteolytic signaling network and enhance inflammation.

A Statistics-based Platform for Quantitative N-terminome Analysis and Identification of Protease Cleavage Products

Terminal amine isotopic labeling of substrates (TAILS), our recently introduced platform for quantitative N-terminome analysis, enables wide dynamic range identification of original mature protein N-termini and protease cleavage products. Modifying TAILS by use of isobaric tag for relative and absolute quantification (iTRAQ)-like labels for quantification together with a robust statistical classifier derived from experimental protease cleavage data, we report reliable and statistically valid identification of proteolytic events in complex biological systems in MS2 mode. The statistical classifier is supported by a novel parameter evaluating ion intensity-dependent quantification confidences of single peptide quantifications, the quantification confidence factor (QCF). Furthermore, the isoform assignment score (IAS) is introduced, a new scoring system for the evaluation of single peptide-to-protein assignments based on high confidence protein identifications in the same sample prior to negative selection enrichment of N-terminal peptides. By these approaches, we identified and validated, in addition to known substrates, low abundance novel bioactive MMP-2 targets including the plasminogen receptor S100A10 (p11) and the proinflammatory cytokine proEMAP/p43 that were previously undescribed.

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

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

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

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

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

Deregulated cathepsin proteolysis occurs across numerous cancers, but inxa0vivo substrates mediating tumorigenesis remain ill-defined. Applying 8-plex iTRAQ terminal amine isotopic labeling of substrates (TAILS), a systems-level N-terminome degradomics approach, we identified cathepsin B, H, L, S, and Zxa0inxa0vivo substrates and cleavage sites with the use ofxa0six different cathepsin knockout genotypes in thexa0Rip1-Tag2 mouse model of pancreatic neuroendocrine tumorigenesis. Among 1,935 proteins and 1,114xa0N termini identified by TAILS, stable proteolyticxa0products were identified in wild-type tumors compared with one or more different cathepsin knockouts (17%-44% of 139 cleavages). This suggests a lack of compensation at the substrate level by other cathepsins. The majority of neo-N termini (56%-83%) for all cathepsins was consistent with protein degradation. We validated substrates, including the glycolytic enzyme pyruvate kinase M2 associated with the Warburg effect, the ER chaperone GRP78, and the oncoprotein prothymosin-alpha. Thus, the identification of cathepsin substrates in tumorigenesis improves the understanding of cathepsin functions in normal physiology and cancer.

Interactome disassembly during apoptosis occurs independent of caspase cleavage

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