Abstract
4.1
Activity-based Proteomics: Applications for Enzyme and Inhibitor Discovery
B. F. Cravatt III
Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA
Genome sequencing projects have revealed that eukaryotic and prokaryotic organisms universally possess a huge number of uncharacterized enzymes. The functional annotation of uncharacterized enzymatic pathways, thus, represents a grand challenge for researchers in the post-genomic era. To address this problem, global molecular profiling methods hold great promise, as they provide a relatively unbiased portrait of the biochemical composition of cells and tissues and can reveal unanticipated alterations in their metabolic and signaling networks. Nonetheless, the identification and functional characterization of enzymatic pathways that support human physiology and pathology have, to date, been hindered by a lack of “systems biology” techniques that can evaluate their activity in complex biological samples. To address this problem, we have introduced functional proteomic and metabolomic technologies that record dynamics in enzyme activity in directly in native biological systems. For example, the activity-based protein profiling (ABPP) technology utilizes active site-directed chemical probes to determine the functional state of large numbers of enzymes in proteomes. In this presentation, I will describe the integrated application of ABPP and complementary functional proteomic/metabolomic methods to discover and functionally annotate enzyme activities in mammalian (patho)physiological processes, including cancer and nervous system signaling. The long-term goal of these studies is to map new biochemical pathways that play important roles in human disease and develop selective chemical tools to perturb these pathways in living systems.
4.2
Global Profiling of Proteolytic Cleavage Sites in Apoptosis
S. Mahrus1, J. Trinidad6, D. Barkan5, H. Nguyen4, A. Sali1,2, A. L. Burlingame6, and J. Wells1,3
Departments of 1Pharmaceutical Chemistry, 2Biopharmaceutical Sciences, 3Cellular and Molecular Pharmacology, 4Hematology/Oncology, and 5Graduate Group in Bioinformatics, University of California, San Francisco, CA; 6Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, University of California, San Francisco, CA
The 600 or so proteases encoded in the human genome are involved in a diversity of biological processes. Some function as nonspecific degradative enzymes associated with protein catabolism, exhaustively cleaving many protein substrates at many sites. In contrast, several others function as selective post-translational modifiers, cleaving only a few protein substrates, usually at only one or a few sites. Apoptosis is an important example of a biological process regulated by widespread but specific intracellular proteolysis, predominantly carried out by the caspase family of proteases. This genetically programmed and non-inflammatory form of cell death is a central component of homeostasis, tissue turnover, and development. Since chemotherapeutics typically kill cells by induction of apoptosis, this process is also highly relevant from a therapeutic standpoint. We have developed a novel method for global profiling of proteolytic cleavage sites in complex biochemical mixtures based on use of an engineered peptide ligase, termed subtiligase, for selective biotinylation of free protein N-termini and positive enrichment of corresponding N-terminal peptides. Using this method to study apoptosis, we have sequenced 333 caspase-like cleavage sites distributed among 292 protein substrates in the acute T cell leukemia cell line Jurkat following treatment with the classic chemotherapeutic etoposide. Surprisingly, these sites are generally not predicted by in vitro caspase substrate specificity, but can be used to predict other physiological caspase cleavage sites. Structural bioinformatic studies show that caspase cleavage sites often appear in surface accessible loops and even occasionally in helical regions. We also find that a disproportionate number of caspase substrates physically interact, suggesting that these dimeric proteases target protein complexes and networks to elicit apoptosis, and that targeting of multiple components in each is required for a full commitment to apoptosis. Our current efforts are focused on quantitative analysis of how proteolysis in apoptosis varies as a function of time, target cell type, and apoptotic inducer.
Support for this research was provided by the Bio-Organic Biomedical Mass Spectrometry Resource at UCSF (A. L. Burlingame, Director) through the Biomedical Research Technology Program of the NIH National Center for Research Resources, NIH NCRR P41RR001614.
4.3
N-terminal & ‘Genome free’ Proteomics; de novo Sequence Analysis by a Combination of LysN Protein Digestion and Electron Transfer Dissociation
A. J. R. Heck
Biomolecular Mass Spectrometry and Proteomics Group, Utrecht University, and Netherlands Proteomics Centre, Utrecht, The Netherlands
In this talk targeted novel targeted proteomics technologies will be discussed used to analyze I) protein N-termini and II) proteomes of species of uncharacterized genomes.
Although N-terminal processing of proteins is an essential process, not many large inventories are available, Indiana, USA particular not for human proteins. Using modern day mass spectrometry based proteomics techniques it is now possible to unravel N-terminal processing in a semi-comprehensive way. Strong cation exchange chromatography with improved separation of singly charged peptides was exploited for the targeted analysis of N-acetylated protein termini from human HEK293 cells. Taking advantage of the complementarity between Lys-N, Lys-C, and trypsin for protein digestion, a total of 1391 non-redundant acetylated protein N-termini could be identified in a multi-protease approach, representing the largest dataset of human acetylated protein N-termini to date. Sequence analysis and comparison of the dataset with related datasets from D. melanogaster, S. cerevisiae and H. salinarum provides new insights into N-terminal processing across these species.
For species with un-sequenced or poorly characterized genomes de novo sequencing of MS/MS fragmentation spectra is essential. However, de novo sequencing is challenging due to the complexity of common CID fragmentation spectra. Lys-N enzymatic cleavage in combination with ETD analysis results in fragmentation spectra almost exclusively containing N-terminal fragment ions. These, easy to interpret, ladder sequences open up a completely new window for de novo sequencing. As a proof of concept we analyzed the proteomes of ostrich muscle and hibernating bear heart. We performed a proteomics study of ostrich through Lys-N proteolytic cleavage followed by low-pH SCX fractionation, RP-nanoLC separation and ETD dissociation. The SCX fractionation is used for isolation of the ‘de novo sequence-able’ peptides. These peptides produce fragmentation spectra, after ETD, dominated by c-type ions, which are relatively easy to interpret. De novo analyses of the ETD spectra is performed by an in-house developed algorithm, called LysNDeNovo, which utilizes the presence of a single fragment ion series to assign the peptide sequence. Our de novo sequencing approach results in a significant higher number of peptides identified than searching the ETD Ostrich proteomics dataset using the Mascot search engine. Moreover, the de novo results allow the determination of point mutations as well as conserved regions between proteins of different species.
References
1. Taouatas, N., Altelaar, A. F. M., Drugan, M. M., Helbig, A. O., Mohammed, S., and Heck, A. J. R. (2009) SCX-based fractionation of Lys-N generated peptides facilitates the targeted analysis of post-translational modifications. Mol. Cell. Proteomics 8, 190–200.
2. Gauci, S., Helbig, A. O., Slijper, M., Krijgsveld, J., Heck, A. J. R., and Mohammed, S. (2009) Lys-N and trypsin cover complementary parts of the phosphoproteome in a refined SCX-based approach. Anal, Chem. 81, 4493–4501.