Investigation of Site-Specific Differences in Glycan Microheterogeneity by N-Glycopeptide Mapping of VEGFR-IgG Fusion Protein
Abstract
:1. Introduction
2. Results and Discussion
2.1. Cleaved Glycan Analysis by MALDI MS
2.2. Protein Sequencing by LC-MS/MS
2.3. LC-MS/MS Glycopeptide Mapping of Fusion Protein
3. Materials and Methods
3.1. Materials
3.2. Stable Cell Line Generation of the VEGFR-IgG Fusion Protein
3.3. Stable Expression and Purification of VEGFR-IgG Fusion Protein
3.4. Cleaved Glycans Preparation
3.5. MALDI MS
3.6. Tryptic Digestion of Fusion Protein
3.7. LC-MS/MS Analysis
3.8. N-Glycopeptide Identification
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Domain | Site | Original Site | Peptide | Fuc% | Sia% |
---|---|---|---|---|---|
VEGFR-1 | Asn57 | VEGFR-1 Domain 2 Asn164 | VTSPNITVTLK | 100.0 | 81.6 |
Asn90 | VEGFR-1 Domain 2 Asn196 | GFIISNATYK | 100.0 | 89.0 | |
VEGFR-2 | Asn149 | VEGFR-2 Domain 3 Asn245 | LVLNCTAR | 3.4 | 42.7 |
Asn224 | VEGFR-2 Domain 4 Asn318 | NSTFVR | 15.2 | 87.8 | |
IgG Fc | Asn316 | IgG Fc Domain Asn297 | EEQYNSTYR | 59.8 | 0.0 |
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Hahm, Y.H.; Lee, J.Y.; Ahn, Y.H. Investigation of Site-Specific Differences in Glycan Microheterogeneity by N-Glycopeptide Mapping of VEGFR-IgG Fusion Protein. Molecules 2019, 24, 3924. https://doi.org/10.3390/molecules24213924
Hahm YH, Lee JY, Ahn YH. Investigation of Site-Specific Differences in Glycan Microheterogeneity by N-Glycopeptide Mapping of VEGFR-IgG Fusion Protein. Molecules. 2019; 24(21):3924. https://doi.org/10.3390/molecules24213924
Chicago/Turabian StyleHahm, Young Hye, Ju Yeon Lee, and Yeong Hee Ahn. 2019. "Investigation of Site-Specific Differences in Glycan Microheterogeneity by N-Glycopeptide Mapping of VEGFR-IgG Fusion Protein" Molecules 24, no. 21: 3924. https://doi.org/10.3390/molecules24213924