The identification of natural substrates and their cleavage sites is pivotal to defining proteolytic pathways. Here we report a novel strategy for the identification of the signature of proteolytic cleavage events based on quantitative proteomics. Lysine residues in proteins are blocked by guanidination so that free N-terminals can be labeled with amine-specific iTRAQ reagents. The quantitative nature of iTRAQ reagents allows us to distinguish N-terminals newly formed by proteolytic treatment (neoepitopes) from original N-terminals in proteins. Proteins are digested with trypsin and analyzed using MALDI-TOF/TOF mass spectrometry. Peptides labeled with iTRAQ reagents are distinguished from other peptides by exhibiting intense signature ions in tandem mass spectrometry analysis. A corresponding data acquisition strategy was developed to specifically analyze iTRAQ tagged N-terminal peptides. To validate the procedure, we examined a set of recombinant Escherichia coli proteins that have predicted caspase-3 cleavage motifs. The protein mixture was treated with active or inactive caspase-3 and subsequently labeled with two different iTRAQ reagents. Mass spectrometric analysis located 10 cleavage sites, all corresponding to caspase-3 consensus. Spiking caspase-cleaved substrate into a human cell lysate demonstrated the high sensitivity of the procedure. Moreover, we were able to identify proteolytic cleavage products associated with the induction of cell-free apoptosis. Together, these data reveal a novel application for iTRAQ technology for the detection of proteolytic substrates.
"Most of the studies utilized this modification on peptide levels.   To consider trypsin-cleaved homoarginine-terminated peptides for our enriched N-terminal peptides search, we performed a trypsin digestion study of two different homoarginine-modified proteins, ubiquitin and β-casein. At first, a small protein, bovine ubiquitin (~8.6 kDa), was used for this study. "
"non-gel-based methods such as combined fractional diagonal chromatography (COFRADIC; Gevaert et al, 2003), proteomic identification of protease cleavage sites (PICS; Schilling and Overall, 2008), terminal amine isotopic labelling of substrates (TAILS; Kleifeld et al, 2010), ICAT and iTRAQ variations (Tam et al, 2004; Dean and Overall, 2007; Enoksson et al, 2007), or genetically engineered enzyme use (Mahrus et al, 2008) were found to be superior, leading to identification of a great number of protease substrates, including those of caspases, granzyme B, and various MMPs (Van Damme et al, 2005, 2009; Mahrus et al, 2008; Morrison et al, 2009). These methods, in addition to identifying cleavage sites within protein substrates, enable determination of protease specificities , especially when used on the larger peptidic fragments that are generated from proteins by trypsin, endo GluC or some other protease cleavage, or on total cellular lysates. "
[Show abstract][Hide abstract] ABSTRACT: Protease research has undergone a major expansion in the last decade, largely due to the extremely rapid development of new technologies, such as quantitative proteomics and in-vivo imaging, as well as an extensive use of in-vivo models. These have led to identification of physiological substrates and resulted in a paradigm shift from the concept of proteases as protein-degrading enzymes to proteases as key signalling molecules. However, we are still at the beginning of an understanding of protease signalling pathways. We have only identified a minor subset of true physiological substrates for a limited number of proteases, and their physiological regulation is still not well understood. Similarly, links with other signalling systems are not well established. Herein, we will highlight current challenges in protease research.
The EMBO Journal 02/2012; 31(7):1630-43. DOI:10.1038/emboj.2012.42 · 10.43 Impact Factor
"Indeed, more than 100 additional putatively cleaved proteins were found in the subthreshold data, including several established markers of apoptosis (e.g., caspase 8, STK3, and CBL) (Graves et al., 1998; Widmann et al., 1998; Table S2), as well as 20 additional explicit cleavage sites (Table S4). N-terminal labeling studies have also uncovered several proteolytic events in apoptotic cells that are non-caspase mediated (Enoksson et al., 2007), and it is possible that some of the cleaved proteins identified by PROTOMAP may result from the activity of other proteases activated during apoptosis (e.g., HtrA1, calpains, cathepsins ). Collectively, these results indicate that further efforts to improve the sensitivity and refine the analysis of PROTOMAP data should yield an even larger number of proteins that can be quantitatively profiled and provide enhanced sequence coverage to facilitate the mapping of explicit sites of proteolysis. "
[Show abstract][Hide abstract] ABSTRACT: Proteolysis is a key regulatory process that promotes the (in)activation, translocation, and/or degradation of proteins. As such, there is considerable interest in methods to comprehensively characterize proteolytic pathways in biological systems. Here, we describe a robust and versatile proteomic platform that enables direct visualization of the topography and magnitude of proteolytic events on a global scale. We use this method to generate a proteome-wide map of proteolytic events induced by the intrinsic apoptotic pathway. This profile contained 91 characterized caspase substrates as well as 170 additional proteins not previously known to be cleaved during apoptosis. Surprisingly, the vast majority of proteolyzed proteins, regardless of the extent of cleavage, yielded persistent fragments that correspond to discrete protein domains, suggesting that the generation of active effector proteins may be a principal function of apoptotic proteolytic cascades.
Keiryn L. Bennett, Xia Wang, Cory E. Bystrom, Matthew C. Chambers, Tracy M. Andacht, Larry J. Dangott, Félix Elortza, John Leszyk, Henrik Molina, Robert L Moritz, Brett S. Phinney, J. Will Thompson, Maureen K. Bunger, David L. Tabb
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