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2017 04-03 Proteomic Forum 2017, Potsdam, Alain van Gool

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Proteomics-focussed lecture, where I outlined the added value of functional omics platforms to complement the next gen sequencing, and the ability of our Translational Metabolic Laboratory to bridge this innovation and translational gap.

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2017 04-03 Proteomic Forum 2017, Potsdam, Alain van Gool

  1. 1. Potsdam, 3 April 2017 Prof Alain van Gool Professor Personalized Healthcare Translational Metabolic Laboratory Radboudumc Technology Centers E: alain.vangool@radboudumc.nl Innovative Top-Down and Glyco- proteomics in Translational Healthcare
  2. 2. • Nijmegen, The Netherlands • Mission: “To have a significant impact on healthcare” • Strategic focus on Personalized Healthcare through “the patient as partner” • Core activities: • Patient care • Research • Education • 11.000 colleagues • 52 departments • 3.300 students • 1.000 beds Radboud university medical center 2 Radboud campus: - Radboudumc - Radboud University - Hogeschool Arnhem Nijmegen - Max Planck Institute - Multiple companies
  3. 3. Patient Radboud Personalized Healthcare A significant impact on healthcare Molecule Population 3 Alain van Gool, Proteomic Forum 2017, Potsdam, 21 Feb 2017
  4. 4. Translational healthcare @ Radboudumc “we create personalized diagnostics” 4 Alain van Gool, Proteomic Forum 2017, Potsdam, 21 Feb 2017
  5. 5. Research Biomarkers Diagnostics Translational Research and Diagnostic Laboratory, 300 fte, yearly budget ~ 28M euro. Integrated diagnostic laboratory with dept of Genetics, Pathology, Pharmacy and Medical Microbiology Specialities: • Proteomics, glycomics, metabolomics • Enzymatic assays • Neurochemistry • Cellulair immunotherapy • Immunomonitoring Areas of disease: • Metabolic diseases • Mitochondrial diseases • Lysosomal /glycosylation disorders • Neuroscience • Nefrology • Iron metabolism • Pediatric oncology • Immunodeficiency • Transplantation In development: • ~500 Biomarkers • Early and late stage • Analytical development • Clinical validation Assay formats: • Immunoassay • Turbidicity assays • Flow cytometry • DNA sequencing • Mass spectrometry • Experimental human (-ized) invitro and invivo models for inflammation and immunosuppression Validated assays*: • ~ 1000 assays • 3.000.000 tests/year Areas of application: • Personalized healthcare • Diagnosis • Prognosis • Mechanism of disease • Mechanism of drug action Department of Laboratory Medicine *CCKL accreditation/RvA/EFI 5 Alain van Gool, Proteomic Forum 2017, Potsdam, 21 Feb 2017
  6. 6. 2012 Patient Targeted Metabolic screen Targeted gene analysis Diagnosis + follow-up 2016 / 2017 Patient Whole exome sequencing Targeted confirmatory gene, metabolite, enzyme testing Diagnosis + follow-up Targeted assays vs holistic approach Functional Omics screening Times are changing… (functional) genome analysis 6 Alain van Gool, Proteomic Forum 2017, Potsdam, 21 Feb 2017
  7. 7. Large-scale implementation of genome diagnostics Agilent V4  V5 0 100 200 300 400 500 600 700 january februari march april may june july august september october november december january februari march april may june july august september october november december january februari march april may june july august september october november december january februari march april may june july 2013 (n=1,533) 2014 (n=4,213) 2015 (n=5,964) Q1-Q2 2016 (n=3,543) #clinicalexomes/month Outsourcing to BGI Agilent V4 Hiseq2000 Hiseq 2000  4000 Human Genetics Nijmegen 7 Alain van Gool, Proteomic Forum 2017, Potsdam, 21 Feb 2017 (Lisenka Vissers, Marcel Nelen, Han Brunner et al)
  8. 8. Added value of WES Total number of patients with a possible diagnosis: 41 Diagnostic yield WES in retrospective cohort (n=150) Whole Exome Sequencing testing Total number of tests: 150 (x3) Average #tests/patient: 1 Genetic cause identified: 44 Standard GENETIC testing Total number of tests: 810 Average #tests/patient: 5.4 (1-28) Genetic cause identified: 11 8 Alain van Gool, Proteomic Forum 2017, Potsdam, 21 Feb 2017 Human Genetics Nijmegen (Lisenka Vissers, Marcel Nelen, Han Brunner et al)
