How Can Glycosylation Sites and the Molecular Weight After Glycosylation Be Analyzed, and Which Prediction Tools Are Available?
Methods for Analyzing Glycosylation Sites
1. Experimental Approaches
(1) Mass Spectrometry (MS) Analysis: Mass spectrometry (MS) is the gold standard for identifying glycosylation sites in proteins. MS/MS analysis of specific peptide fragments enables precise localization of glycosylation sites.
(2) Immunoblotting: Detection of glycosylated proteins can be achieved using antibodies specific to glycosylation sites.
2. Bioinformatics-Based Prediction
(1) Prediction Tools: Computational tools such as NetNGlyc (N-glycosylation site prediction), NetOGlyc (O-glycosylation site prediction), and GlycoEP are widely used for glycosylation site prediction.
(2) Typical Workflow
①Input the amino acid sequence of the target protein.
②Specify the glycosylation type (N- or O-glycosylation).
③Execute the prediction algorithm to identify potential glycosylation sites.
④Assess the prediction results, which are typically provided with confidence scores.
Prediction of Molecular Weight After Glycosylation
1. Theoretical Estimation
Once glycosylation sites and types are identified, the molecular weight change can be estimated according to the type and quantity of attached glycans.
2. Mass Spectrometry Measurement
The overall molecular weight of glycosylated proteins can be directly determined by mass spectrometry. The mass shift due to glycosylation can be determined by comparing peak differences between glycosylated and non-glycosylated forms.
By combining experimental validation with bioinformatics predictions, a comprehensive analysis of protein glycosylation sites and molecular weight changes can be achieved.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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