October 2014 Meeting Announcement, Delaware Valley Mass Spectrometry Discussion Group
PLEASE NOTE: We will meet in Mendel 101.
- Topic: "Outfoxing Foxy Phosphohistidine: New Strategies for Global Phosphohistidinomics Reveal Old and New Targets Among Enzymes of Central Metabolism."
- Speaker: David Perlman, Princeton University
- Date: Monday, October 13, 2014. 6:00 PM
- Time: Social Hour: 6:00 PM.
Talk: 7:00 PM.
Please RSVP to John Masucci JMasucci@its.jnj.com by Thursday October 9.
- Place: Department of Chemistry, Villanova University (Room 101, Mendel Hall)
- Abstract: Protein histidine phosphorylation is increasingly recognized as a key posttranslational modification (PTM) in central metabolism and cell signaling. Most is currently known about its role in bacteria, where it has been shown to be critical in two-component regulatory systems and in the phosphotransferase system (PTS) involved in sugar uptake and utilization, as well as in other regulatory activities. Although it was discovered more than 50 years ago, much initial progress came from “classic” radiolabeling experiments, and the field has advanced little in the last three decades due to the difficulty of detecting this modification in biological samples by modern mass spectrometric techniques. These difficulties arise from the labile nature of the phosphohistidine (pHis) moiety and the paucity of tools to enrich and detect this labile PTM. To address this, we pursued a methodology for global proteomic analysis of pHis proteins (Phosphohistidinomics), which has involved the development of pan-specific pHis antibodies raised against a stable pHis antigen mimetic and their utilization for selective immunoenrichment of pHis-containing peptides. Through MS study of synthetic pHis peptides and peptides from known pHis proteins, we have observed that they produce prominent characteristic neutral losses of 98, 80, and 116 Da when subjected to collision-induced dissociation (CID). Using stable isotopic labeling, we have also demonstrated that the main 98 Da neutral loss occurs by gas-phase phosphoryl transfer from pHis to the peptide C-terminal a-carboxylate or to any available Glu/Asp side-chain residues. We have exploited this gas-phase fragmentation behavior in a two-step MS-based strategy in which we initially screen enriched peptides from cellular lysates for the pHis neutral loss triplet signature, then subject these triplet-positive peptides to subsequent analyses using alternative fragmentation techniques. Using this method to analyze pHis peptides from glycerol-fed and mannitol-fed E. coli cells, we have been able to directly detect and characterize unparalleled numbers of pHis sites, including many known and several previously speculative pHis sites, largely among enzymes of the PTS. Furthermore, we identified two new sites of histidine phosphorylation on the enzymes of central metabolism, aldehyde-alcohol dehydrogenase (AdhE) and pyruvate kinase (PykF), not previously known to bear this modification. These findings may have important regulatory implications for these new proteins and demonstrate the efficacy of this approach for future Phosphohistidinomic studies in bacterial and mammalian systems.
Dr. David H. Perlman is currently a Research Scientist and Proteomic Specialist within the Princeton University Department of Chemistry, where he conducts Integrative Omics and Systems Biology research, specializing on cancer pathway analysis and protein post-translational modification characterization. Dr. Perlman previously served as the founding director of the Princeton Collaborative Proteomics Center where he pioneered integrative MS-based proteomics research at Princeton in which biologists, chemists, mass spectrometrists, and bioinformaticians work together toward common research goals. During his leadership, the Center has played a principal role in grant-funded projects totaling in the 10s of millions of dollars, and he brought in nearly $4M in MS instrumentation and capital equipment into the core lab through fund raising, academic-industry collaborations, and negotiated gift-giving, establishing a Technology Alliance Partnership with ThermoFisher Scientific and playing an instrumental role in an Princeton-Agilent Thought Leader Grant relationship. Dr. Perlman has trained and mentored numerous undergraduates, graduate students, and post-docs in Biological Mass Spectrometry, and has taught in several graduate and undergraduate Princeton University courses spanning the departments of Chemistry, Molecular Biology, Genomics, and Physics. Prior to this, Dr. Perlman received a Ph.D. Molecular and Cell Biology from Boston University School of Medicine in and served as a Post-doctoral Fellow and Senior Research Associate with renowned mass spectrometrist, Catherine E. Costello. Dr. Perlman has a BA in Physics from Carleton College.
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