Protein Characterization: Testing to Triage Lead Candidates

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


Digest by Specific Proteases (pH<6.7)

Non-reduced Fraction


Deglycosylation if Necessary


Profile Comparison to Find Different Peaks

Reduced Fraction


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


high mannose hybrid complex



Fluorescent 2-AB/Procainamide Labeling

Labeled Glycan Pool + Dye

Glycans with Free Reducing End


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


Peak feature detection based on MS1 only



Un-identified Small Peaks

Manual Assignments


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. |


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