Structural Characterization of Glycoproteins using Agilent 6210 ESITOF: Analysis of PostTranslationa

1
Structural Characterization of Glycoproteins
using Agilent 6210 ESI-TOFAnalysis 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
• lt3 ppm mass accuracy
• gt17000 resolution

5
Case Studies (1)

• Intact Molecular Weight Determination

6
Experimental conditions

• Injection of 0.3 ug protein in 50 ACN/0.1TFA
• 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
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 lt2 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
(No Transcript)
21
TIC of Large Intact Protein
22
ESI-ToF of Large Intact Protein Survey Spectrum
23
ESI-ToF of Intact Protein Deconvolution Results
Predicted average mass 82958.07
24
ESI-ToF of Intact Protein Deconvolution Results
(2)
25
Case Studies (2)
Sequence Confirmation and Disulfide Linkage
Mapping
26
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 lt5 ppm mass accuracy

27
Fusion protein peptide map UV chromatogram
(overlay)
28
Fusion protein predicted disulfide linkages and
associated tryptic peptides
29
Fusion protein identified disulfide linkages and
associated tryptic peptides
30
Fusion protein non-reduced peptide mapPeptides
ALCGGELVDTLQFVCGDRGFYFSRPASR GIVEECCFRSCDLALLETY
CATPAKresidues 1-28 35-59 at retention time
109.9494molecular mass 5791.7 Daltons (three
disulfide linkages detected)
31
Fusion protein non-reduced peptide mapPeptides
ALCGGELVDTLQFVCGDRGFYFSRPASR GIVEECCFRSCDLALLETY
CATPAK residues 1-28 35-59 4 charge state
displayedmolecular mass 5791.7 Daltons (three
disulfide linkages detected)
32
Case Studies (3)

• Relative quantitation of methionine oxidation

33
Experimental conditions

• Client requires quantitation of methionine
  oxidation on three residues to release product
  (650-residueltltcheck this, not exactgtgt 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 lt5 ppm mass
  accuracy
• Relative quantitation using volume of
  deconvoluted oxidized and non-oxidized peptide
  peaks via BioConfirm

34
Monoclonal Ab digest UV chromatogram
oxidated
Non-oxidated
35
Oxidated peptide spectra
36
Non-oxidated peptide spectra
37
Compound detection by Qualitative Data Analysis
38
Sequence Matching results
39
Quantitation Results
40
Acknowledgements/Contact Info
41
Blue Stream LaboratoriesContact Information

• Contact

• Email

• Mario DiPaola PhD / MBA
• CSO/COO
• William C Rash
• VP Business Development

• mdipaola_at_bluestreamlabs.com
• Brash_at_bluestreamlabs.com