Agilent Technologies. Carl Myerholtz, Ph.D. Senior Director of QTOF Technology. FAPESP/Agilent Workshop August 30,

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1 Agilent Technologies Carl Myerholtz, Ph.D. Senior Director of QTOF Technology FAPESP/Agilent Workshop August 30,

2 FAPESP/Agilent - Call for Research Proposals Focus Areas Biopharma Metabolomics In both focus areas, successful proposals could include refinement of instruments, development of new measurement protocols, new ways to analyze data, invention of new workflows, or applications of existing techniques to yield new biological understanding within the context of the specific focus. Proposals Due: 29 September 2017 For more information: FAPESP/Agilent Workshop August 30,

3 BioPharma We live in a time of rapid advances in the study of disease and the development of new therapies. New tools lead to new understanding that is revolutionizing the development of biotherapeutics. Companion diagnostics are leading us into the era of personalized medicine. New approaches based on a sound understanding of biology may lead to exciting new cures for some of our most intractable diseases. Within this focus area, the goal is to fund projects that can advance the state of the art of measurements for BioPharma, for human health, veterinary medicine and agricultural research. Specifically, we are interested in finding ways to make the discovery, development, and manufacture of biologics and biosimilars faster, easier, more robust and higher in quality. This includes characterization of monoclonal antibodies and addressing all phases of the manufacturing process: QC of inputs, process control, characterization of outputs, and authentication. Our interests span sample prep, LC, LC/MS, Columns, cellular analysis, informatics, database development and management, workflow development and standardization, and effective integration of services in support of robust and efficient analytical measurements. Research which combines leading academic work, a Biopharma partner with analytical challenges, and Agilent s unique measurement capabilities would be of added interest. FAPESP/Agilent Workshop August 30,

4 Metabolomics Small molecules have been measured in biological system for centuries; but new measurement tools have led to a renaissance in our understanding of metabolites and metabolism. Metabolomics is now one of the fastest growing areas in life science research. New tools enable identification, quantification, and structure determination of small molecules with unprecedented sensitivity, accuracy, and throughput. Increasingly, tools are being used to measure not just the presence of metabolites, but also their flux in a cell, tissue, or organism. Human health, veterinary medicine, and agricultural research are each areas of interest. The goal for this focus area is to fund projects that lead to improved tools for metabolomics or to use them to increase the understanding of biological systems to improve the quality of life. FAPESP/Agilent Workshop August 30,

5 Intact Protein Characterization Workflow Sample Prep Separation Detection Analysis Intact mab HC & LC Deglycosylated mab Agilent AssayMAP Bravo Deglycosylated HC & LC Fc, F(ab) 2 Fc, Fd & LC For Research Use Only. Not for use in diagnostic procedures. 5

6 Know More at the Intact Level Agilent 6545XT Agilent 6545XT In a previously published multi-lab study 1 characterizing the NISTmAb, 18 proteoforms are described by three labs using different HRAM systems. Each lab missed some of the proteoforms. The 6545XT AdvanceBio LC/Q-TOF detects every proteoform described, while injecting less material, and with the best mass accuracy for 13 of the 18 peaks 1. Schiel et al.; State-of-the-Art and Emerging Technologies for Therapeutic Monoclonal Antibody Characterization Volume ACS Symposium Series; American Chemical Society: Washington, DC, For Research Use Only. Not for use in diagnostic procedures. 6

7 Peptide Sequence Mapping and PTM Identification Sample Prep Separation Detection Analysis Red/Yellow/Green Confirmation of Protein Chromatogram MassHunter BioConfirm -Find Peptides -Identify Peptides -Identify PTMs -Confirm Sequence MS Spectrum Annotated MS/MS Spectrum AUTOMATICALLY Sequence Match Peptides Found For Research Use Only. Not for use in diagnostic procedures. 7

8 Methionine Oxidation Localization by MS/MS native peptide DIQMTQSPSTLSASVGDR b 1 b 3 b 4 b b b DIQMTQSPSTLSASVGDR b 1 b 3 b 4 b b b oxidized peptide b For Research Use Only. Not for use in diagnostic procedures. 8

