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

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Structural Characterization of Glycoproteins
using Agilent 6210 ESI-TOFAnalysis of
Post-Translational Modifications

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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

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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

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Agilent 6210 ToF

• ESI-ToF coupled to Agilent 1200 binary LC
• AP-MALDI (MassTech) source available
• lt3 ppm mass accuracy
• gt17000 resolution

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Case Studies (1)

• Intact Molecular Weight Determination

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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

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TIC, sample 1 Small Protein
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Survey spectrum of protein peak
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Charge state envelope of protein
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Deconvolution results
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Deconvolution results (zoom)
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Deconvolution evidence
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MALDI-ToF of Intact Protein
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ESI-ToF TIC of Intact Protein
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ESI-ToF of Intact Protein Survey Spectrum
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ESI-ToF of Intact Protein Deconvolution Results
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ESI-ToF of Intact Protein Deconvolution Results
Multiple Phosphorylations (7 predicted)
Average mass peak
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ESI-ToF of Intact Protein Deconvolution Evidence
(Average Mass Peak)
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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

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(No Transcript)
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TIC of Large Intact Protein
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ESI-ToF of Large Intact Protein Survey Spectrum
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ESI-ToF of Intact Protein Deconvolution Results
Predicted average mass 82958.07
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ESI-ToF of Intact Protein Deconvolution Results
(2)
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Case Studies (2)
Sequence Confirmation and Disulfide Linkage
Mapping
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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

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Fusion protein peptide map UV chromatogram
(overlay)
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Fusion protein predicted disulfide linkages and
associated tryptic peptides
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Fusion protein identified disulfide linkages and
associated tryptic peptides
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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)
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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)
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Case Studies (3)

• Relative quantitation of methionine oxidation

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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

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Monoclonal Ab digest UV chromatogram
oxidated
Non-oxidated
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Oxidated peptide spectra
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Non-oxidated peptide spectra
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Compound detection by Qualitative Data Analysis
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Sequence Matching results
Data File C\Documents and Settings\bhekking\Desktop\28feb08\28FEB08_ref2.csv Data File C\Documents and Settings\bhekking\Desktop\28feb08\28FEB08_ref2.csv Data File C\Documents and Settings\bhekking\Desktop\28feb08\28FEB08_ref2.csv Data File C\Documents and Settings\bhekking\Desktop\28feb08\28FEB08_ref2.csv Data File C\Documents and Settings\bhekking\Desktop\28feb08\28FEB08_ref2.csv Data File C\Documents and Settings\bhekking\Desktop\28feb08\28FEB08_ref2.csv Data File C\Documents and Settings\bhekking\Desktop\28feb08\28FEB08_ref2.csv Data File C\Documents and Settings\bhekking\Desktop\28feb08\28FEB08_ref2.csv
Find Compounds Settings Start retention time (min) 0.0000 End retention time (min) -1.0000 M/z from (Da) 100.0000 M/z to (Da) 3500.0000 S/N threshold 3.0000 Number of Compounds 500.0000 Find Compounds Settings Start retention time (min) 0.0000 End retention time (min) -1.0000 M/z from (Da) 100.0000 M/z to (Da) 3500.0000 S/N threshold 3.0000 Number of Compounds 500.0000 Find Compounds Settings Start retention time (min) 0.0000 End retention time (min) -1.0000 M/z from (Da) 100.0000 M/z to (Da) 3500.0000 S/N threshold 3.0000 Number of Compounds 500.0000 Find Compounds Settings Start retention time (min) 0.0000 End retention time (min) -1.0000 M/z from (Da) 100.0000 M/z to (Da) 3500.0000 S/N threshold 3.0000 Number of Compounds 500.0000 Find Compounds Settings Start retention time (min) 0.0000 End retention time (min) -1.0000 M/z from (Da) 100.0000 M/z to (Da) 3500.0000 S/N threshold 3.0000 Number of Compounds 500.0000 Find Compounds Settings Start retention time (min) 0.0000 End retention time (min) -1.0000 M/z from (Da) 100.0000 M/z to (Da) 3500.0000 S/N threshold 3.0000 Number of Compounds 500.0000 Find Compounds Settings Start retention time (min) 0.0000 End retention time (min) -1.0000 M/z from (Da) 100.0000 M/z to (Da) 3500.0000 S/N threshold 3.0000 Number of Compounds 500.0000 Find Compounds Settings Start retention time (min) 0.0000 End retention time (min) -1.0000 M/z from (Da) 100.0000 M/z to (Da) 3500.0000 S/N threshold 3.0000 Number of Compounds 500.0000 Find Compounds Settings Start retention time (min) 0.0000 End retention time (min) -1.0000 M/z from (Da) 100.0000 M/z to (Da) 3500.0000 S/N threshold 3.0000 Number of Compounds 500.0000 Find Compounds Settings Start retention time (min) 0.0000 End retention time (min) -1.0000 M/z from (Da) 100.0000 M/z to (Da) 3500.0000 S/N threshold 3.0000 Number of Compounds 500.0000
Sequence File C\Program Files\Agilent\TOF Software\ProteinSequences\CTM0016.psq Sequence File C\Program Files\Agilent\TOF Software\ProteinSequences\CTM0016.psq Sequence File C\Program Files\Agilent\TOF Software\ProteinSequences\CTM0016.psq Sequence File C\Program Files\Agilent\TOF Software\ProteinSequences\CTM0016.psq Sequence File C\Program Files\Agilent\TOF Software\ProteinSequences\CTM0016.psq Sequence File C\Program Files\Agilent\TOF Software\ProteinSequences\CTM0016.psq Sequence File C\Program Files\Agilent\TOF Software\ProteinSequences\CTM0016.psq Sequence File C\Program Files\Agilent\TOF Software\ProteinSequences\CTM0016.psq
Selected Match Sets Complete Digest Incomplete Digest Sample handling artifacts Selected Match Sets Complete Digest Incomplete Digest Sample handling artifacts Selected Match Sets Complete Digest Incomplete Digest Sample handling artifacts Selected Match Sets Complete Digest Incomplete Digest Sample handling artifacts Selected Match Sets Complete Digest Incomplete Digest Sample handling artifacts Selected Match Sets Complete Digest Incomplete Digest Sample handling artifacts Selected Match Sets Complete Digest Incomplete Digest Sample handling artifacts Selected Match Sets Complete Digest Incomplete Digest Sample handling artifacts
Sequence Coverage 38.3073 Sequence Coverage 38.3073 Sequence Coverage 38.3073

