Disulfide mapping analysis to determine unknown disulfide linkage
Deliverables: • Confirm known disulfide linkage information • Report free cysteine amount and any mis-linked disulfide bond with >5% abundance • Determine unknown disulfide linkage for every cysteine
Purified Protein Samples
Ethanol Precipitation
Alkylation
Digest by Specific Proteases (pH<6.7)
Non-reduced Fraction
LC-MS
Deglycosylation if Necessary
DTT
Profile Comparison to Find Different Peaks
Reduced Fraction
LC-MS
N-glycan analysis by LC-Fluorescence-MS/MS for complex glycosylation
Deliverables: • Report any glycan relative abundance >1% based on fluorescence • Report glycan ID based on mass spec results
N-glycanase Release Glycans HLB Plates
Glycan Pool
Glycoprotein
high mannose hybrid complex
M6P
Convert
Fluorescent 2-AB/Procainamide Labeling
Labeled Glycan Pool + Dye
Glycans with Free Reducing End
Clean
Identification Software Search Manual Interpretation
Liquid Chromatography MS/MS
Component Identification
Glycan Distribution
Quantification by Byomap
Detailed peptide mapping for sequence coverage, PTM, sequence variant analysis
Deliverables: • Confirm protein sequence: sequence coverage map • Software-based identification for PTM, SV and fragment et al analysis • Check sequence homogeneity: manual check for all components with user defined intensity threshold (>1%) for detailed PTM, SV and fragment et al analysis
Identified Major Peaks
Confirmed Peak Identity Sequence Coverage
Database Search
LC-MS/MS
Peak feature detection based on MS1 only
MS2/MS3
LC-MS/MS (tMS2) CID/HCD/ETD
Un-identified Small Peaks
Manual Assignments
m/z
PTM 9%
Variants 2%
Adducts, non-specific digest 89%
1000-2000 components
Learn more: Protein Sciences: Early-Stage R&D Protein Characterization Services
About WuXi Biologics
WuXi Biologics is a leading contract research, development and manufacturing organization (CRDMO) that provides end-to-end capabilities to healthcare organizations worldwide. With operations in China, the United States, Ireland, Germany, and Singapore, we enable our partners to effectively and efficiently bring biologics and vaccines to patients worldwide through our comprehensive and high-quality drug development model.
The world’s leading global single-source platform from concept to commercialization.
wuxibiologics.com | info@wuxibiologics.com
5-9-2024
Powered by FlippingBook