CF@LANTA RDP Cores Highlights: the CF Analytics Core Facundo M. Fernández School of Chemistry and Biochemistry Georgia Institute of Technology
CF@LANTA RDP Center The CF@LANTA RDP Center at Emory University and Georgia Tech will promote interdisciplinary CF research and translate this knowledge into new therapeutic strategies.
Organizational structure of the CF@LANTA RDP Center Each of the dark blue boxes represents a Research Core; these are supported by the cores for administration and training, and the P&F program (light blue boxes). Green lines represent definition of research problems and acquisition of samples from our large patient population and translation of new knowledge back to the patients. Co-operated by Rabin Tirouvanziam, LouAnn Brown, Ronghu Wu, Facundo M. Fernández
Organizational structure of the CF@LANTA RDP Center Multi-institutional CFAC Core : Mass spectrometry-based high resolution accurate mass targeted metabolomic assays. Quant. panel Lipidomics GT Standard protein ID etc. Advanced assays GT : Mass spectrometry-based assays for quantitative proteomics. Redox: Analyses of GSH:GSSG, Cys:CySS and other antioxidant systems, and oxidative stress measures, in cells, tissues, and aqueous samples. Immunoinflammation + Redox Emory Immunoinflammation: State-of-the-art methods and consultation for profiling of cellular and molecular outcomes related to airway inflammation and immunological imbalance. Co-operated by Rabin Tirouvanziam, LouAnn Brown, Ronghu Wu, Facundo Fernandez
Organizational structure of the CF@LANTA RDP Center Multi-institutional CFAC Core : Mass spectrometry-based high resolution accurate mass targeted metabolomic assays. Quant. panel Lipidomics GT Standard protein ID etc. Advanced assays GT : Mass spectrometry-based assays for quantitative proteomics. Redox: Analyses of GSH:GSSG, Cys:CySS and other antioxidant systems, and oxidative stress measures, in cells, tissues, and aqueous samples. Immunoinflammation + Redox Emory Immunoinflammation: State-of-the-art methods and consultation for profiling of cellular and molecular outcomes related to airway inflammation and immunological imbalance. In the case of CFAC, the RDP Center does not subsidize cost/sample Co-operated by Rabin Tirouvanziam, LouAnn Brown, Ronghu Wu, Facundo Fernandez
Redox and Immunoinflamation subcore
Collaborative Emory and GT Cores Quant. panel Lipidomics Protein ID and Quantitation Advanced assays Discovery-Orbitrap based Network analysis Protein ID & Quantitation Systems Mass Spectrometry Center (SyMS-C) @ GT David Gaul Core-Research Scientist David Smalley Center Technical/Managing Director Emory Facundo Fernandez Faculty Director Matt Torres Faculty Co- Director Dean Jones Shuzhao Li Nick Seyfried Ronghu Wu Faculty Member
Collaborative Emory and GT Cores Quant. panel Lipidomics Protein ID and Quantitation Advanced assays Discovery-Orbitrap based Network analysis Protein ID & Quantitation Systems Mass Spectrometry Center (SyMS-C) @ GT David Gaul Core-Research Scientist David Smalley Center Technical/Managing Director Emory Facundo Fernandez Faculty Director Matt Torres Faculty Co- Director Dean Jones Shuzhao Li Nick Seyfried Ronghu Wu Faculty Member
Collaborative Emory and GT Cores Quant. panel Lipidomics Protein ID and Quantitation Advanced assays Discovery-Orbitrap based Network analysis Protein ID & Quantitation Systems Mass Spectrometry Center (SyMS-C) @ GT Protein ID Protein Quantitation Top-Down Protein Characterization (Smalley/Torres) Kinomics (Smalley) Glycosylation, Phosphorylation (Wu) Emory Protein ID Protein Quantitation Deep