Index RT (min) Abundance Measured Mass (Da) Theoretical Mass (Da) Delta ppm Location Reagent Description Predicted Modifications
1 25.923 2426833 2315.1275 2315.1256 0.7926 A(44-64) LysC Complete Digest
2 21.877 202966 2331.1229 2331.1205 0.9909 A(44-64) LysC Sample handling artifacts Oxidation
3 10.717 2846306 1263.652 1263.6684 -12.9828 A(65-75) LysC Complete Digest
4 12.137 3528585 1185.6436 1185.6394 3.5871 A(125-136) LysC Complete Digest
5 10.717 2846306 1263.652 1263.6493 2.128 A(137-150) LysC Complete Digest
6 26.823 3115053 2897.4187 2897.4151 1.2459 A(252-277) LysC Complete Digest
7 23.4 205399 2913.414 2913.41 1.385 A(252-277) LysC Sample handling artifacts Oxidation
8 14.946 60609 1676.7946 1676.7947 -0.0716 A(278-291) LysC Complete Digest
9 14.949 2613726 1676.7989 1676.7947 2.5048 A(278-291) LysC Complete Digest
10 14.298 2248891 1103.6029 1103.6009 1.8548 A(364-373) LysC Complete Digest
11 21.139 1994630 2543.1289 2543.1241 1.9004 A(374-395) LysC Complete Digest
12 25.668 3405231 1872.9166 1872.9146 1.0988 A(396-412) LysC Complete Digest
13 18.954 2032947 2986.3766 2986.3715 1.7141 A(418-442) LysC Complete Digest
14 10.401 51673 3002.3715 3002.3664 1.6767 A(418-442) LysC Sample handling artifacts Oxidation
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Quantitation Results
Peptide Measured mass Theoretical Mass Relative abundance Percent Oxidation
Met57 2315.1275 2315.1256 2426833.00 7.7
Met57ox 2331.1229 2331.1205 202966.00
Met255 2897.4187 2897.4151 3115053.00 6.2
Met255ox 2913.4140 2913.41 205399.00
Met431 2986.3766 2986.3715 2032947.00 2.5
Met431ox 3002.3715 3002.3664 51673.00
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Acknowledgements/Contact Info
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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