9 Deamidation Localization by MS/MS (Asparagine) native peptide NQVSLTOLVK b 1 b 3 b b b deamidated peptide NQVSLTCLVK b 1 b 3 b b b For Research Use Only. Not for use in diagnostic procedures. 9

10 Qualitative Flux Analysis of a Cancer Cell Line with Enzyme Mutation (IDH2) Goal: Understand impact of IDH2 mutation on glutamate metabolism in chondrosarcoma cell line Tracer experiment with U-13C-Glutamine (Gln) Monitoring metabolites labeled in green Triplicate analysis for each time point (0, 0.5, 1, 3 and 8 hr) Courtesy of Justin Cross, PhD., Donald B. and Catherine C. Marron Cancer Metabolism Center, Memorial Sloan Kettering Cancer Center 10

11 Qualitative Flux Results: Isotopologue Extraction Oxoglutarate MS Spectra Isotopologue Results 2-hydroxyglutarate M+0 oxoglutarate M+5 succinate Courtesy of Justin Cross, PhD., Donald B. and Catherine C. Marron Cancer Metabolism Center, Memorial Sloan Kettering Cancer Center 11

12 Qualitative Flux Results: Isotopologue Extraction 2-Hydroxyglutarate MS Spectra Isotopologue Results M+0 2-hydroxyglutarate oxoglutarate M+5 succinate Courtesy of Justin Cross, PhD., Donald B. and Catherine C. Marron Cancer Metabolism Center, Memorial Sloan Kettering Cancer Center 12

13 Qualitative Flux Results: Isotopologue Extraction Succinate MS Spectra Isotopologue Results 2-hydroxyglutarate M+0 oxoglutarate M+4 succinate Courtesy of Justin Cross, PhD., Donald B. and Catherine C. Marron Cancer Metabolism Center, Memorial Sloan Kettering Cancer Center 13

14 Visualizing Qualitative Flux Results Quick Summary of Isotopologue Results on Network Metabolite node coloring gives quick summary of results on network Fold-change (left), label incorporation (middle) and fractional labeling (right) are shown here 2-HG is produced from glutamine by the mutant IDH2 enzyme via oxoglutarate as shown by label incorporation and fractional labeling Courtesy of Justin Cross, PhD., Donald B. and Catherine C. Marron Cancer Metabolism Center, Memorial Sloan Kettering Cancer Center 14

15 Visualizing Qualitative Flux Results Quilt Plot Display of Isotopologue Results Quilt plot displays all time points and isotopologue results Statistically significant (*) label incorporation in M+5 isotopologue of 2-HG at 3 and 8 hours Courtesy of Justin Cross, PhD., Donald B. and Catherine C. Marron Cancer Metabolism Center, Memorial Sloan Kettering Cancer Center 15

16 Visualizing Qualitative Flux Results Label Incorporation Display of Isotopologue Results Percent unlabeled and labeled for each metabolite Significant label incorporation into M+5 isotopologue of 2-HG at 3 and 8 hours Courtesy of Justin Cross, PhD., Donald B. and Catherine C. Marron Cancer Metabolism Center, Memorial Sloan Kettering Cancer Center 16

17 Qualitative Flux Results Isotopologue Plot of Oxidized Glutathione Shows cellular glutathione pool becomes labeled with first one and then two glutamine-derived 5 carbon building blocks. Due to large cellular pool, replacement most apparent at 3 and 8 hours Courtesy of Justin Cross, PhD., Donald B. and Catherine C. Marron Cancer Metabolism Center, Memorial Sloan Kettering Cancer Center 17

18 Qualitative Flux Analysis Experiment Comprehensive View of Isotopologue Results on Network Courtesy of Justin Cross, PhD., Donald B. and Catherine C. Marron Cancer Metabolism Center, Memorial Sloan Kettering Cancer Center 18

19 Summary This experiment confirms glutamine is the major substrate used to produce 2-HG in IDH2-mutant CS-1 cells For 2-HG, the presence of the M+5 isotopologue instead of the M+2 or M+4 isotopologue demonstrates the direction of the reaction The new Agilent VistaFlux workflow easily extracts isotopologue information and visualizes results on pathways Manual EIC extraction for each isotopologue and natural isotope correction for 5 metabolites at all time points required 2 weeks The VistaFlux workflow performed the same analysis for 17 metabolites in about 2 minutes! 19