Collaborative Emory and GT Cores Quant. panel Lipidomics Protein ID and Quantitation Advanced assays Discovery-Orbitrap based Network analysis Protein ID & Quantitation Systems Mass Spectrometry Center (SyMS-C) @ GT Discovery (Fernandez Lab) Targeted 1. Lipidomics (relative quantitation, SyMS-C 2015) 2. Biocrates p180 Kit (absolute quantitation, SyMS-C 2016) 3. Lipid/Metabolite Imaging MS (SyMS-C in 2016, Fernandez Lab) Discovery Emory
Basic Services - 1) Protein Identification of Isolated Proteins 2) Protein Characterization 3) Intact Mass 4) Analysis of Complex Protein Mixtures including Relative Quantitation 5) Phosphopeptide Enrichment 6) Kinome Enrichment (Future) Basic Services - 1) Lipidomics 2) Biocrates p180 3) MS Imaging (Future Core)
Protein Characterization 1) PTMs a) Phosphorylation b) Glycosylation c) Ubiquitination d) N-Acetylation e) Methylation f) S-Nitrosylation g) Lipidation h) Deamidation i) Others 2) Oxidation 3) Cleavage Site 4) Amino Acid Substitution 5) Disulfide Crosslinking 6) Chemical Modification 7) Others
Relative Quantitation of Complex Mixtures Label Free Approaches 1) Spectral Counting (PSM) 2) Ion Intensity (LFQ) 3) MRM (Multiple Reaction Monitoring) Label Approaches 1) Isobaric Tags (itraq and TMT) 2) Metabolic Labeling (SILAC and Super-SILAC) 3) ICAT (Cys Residues) 4) Other (Turowec, MCP, 2014)
MW Determination by Electrospray (Ribonuclease A) Charge +8 1711.3 +9 1521.2 +10 1369.2 Relative Abundance 100 95 90 85 80 75 70 65 60 55 50 45 40 +10 +11 +12 1141.20288 R=39100 z=12 1244.76721 R=38000 z=11 1369.24243 R=36300 +9 R=33800 z=9 +8 1711.30273 R=32100 z=8 Relative Abundance 105 100 95 90 85 80 75 70 65 60 55 50 45 40 1369.24243 R=36300 1369.54163 1368.74353 R=35704 R=36004 1369.84167 R=35204 +10 600 +11 1244.7 +12 1141.2 35 30 1750.69861 1955.34521 25 R=31700 R=30100 z=8 z=7 20 1053.49536 1413.75708 R=40500 R=29304 15 z=13 z=? 1567.51111 10 R=31800 669.58838 816.90942 978.53229 z=9 1827.65637 5 R=43504 R=43904 R=41900 R=29604 z=? z=? z=14 z=? 0 800 1000 1200 1400 1600 1800 2000 m/z 35 30 25 1368.45178 1373.03894 20 R=33304 1370.14197 R=35300 R=34904 1372.34021 15 R=35204 1373.54028 1367.53943 1370.84167 R=33300 10 R=34304 R=34504 1374.73413 5 R=36004 0 1368 1369 1370 1371 1372 1373 1374 1375 m/z + + + + + + Ribonuclease A Protein + 10 H + m/z (Mass/charge) = 1369.2 mass = 13692 Protein + 0 H + (native protein) Mass = 13682 Intensity x 10^6 140 120 100 80 60 40 20 0 Deconvoluted 13682 13948 10000 11000 12000 13000 14000 15000 16000 17000 18000 Mass, Da
Basic Services - 1) Protein Identification of Isolated Proteins 2) Protein Characterization 3) Intact Mass 4) Analysis of Complex Protein Mixtures including Relative Quantitation 5) Phosphopeptide Enrichment 6) Kinome Enrichment (Future) Basic Services - 1) Lipidomics 2) Biocrates p180 3) MS Imaging (Future Core)
Shotgun Lipidomics
Quantitative Targeted Biocrates p180 Panel Quantifies up to 186 metabolites in 5 compound classes 1. Acylcarnitines 2. Amino acids 3. Biogenic amines 4. Sum of hexoses 5. Phospho- and Sphingolipids
MS Imaging Desorption and Ionization beams Biomarker 1 m/z=m 1 Biomarker 2 m/z=m 2 x y Tissue section on x-y stage General process for obtaining a tumor tissue image using Imaging MS. Abundance m/z Mass spectrum