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Structural Characterization of Glycoproteins using Agilent 6210 ESI-TOF: Analysis of Post-Translational Modifications

Structural Characterization of Glycoproteins using Agilent 6210 ESI-TOF: Analysis of Post-Translational Modifications. Overview. Profile of Blue Stream Laboratories Case studies of protein characterization using the Agilent 6210

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Structural Characterization of Glycoproteins using Agilent 6210 ESI-TOF: Analysis of Post-Translational Modifications

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  1. Structural Characterization of Glycoproteins using Agilent 6210 ESI-TOF:Analysis of Post-Translational Modifications

  2. Overview • Profile of Blue Stream Laboratories • Case studies of protein characterization using the Agilent 6210 • Molecular weight determination of intact small/medium/large peptides and proteins • Sequence confirmation and disulfide linkage mapping of protein • Relative quantitation of methionine oxidation

  3. Blue Stream Laboratories • Established Q1 2006 • Located in Woburn, MA • Well equipped • Glycoprotein / Peptide / Small Molecule Advanced Analytical lab • Mass Spectrometry lab • Service contracts include • Glycoprotein Structural Characterization Programs • CMC Analytical (Structure / Function) Support • Process / Comparability Programs • Large and Small Molecule Technology Transfer • Method Development / Optimization • Method Validation (ICH) • GMP Stability and Release Testing • Consulting

  4. Agilent 6210 ToF • ESI-ToF coupled to Agilent 1200 binary LC • AP-MALDI (MassTech) source available • <3 ppm mass accuracy • >17000 resolution

  5. Case Studies (1) Intact Molecular Weight Determination

  6. Experimental conditions • Injection of 0.3 ug protein in 50% ACN/0.1%TFA • 200 uL/minute flow rate • 100-3200 m/z acquisition range • Extraction of survey spectrum from TIC • Deconvolution via BioConfirm/Protein Apps

  7. TIC, sample 1: Small Protein

  8. Survey spectrum of protein peak

  9. Charge state envelope of protein

  10. Deconvolution results

  11. Deconvolution results (zoom)

  12. Deconvolution evidence

  13. MALDI-ToF of Intact Protein

  14. ESI-ToF: TIC of Intact Protein

  15. ESI-ToF of Intact Protein: Survey Spectrum

  16. ESI-ToF of Intact Protein: Deconvolution Results

  17. MALDI ESI-ToF of Intact Protein: Deconvolution Results Multiple Phosphorylations (7 predicted) Average mass peak

  18. ESI-ToF of Intact Protein: Deconvolution Evidence (Average Mass Peak)

  19. MS-ToF Of Large Intact Protein (Agilent 6220) • Agilent 6220 ToF • mass accuracy: <2 ppm • Resolution 20,000 • Up to 10 spectra/second • M/z range of 25 to 20,000 • New MassHunter Workstation • Integrated deconvolution and molecular feature extraction

  20. TIC of Large Intact Protein

  21. ESI-ToF of Large Intact Protein: Survey Spectrum

  22. ESI-ToF of Intact Protein: Deconvolution Results Predicted average mass: 82958.07

  23. ESI-ToF of Intact Protein: Deconvolution Results (2)

  24. Case Studies (2) Sequence Confirmation and Disulfide Linkage Mapping

  25. Experimental conditions • 947-residue fusion protein, requiring sequence confirmation with disulfide linkages mapped • Sample split, one aliquot reduced/alkylated, other alkylated only, followed by proteolytic digest of reduced and non-reduced preps (trypsin/endo-Lys-C) • Injection of 3-5 µg of proteolytic digest over analytical C18 (reversed phase) • 180 min. linear gradient, 200 µL/minute flow rate • 100-3200 m/z acquisition range, 215, 254, 280 nm UV • Deconvolution of peptides and sequence matching via BioConfirm with <5 ppm mass accuracy

  26. Fusion protein peptide map UV chromatogram (overlay)

  27. Fusion protein predicted disulfide linkages and associated tryptic peptides AA(1-28) + AA(35-59) ALC3GGELVDTLQFVC15GDRGFYFSRPASR + GIVEEC40C41FRSC45DLALLETYC54ATPAK - readily detected in non-reduced sample AA(72-91) + AA(102-109) AVPTQC77DVPPNSR*FDC87APDK + GC103C104YIPAK - only detected with Arg to Ser point mutation at Arg84 AA(92-101) + AA(110-132) AITQEQC98EAR + QGLQGAQMGQPWC122FFPPSYPSYK • linkage not detected

  28. AA(1-28) + AA(35-59) ALC3GGELVDTLQFVC15GDRGFYFSRPASR + GIVEEC40C41FRSC45DLALLETYC54ATPAK AA(523-580) GSEDGC528PNNELENPPYVPGVVGGTLQAATIC553ASSHQFLSTHYNLHNLYGLTEAIASHR AA(604-655) YAGHWTGDVWSSWEQLASSVPEILQFNLLGVPLVGADVC642GFLGNTSEELC653VR - all linkages readily detected in non-reduced sample Fusion protein identified disulfide linkages and associated tryptic peptides

  29. Fusion protein non-reduced peptide mapPeptides ALCGGELVDTLQFVCGDRGFYFSRPASR + GIVEECCFRSCDLALLETYCATPAK[residues 1-28 + 35-59] at retention time 109.9494molecular mass: 5791.7 Daltons (three disulfide linkages detected) (M+4H)+ (M+3H)+

  30. Fusion protein non-reduced peptide mapPeptides ALCGGELVDTLQFVCGDRGFYFSRPASR + GIVEECCFRSCDLALLETYCATPAK [residues 1-28 + 35-59] 4+ charge state displayedmolecular mass: 5791.7 Daltons (three disulfide linkages detected)

  31. Case Studies (3) • Relative quantitation of methionine oxidation

  32. Experimental conditions • Client requires quantitation of methionine oxidation on three residues to release product (650-residue<<check this, not exact>> monoclonal Ab) • Sample reduced and digested using endo-Lys-C • Injection of 5 µg proteolytic digest over analytical C18 (reversed phase) • 40 min. linear gradient, 200 µL/minute flow rate • 100-3200 m/z acquisition range, 215 nm UV • Deconvolution of peptides and sequence matching via Qualitative Data Analysis with <5 ppm mass accuracy • Relative quantitation using “volume” of deconvoluted oxidized and non-oxidized peptide peaks via BioConfirm

  33. Monoclonal Ab digest UV chromatogram oxidated Non-oxidated

  34. Oxidated peptide spectra

  35. Non-oxidated peptide spectra

  36. Compound detection by Qualitative Data Analysis

  37. Sequence Matching results

  38. Quantitation Results

  39. Acknowledgements/Contact Info

  40. Blue Stream LaboratoriesContact Information • Contact • Email • Mario DiPaola PhD / MBA • CSO/COO • William C Rash • VP Business Development • mdipaola@bluestreamlabs.com • Brash@bluestreamlabs.com

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