  9. 9. One genome → multiple proteomes Genes Transcripts Proteins (21.000) (1.000.000?) Metabolites Our approach: innovate diagnosis by combining Genomics + other Omics 9 Alain van Gool, Proteomic Forum 2017, Potsdam, 21 Feb 2017
  10. 10. Our Functional Omics platforms Research Biomarkers Diagnostics Translational Metabolic Laboratory (www.youtube.com/watch?v=yhLbuX0H7rg) Targeted proteomics Glycopeptidomics 500 1000 1500 2000 m/z 5 10 15 20 25 30 35 40 Time [min] Metabolomics 400 600 800 1000 1200 1400 m/z 10 20 30 40 50 60 Time [min] Bottom-up proteomics Glycomics Top-down proteomics 10 Alain van Gool, Proteomic Forum 2017, Potsdam, 21 Feb 2017
  11. 11. Innovation in protein biomarker diagnostics Current diagnostic protein assays: • Focus on assay simplicity, robustness, throughput, costs • Often sandwich/turbidity-immunoassay format. MRM MS slowly emerging. • Mostly protein abundance • Often unknown epitope of detection • Ignore occurence of proteoforms Room for innovation ! 11 Alain van Gool, Proteomic Forum 2017, Potsdam, 21 Feb 2017
  12. 12. 12 Alain van Gool, Proteomic Forum 2017, Potsdam, 21 Feb 2017 Intact protein biomarker analysis Bottom-up proteomics Top-down proteomics
  13. 13. Top-Down proteomics: analysis of in-vitro protein processing HIS-tagremovalbyprotease 13 Alain van Gool, Proteomic Forum 2017, Potsdam, 21 Feb 2017
  14. 14. Top-Down proteomics: analysis of highly purified complex I Sequence coverage of NIDM subunit MS/MS sequence coverage: 85.7% MS sequence coverage: 100% m/z z MASCOT Score Residues Fragmentation Modifications 841.2617 13 128 S2-K92 ETD Truncated Met, Acetyl: 1 16 29 19 7411 8860 18355 10810977 155137 17598 1406 1431411381138 13146 128 164146123 170149134 17851 120 187191 13 39 23 18 70 64 61 43 36 34 66 27 42 89 21 49 93 99 173104 7231 83 1855838 91102 1111019679 161152117474 135 168121 125 15786 167158144 1627525 18815013082 18154 177103 115 CI filtered Captive 3ul 05FA_Tray02-E1_01_1071.d 0.5 1.0 1.5 2.0 7x10 Intens. 10 20 30 40 50 Time [min] Extracted Ion Chromatograms '10923.3198 Mr CI filtered Captive 3ul 05FA_Tray02-E1_01_1071.d: +MS, 11.2-12.1min, Deconvoluted (MaxEnt, 503.10-2187.28, *0.063125, 50000) 10924.3133 1+ 10925.3161 1+ 10926.3187 1+ 10927.3213 1+ 10928.3238 1+ 10929.3263 1+ 10930.3287 1+ 10931.3311 1+ 10932.3335 1+ 10933.3359 1+ 10934.3383 1+ 10935.3408 1+ 10936.3435 1+ 10937.3460 1+ 10938.3489 1+ CIfilteredCaptive₃ul₀₅FA_Tray₀₂-E₁₀1₁071.d:C₄₇₇H₇₃₄N₁₃₈O₁₅₂S₃, , 10923.3105 10922 10924 10926 10928 10930 10932 10934 10936 10938 10940 m/z NIDM subunit Mr 10.923,3198 Da Mass error: 0.0088Da (0.81 ppm) • Unambiguous identification and quantitation of mature molecular forms of Complex I subunits • 42 subunits but >250 proteoforms detected ! • Insights into the combined dynamics of all available PTMs in a single experiment (Complex I crystal-grade purified from Yarrowia lipolytica) 14 Alain van Gool, Proteomic Forum 2017, Potsdam, 21 Feb 2017 (Hans Wessels, Alain van Gool, Ulrich Brandt unpublished data)
  15. 15. Chromatographic separation of complex V subunits Nativegelelectrophoresis Top-Down proteomics: analysis of isolated membrane complexes 15 Alain van Gool, Proteomic Forum 2017, Potsdam, 21 Feb 2017 (Hans Wessels, Sergio Guerrero-Castillo, Susanne Arnold, Ulrich Brandt, Alain van Gool unpublished data)
  16. 16. Top-Down proteomics: analysis of isolated membrane complexes Chromatographic separation of complex V and identification of subunit ATP5J Ragged N-terminus of ATP5J Nativegelelectrophoresis 16 Alain van Gool, Proteomic Forum 2017, Potsdam, 21 Feb 2017
  17. 17. Top-Down proteomics: map subunit-PTMs in specific complexes Different proteoforms of subunit COX5A within complex IV Nativegelelectrophoresis Annotation Mr (Da) Relative occurrence Proteoform ID by top down MS2? Modification ID by bottom-up MS2? N terminus ID by bottom-up MS2? 44-END phospho 12.509,3623 52,1 % Yes - Yes 44-END 12.429,3950 41,6 % Yes Yes 46-END phospho 12.285,2733 2,9 % - - - 43-END deamidated 12.592,4715 1,4 % - Deamidation N77 and N102 - 43-END 12.592,4715 1,1 % - - 44-END acetyl 12.471,3661 0,5 % - Acetyl N-term Acetyl K89 Yes 44-END acetyl+acetyl 12.513,3739 0,4% - Acetyl N-term Acetyl K89 Yes (Sequence 100% proteoforms identified using top down and bottom-up MS and MS/MS) (Mass error top-down MS2  0,6-0,7 ppm) • Accurate relative quantitation of all proteoforms of one subunit in complex IV • Much better insight as compared to: • Bottom-up analysis of peptide fragments alone (missing protein data) • Analysis after enrichment for specific PTMs (eg phosphoproteomics) 17 Alain van Gool, Proteomic Forum 2017, Potsdam, 21 Feb 2017
