We offer the following types of analyses: Protein identification (from in-solution, 1D/2D gel) Relative protein quantitation (label-free, SILAC, TMT, DIGE) Peptide/protein fractionation using gels, offline basic reversed LC, IEF or SCX Protein post-translational modifications identification and relative quantification (phosphorylation, acetylation, succinylation, malonylation, nitrosylation, ubiquitination, nitration, SUMO, sulfhydration) We work with investigators on custom projects (either targeted proteins or systems biology approaches) We support over 40 investigators within NHLBI and perform around 5000 mass spectrometry hours per year. In addition to helping the NHLBI investigators, our research involves developing new approaches for PTM characterization and absolute protein quantitation (e.g. acetylation, tissue ubiquitination and absolute quantification of a mitochondrial protein panel). 1.Acetylation and succynilation on lysine: We developed the workflows for identification, relative quantification and occupancy measurements. Several NHLBI investigators have taken advantage of this research and applied it to e.g. heart tissue. 2.Phosphotyrosine modifications in patients with CLL: We used a mass spectrometry-based phosphoproteomic analysis of cells isolated from both peripheral blood and lymph nodes of CLL patients to better understand the BCR phosphorylation signaling pathway and others that may potentially play a significant role in the progression of CLL (collaboration with Wiestner lab). For example, 4382 proteins were identified from peripheral blood (PB) and lymph node (LN) derived B-cells in a 9-plex TMT experiment. 10,808 phosphopeptides were identified using TMT labeling combined with IMAC enrichment and more than 1,100 phospho-tyrosine containing peptides were identified without the need for phospho-tyrosine anti-body enrichment. 3.Software development: We designed an improved random decoy strategy to overcome some of the weaknesses associated with current decoy construction methods: reduced sequence redundancy (increased effective database size), presence of target sequences, or lacking a stochastic component. Having a stochastic component, the iRanD strategy generates decoy databases that faithfully mimic the characteristics of a target database in sequence redundancy, peptide length and mass distributions, and # of theoretical peptides, without the presence of target sequences. In target-decoy database searches, iRanD decoys perform similarly or better than current methods and can potentially lead to improved outcomes in large-scale proteomic studies. 4.Development of mitochondrial protein panel: We are developing targeted peptide analysis on Orbitrap Elite to simultaneously follow specific mitochondrial proteins in healthy and diseased platelets (working with Drs. Balaban and Sack). The targeted method is able to quantify 11 mitochondrial proteins in patient platelets with CVs less than 20%. 5.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). Serum proteomics and effect of fasting (Dr. Sack lab).

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Project End
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Budget End
Support Year
8
Fiscal Year
2015
Total Cost
Indirect Cost
Name
U.S. National Heart Lung and Blood Inst
Department
Type
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