In addition to helping the NHLBI investigators, our research involves developing new approaches for PTM characterization and absolute protein quantitation (e.g. measuring occupancy of nitrosylation with cys-TMT tags, acetylation occupancy, tissue ubiquitination and absolute quantification of a mitochondrial protein panel). The main accomplishments for the Proteomics Core individual research are the following: 1. Acetylation on lysine: We developed the workflows for identification, relative quantification and occupancy measurements (Chen et al, ASMS 2012, poster). Several NHLBI investigators have taken advantage of this research and applied it to e.g. heart tissue (Murphy lab, paper in preparation), identifying RIP1 acetylation (Finkel lab, Nature paper) and liver tissue (Sack lab, EMBO paper). 2. Nitrosylation identification and occupancy measurements: We developed the workflows for identification of nitrosylation sites after SNO-RAC enrichment and worked out the details for occupancy measurements using cys-TMT tags. Dr. Murphys lab utilized these techniques to identify nitrosylation sites in the mouse heart tissue (Kohr et al, Circulation Research, 2011) and to measure occupancy of nitrosylation (Kohr et al, Circulation Research, 2013). 3. Ubiquitination: We are developing ubiquitination and SUMOylation workflows for identification and quantitation. We have applied this technology to analyze heart tissue (Swatkoski et al, ASMS, 2012, oral presentation) 4. Software development: The PCF has improved the software for relative quantification QUOIL to be able to accommodate new types of raw data files. Dr. Wang also developed software for iTRAQ data analysis (QUARI) whose integral part is the ability to correct the iTRAQ ratios based on the isolation purity of precursor ions (Wang et al, ASMS 2012, poster). 5. Absolute protein quantitation: We developed a targeted peptide method taking advantage of the sensitivity of Orbitrap Velos to measure absolute abundance of STAT3 transcription factor in the pig heart tissue (Phillips et al, JBC, 2010). 6. Development of mitochondrial protein panel: We are developing targeted peptide analysis on Velos Elite to simultaneously follow specific mitochondrial proteins in healthy and diseased platelets (working with Drs. Balaban and Sack). 7. Clinical proteomics: Depletion of the most abundant serum proteins to study the protein differences in patients samples (collaboration with Dr. Saligan from NINR). Using saliva as a possible tool for biomarker discovery (collaboration with Dr. Melvin from NIDCR).

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