RDP Cores Highlights: the CF Analytics Core. Facundo M. Fernández School of Chemistry and Biochemistry Georgia Institute of Technology

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1 RDP Cores Highlights: the CF Analytics Core Facundo M. Fernández School of Chemistry and Biochemistry Georgia Institute of Technology

2 RDP Center The RDP Center at Emory University and Georgia Tech will promote interdisciplinary CF research and translate this knowledge into new therapeutic strategies.

3 Organizational structure of the 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

4 Organizational structure of the 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

5 Organizational structure of the 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

6 Redox and Immunoinflamation subcore

7 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 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

8 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 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

9 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 GT Protein ID Protein Quantitation Top-Down Protein Characterization (Smalley/Torres) Kinomics (Smalley) Glycosylation, Phosphorylation (Wu) Emory Protein ID Protein Quantitation Deep

10 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 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

11 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)

12 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

13 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)

14 MW Determination by Electrospray (Ribonuclease A) Charge Relative Abundance R=39100 z= R=38000 z= R= R=33800 z= R=32100 z=8 Relative Abundance R= R=35704 R= R= R=31700 R=30100 z=8 z= R=40500 R= z=13 z=? R= z= R=43504 R=43904 R=41900 R=29604 z=? z=? z=14 z=? m/z R= R=35300 R= R= R= R=34304 R= R= m/z Ribonuclease A Protein + 10 H + m/z (Mass/charge) = mass = Protein + 0 H + (native protein) Mass = Intensity x 10^ Deconvoluted Mass, Da

15 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)

16 Shotgun Lipidomics

17 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

18 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