  18. 18. Next: intact complexome proteins as new biomarkers? • Native tissue biopsies • Isolate intact membrane complexes • Separate and isolate complexes using native gels • LC-MS/MS analysis of intact proteins • Data analysis Tissue 1 (n=3) Tissue 2 (n=3) Subunit Subunit – tissue 1 Subunit – tissue 2 • Identified protein sequence of subunit • Deduce simulated sequences from database • Determine fit with experimental data 18
  19. 19. Protein(s)DNA Particular interest in protein glycosylation metabolites PTMs 500 750 1000 1250 1500 1750 m/z 10 15 20 25 30 35 40 Time [min] Q What are the factors that determine the GlycoCode? Can we embed Glycosylation in actual patient care? glycoproteins {Dirk Lefeber, Monique van Scherpenzeel} 19 Alain van Gool, Proteomic Forum 2017, Potsdam, 21 Feb 2017
  20. 20. Extracellular matrix Secreted proteins Membrane receptors, Channels, etc. -- N ER Golgi Congenital Disorders of Glycosylation Glycosyltransferases Primary factors Pediatrics 2011 Hum Mol Genet 2012 Hum Mol Genet 2013 Neurology 2012 Brain 2014 1. Golgi homeostasis 2. Sugar metabolism Secondary factors Am J Hum Gen 2009 Cell & Brain 2010 PLosGenetics 2011 Ann Neurol 2012 Nature Genetics 2012 New Eng J Med 2014 Nature Genetics 2016 Nature Comm 2016 Am J Hum Gen 2016 Am J Hum Gen 2016 {Dirk Lefeber, Monique van Scherpenzeel} 20 Alain van Gool, Proteomic Forum 2017, Potsdam, 21 Feb 2017
  21. 21. Analyse glycosylation at different levels 1. Intact glycoprotein 3. Free glycans 2. Glycopeptides 500 750 1000 1250 1500 1750 m/z 10 15 20 25 30 35 40 Time [min] Glycopeptide spectrum Nanochip-PGC-QTOF Nanochip-C8-QTOF 21 Alain van Gool, Proteomic Forum 2017, Potsdam, 21 Feb 2017
  22. 22. Complementary approaches to get the full picture P Ac Top-Down proteomics: • Quantitative information • Amino acid backbone: Q21 – H132 • Phosphoryl @ S61 • Acetyl @ K80 • Unknown mod @ N121 Glycopeptide analysis: • Glycan structure: Hex5HexNAc4NeuAc2 • Glycosylation site: N121 LC-MS/MS Digest + enrichment LC-MS/MS Simple modifications Complex modifications 22 Alain van Gool, Proteomic Forum 2017, Potsdam, 21 Feb 2017
  23. 23. Biomarker discovery by glycopeptide profiling • Mass spectrometry analysis of glycoproteins in human plasma • 1/20 microliter analysis: detection of 1.000.000 signals in one scan (1,4 Gb) • ~40.000 peptides of which >80% contain sugar modification • Potential to screen patients and identify new biomarkers? 500 1000 1500 2000 m/z 5 10 15 20 25 30 35 40 Time [min] Proof of principle study: Biomarkers !? 23 Alain van Gool, Proteomic Forum 2017, Potsdam, 21 Feb 2017
  24. 24. Comprehensive glycopeptide profiling workflow
  25. 25. Comprehensive glycopeptide profiling workflow Comprehensive QC analytics
  26. 26. New diagnostic intact glycoprotein biomarker • Glycoprofile of intact transferrin protein (80 kDa) • Identified through combination glycoproteomics and exome sequencing • Is linked to specific glycosylation disorders (rare metabolic diseases) • Implemented now in clinical care as diagnostic mass spec test 26 Alain van Gool, Proteomic Forum 2017, Potsdam, 21 Feb 2017
  27. 27. Acknowledgments Translational Metabolic Laboratory Hans Wessels Anouk Suppers Maurice van Dael Dirk Lefeber Monique van Scherpenzeel Nurulamin Bin Abu Bakar Esther Willems Sergio Guerrero-Castillo Susanne Arnold Ulrich Brandt Wynand Alkema Jenneke Keizer-Garritsen Jolein Gloerich Leo Kluijtmans Alain van Gool Ron Wevers and others Collaborators/funders alain.vangool@radboudumc.nl www.slideshare.net/alainvangool Bruker Daltonics Kristina Marx Pierre-Olivier Schmit Stuart Pengelley

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