Global Mapping of the Topography and Magnitude of Proteolytic Events in Biological Systems Melissa M. Dix *, Gabriel M. Simon *, Benjamin F. Cravatt † * These authors contributed equally; † To whom correspondence should be addressed: [email protected] Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California 92037 Introduction Persistent Fragments are Diverse Identification of Explicit Sites of Proteases play central roles in numerous biological and Identification of Known Markers of pathological processes. Their importance is underscored by the fact Apoptosis with PROTOMAP and Often Correlate with Functional Caspase Cleavage that roughly 2% of the genes in the human genome encode Domains proteases . Despite their essential functions, even the most well- Apoptosis was induced by incubation with the pan-kinase inhibitor studied proteolytic cascades remain only partially understood, and a staurosporine ( STS ) for 4 hours large portion of human proteases are wholly uncharacterized with B respect to endogenous substrates and biological function. Control Introduction Various methods have been devised to identify protease Apoptotic (STS) substratesProteolytic. enzymesThese methods are ubiquitious can ingenerally biological be placed into three systems, comprising anywhere from 1-5% of a categories:organisms genome.(1) DNA/RNA Proteolysis isdisplay, a key regulatory (2) 2-dimensional gel electrophoresis (2D-GE) and (3) N-terminal labeling strategies. These methods have all been applied, with varying degrees of success, to identify protease substrates in numerous biological CD contexts. While protease substrates can, indeed, be identified using these methods, they all suffer from serious drawbacks. Iterative display techniques such as DNA- or RNA-display, rely on ex-vivo expression of proteins and, therefore, often result in a high rate of false-positives due to expression of substrates at superphysiological concentrations. 2D-GE is conceptually straightforward, but is In this example, a peptide was detected that spans the precise site-of - severely limited by sensitivity and reproducibility. Furthermore, cleavage (B) in U2AF2 (MTPD128). Furthermore, spectra matching the substrates identified by iterative display or 2D-GE require laborious nascent N- and C-terminal peptides (C and D, respectively) were detected. follow-up experiments involving mutagenesis and immunoblotting to identify the topography and sites of proteolysis. N-terminal labeling approaches, in which nascent N-termini are chemically captured and enriched, always identify sites of proteolysis, but detection of cleavage relies on proteomic identification of a single peptide which is frequently difficult or ambiguous. Here, we present a novel proteomic method for the global identification and description of proteolytic events in native biological systems. Termed PROTOMAP , for PRO tein TO pography and Migration Analysis Platform, this system relies on comparitive 1D SDS-PAGE fractionation followed by 1D LC-MS/MS to identify Over 70 precise sites-of-cleavage were detected in this study, the majority proteomie-wide shifts in gel-migration . These data are Time-course Demonstrates Temporal of which were previously unreported. Additionally, for many proteins in assembled into “peptographs ” which enable intuitive visualization of Stability of Persistent Fragments which explicit cleavage-sites were not detected, the site-of-cleavage could the topography and magnitude of proteolysis for each protein. We be inferred from the topography of the persistent fragments applied PROTOMAP to the intrinsic apoptotic pathway in Jurkat T- cells and identify hundreds of cleaved-proteins, the majority of which were not previously reported. Many Novel Cleavage Events were PARP1 Cleavage Discovered using PROTOMAP Caspase 3 Activation Experimental Design DNA Fragmentation Conclusions PROTOMAP is a novel 1D-GE/LC-MS/MS-based method for PROTOMAP identified 170 proteins that were not previously reported to the detection of proteolysis on a proteome-wide scale. Proteins are be cleaved. Many previously-reported proteins were highly abundant size-fractionated by SDS-PAGE and then analyzed by 1D-LC-MS/MS suggesting that previous approaches were limited by sensitivity. to detect shifts in gel-migration between control and experimental samples. Peptides from each protein are assembled into peptographs -- a unique data format that provides a detailed visual Anatomy of a Peptograph description of the topography and magnitude of proteolytic events. PROTOMAP was used to globally analyze cleavage events associated with the intrinsic apoptotic pathway in Jurkat T-cells . This approach led to identification of many proteins not previously reported as being cleaved. Furthermore, in nearly every case, detailed information about the topography of cleavage was generated, Two examples: JMJD1B and MAP2K2 were not previously reported to often yielding explicit or implicit sites-of-cleavage . Our data revealed be cleaved during apoptosis. that the vast majority of proteins cleaved during apoptosis generate persistent fragments that often correlate with functional domains. Furthermore, a time-course analysis demonstrated that the majority of these proteolytic fragments persist well beyond the half-lives of the PROTOMAP Enables Estimation of parental proteins. Collectively, these data indicate that generation of persistent fragments, corresponding to discrete protein domains, may the Magnitude of Cleavage be much more common during apoptosis than originally appreciated, and that generation of active effectors may be a principal function of apoptotic cascades. Based on the quantity of parental species remaining at the 4 hr time- In summary, PROTOMAP has several advantages over other point, proteins were grouped into 2 general categories for their rates of methods currently in use, namely: degradation; rapid (>50% degraded) and slow (<50% degraded). • Exquisite sensitivity 1D-GE is Accurate and Reproducible • Magnitude of cleavage and abundance of parent protein can A B both be estimated • Detailed topography of cleavage event is generated, often yielding explicit or implicit sites-of-cleavage We believe that PROTOMAP will serve as a useful platform which can be applied to map the substrate profiles of any protease directly within the confines of the environment in which the enzyme is naturally expressed. (A) Predicted vs observed molecular weight demonstrates accurate Slow Degradation of Rapid Degradation of The complete apoptosis dataset, as well as software and migration of proteins by 1D-GE. Each circle represents the average Parental Parental documentation for running PROTOMAP are available at: observed molecular weight of a protein. By comparing the spectral counts of the parental form of the protein (B) The average spectral-counts-per-band was consistent across in control vs. apoptotic, the magnitude of cleavage can be >80% of cleaved proteins displayed fragments that persist for http://www.scripps.edu/chemphys/cravatt/protomap multiple gels demonstrating high reproducibility. estimated. at least 2 consecutive time-points (> 4 hours). .