Title : Determination of site-specific glycan heterogeneity on
glycoproteins
Abstract :
- The comprehensive analysis of protein glycosylation is a major requirement for understanding glycoprotein function in biological systems, and is a prerequisite for producing recombinant glycoprotein therapeutics
- This protocol describes workflows for the characterization of glycopeptides and their site-specific heterogeneity, showing examples of the analysis of recombinant human erythropoietin ( rHuEPO ), α1-proteinase inhibitor (A1PI) and immunoglobulin ( IgG )
- Glycoproteins of interest can be proteolytically digested either in solution or in-gel after electrophoretic separation, and the (glyco) peptides are analyzed by capillary/nano-liquid chromatography-electrospray ionization tandem mass spectrometry (LC- ESI-MS/MS)
- If required, specific glycopeptide enrichment steps, such as hydrophilic interaction liquid chromatography (HILIC), can also be performed
- Particular emphasis is placed on data interpretation and the determination of site-specific glycan heterogeneity
- The described workflow takes approximately 3-5 d, including sample preparation and data analysis
- The data obtained from analyzing released glycans of rHuEPO and IgG , described in the second protocol of this series (10.1038/nprot.2012.063), provide complementary detailed glycan structural information that facilitates characterization of the glycopeptides
Output (sent_index, trigger,
protein,
sugar,
site):
- 0. glycoproteins, , glycoproteins, -, -
- 1. glycoprotein, , glycoprotein, -, -
- 2. glycopeptides, , -, -, glycopeptides
- 4. glycopeptide, , -, -, glycopeptide
- 7. IgG, , IgG, released glycans, -
- 7. glycopeptides, , -, -, glycopeptides
- 7. rHuEPO, , rHuEPO, released glycans, -
Output(Part-Of) (sent_index,
protein,
site):
*Output_Site_Fusion* (sent_index,
protein,
sugar,
site):