Glycopeptides analysis links the glycosylation information to exact locations (glycosylation sites) on proteins. The analysis is designed to detect and characterize novel target molecules, which might lead to the development of new products. Comparative glycopeptides analysis can be helpful both in diagnosis and disease treatment through the understanding the mechanism of disease progression and pathology.
Figure 1. Schematic diagram of the steps involved in glycopeptides analysis
The analysis employs a bottom-up approach. This method typically presents a combination of specific enzymatic proteolysis followed by fractionation of glycopeptides via affinity chromatography or liquid chromatography and eventually identified through 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) were introduced as a novel approach used for the glycopeptide enrichment.
Figure 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 procedure as well as HILIC columns method. Reverse phase columns typically separate the glycopeptides based on the peptide portion, although glycoforms would contain 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 a disturbance that abundant glycoforms might suppress the signals of less abundant species. HILIC columns provide an alternative separation strategy in which 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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