The overall focus of this research project is to develop methods for characterizing protein post-translational modifications, particularly those that exist in combination on the same protein molecule and together regulate its activity. Examples include the hundreds, if not thousands, of different combinations of methyl, acetyl and phoshorylation marks that exist on histones (the "histone code"), and the crosstalk between phosphorylation and O-GlcNAcylation that occurs on many other proteins. Because only a small fraction of particular protein molecules will be post-translationally modified at any given time, protocols are required that include;(a) enrichment of the sample for the posttranslational modification of interest;(b) enzymatic digestion that converts the modified protein to the largest possible fragments that still cover the complete sequence of the protein;and (c) mass spectrometry technology that facilitates sequence analysis of fragments that contain 60-100 residues (or more) at a time and is able to assign the location of the most labile of posttranslational modifications to a single, amino-acid residue. Electron transfer dissociation (ETD) mass spectrometry, a technique developed in the previous grant period, is ideally suited for this purpose. Since ETD works best on multiply charged protein fragments, we will evaluate several enzymatic and chemical methods for increasing the charge state of peptides and proteins by converting neutral or acidic, amino-acid residues to amines that can then be protonated and function as hydrogen donors. Also proposed are several new ways of tagging OGlcNAcylated proteins so that they can be enriched from complex sample mixtures by immobilized metal affinity chromatography. In addition to the above work, we will conduct experiments to define the posttranslational modifications that exist on the histone, H3 variant, CENP-A, that is located in nucleosomes at the centromere and also continue our efforts to characterize post-translational modifications on histone and histone binding partners involved in establishing the epigenetic state (modified chromatin) in the fertilized egg. We will also characterize the substrate motif for a protein n-terminal methyltransferase, NRMT, identify human nuclear proteins that are trimethylated on the n-terminus, and identify and characterize the demethylase that removes this modification. We have already determined that RCC1, a nucleotide exchange factor that binds to histones and DNA, is methylated on its n-terminus. This modification is required for the prevention of genomic instability (polyploidy nuclei). Two other important proteins, retoblastoma B and CENP-A, are both trimethylated as well. We believe that several hundred DNA and RNA binding proteins are likely to be modified in the same way. Accordingly, we will conduct experiments to identify these proteins and determine the n-terminal motif required for trimethylation by NRMT.
Proposed here is research to develop mass spectrometry methods to identify combinations of posttranslational modifications on proteins that are involved in cell signaling. Targets for this research are proteins that regulate DNA transcription, silencing, and repair plus proper segregation of chromosomes during cell division. Proteins that regulate the cellular response to stress and nutrient availability, and those involved in establishing the embryonic epigenetic state, will also be targeted. Dysregulation of cell signaling is a hallmark of many disease states, including diabetes, cancer, and numerous mental disorders.
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|Anderson, Lissa C; Karch, Kelly R; Ugrin, Scott A et al. (2016) Analyses of Histone Proteoforms Using Front-end Electron Transfer Dissociation-enabled Orbitrap Instruments. Mol Cell Proteomics 15:975-88|
|Zentella, Rodolfo; Hu, Jianhong; Hsieh, Wen-Ping et al. (2016) O-GlcNAcylation of master growth repressor DELLA by SECRET AGENT modulates multiple signaling pathways in Arabidopsis. Genes Dev 30:164-76|
|Bailey, Aaron O; Panchenko, Tanya; Shabanowitz, Jeffrey et al. (2016) Identification of the Post-translational Modifications Present in Centromeric Chromatin. Mol Cell Proteomics 15:918-31|
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|Bhattacharjee, Somnath; Liu, Wenge; Wang, Wei-Han et al. (2015) Site-Specific Zwitterionic Polymer Conjugates of a Protein Have Long Plasma Circulation. Chembiochem 16:2451-5|
|Onikubo, Takashi; Nicklay, Joshua J; Xing, Li et al. (2015) Developmentally Regulated Post-translational Modification of Nucleoplasmin Controls Histone Sequestration and Deposition. Cell Rep :|
|Hunt, Donald F; Shabanowitz, Jeffrey; Bai, Dina L (2015) Peptide Sequence Analysis by Electron Transfer Dissociation Mass Spectrometry: A Web-Based Tutorial. J Am Soc Mass Spectrom 26:1256-8|
|Albrecht, Lauren V; Zhang, Lichao; Shabanowitz, Jeffrey et al. (2015) GSK3- and PRMT-1-dependent modifications of desmoplakin control desmoplakin-cytoskeleton dynamics. J Cell Biol 208:597-612|
|Abelin, Jennifer G; Trantham, Paisley D; Penny, Sarah A et al. (2015) Complementary IMAC enrichment methods for HLA-associated phosphopeptide identification by mass spectrometry. Nat Protoc 10:1308-18|
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