Glycopeptides analysis is a very convenient method to link the glycosylation information to exact locations (glycosylation sites) on proteins. It provides very helpful information about the glycans on one or more proteins.
The analysis of glycopeptides can be used to discover and characterize some novel and interesting molecules, which leads to the development of new products. Comparative glycopeptides analysis can be helpful both in diagnosing or treating disease and also in developing a better understanding of disease progression and pathology.
Glycopeptides AnalysisFigure 1. Schematic diagram of the steps involved in glycopeptides analysis
Currently glycopeptides analysis commonly employs a bottom-up approach. This method typically involves a combination of specific enzymatic proteolysis followed by fractionation of glycopeptides by affinity chromatography or liquid chromatography and eventually glycopeptide analysis by mass spectrometry.
Sample Enrichment of glycopeptides is a crucial step before acquiring useful data for glycopeptide analysis. Usually an antibody is used to recognize the protein of interest if just one glycoprotein is to be analyzed. Lectin affinity chromatography is used to select for a group of proteins that are glycosylated. More recently, hydrophilic interaction liquid chromatography (HILIC) is a very advantageous approach used for the glycopeptide enrichment.
Glycopeptides AnalysisFigure 2. HILIC separation of glycopeptides with the same peptide backbone
Glycopeptides analysis often uses on-line separation and enrichment in addition to the off-line separation methods. LC-MS separation of glycopeptides with reverse phase columns is the most widely adopted pre-MS separation method. HILIC columns are also becoming more commonly used as well. Reverse phase columns typically separate the glycopeptides based on the peptide portion, although most of the glycoforms containing the same peptide sequence co-elute. This co-elution can be an advantage, in terms of data analysis, because all the glycoforms of a given glycopeptide are easier to find in the data set. However, co-elution is also disadvantageous in that abundant glycoforms can suppress the signals of lesser abundant species. HILIC columns provide an alternative separation strategy in that the glycoforms interact with the hydrophilic column; thus, the glycoforms for a given peptide are more readily separated. Graphitized carbon column can be used to separate glycopeptides isomers.
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