Abstract
Efficient glycopeptides enrichment prior to mass spectrometry analysis is essential for glycoproteome study. ZIC-HILIC (zwitterionic hydrophilic interaction liquid chromatography) based glycopeptides enrichment approaches have been attracting more attention for several benefits like easy operating, high enrichment specificity and intact glycopeptide retained. In this study, Poly (amidoamine) dendrimer (PAMAM) was adopted for the synthesis of zwitterionically functionalized (ZICF) materials for glycopeptide enrichment. The multiple branched structure and good solubility of ZICF-PAMAM enables a sufficient interaction with glycopeptides. The ZICF-PAMAM combined with the FASP-mode enrichment strategy exhibits more superior performance compared with the existing methods. It has the minimum detectable concentration of femtomolar level and high recovery rate of over 90.01%, and can efficiently enrich glycopeptides from complex biological samples even for merely 0.1 μL human serum. The remarkable glycopeptides enrichment capacity of ZICF-PAMAM highlights the potential application in in-depth glycoproteome research, which may open up new opportunities for the development of glycoproteomics.
Highlights
Selective enrichment of glycopeptides/glycoproteins prior to mass spectrometry (MS) analysis is a prerequisite for in-depth glycoproteome research[1], because of the inherent low stoichiometry and microheterogeneity of glycosylation and serious signal suppression of glycopeptides by abundant non-glycopeptides in MS identification[2]
With this method we successfully enriched and identified all the glycopeptides from standard glycoproteins with high selectivity and sensitivity. We further applied this strategy to the investigation of human serum glycoproteins and successfully identified 395 unique glycopeptides with 417 glycosylation sites mapped to 178 glycoproteins in three replicates
What’s more, we identified 44 unique glycopeptides with 48 glycosylation sites from 28 glycoproteins with only 0.1 μL human serum, demonstrating that this strategy can be a powerful tool in the study of protein glycosylation
Summary
Weiqian Cao1,2,*, Jiangming Huang1,*, Biyun Jiang[1,2], Xing Gao1 & Pengyuan Yang[1,2]. ZIC-HILIC (zwitterionic hydrophilic interaction liquid chromatography) based glycopeptides enrichment approaches have been attracting more attention for several benefits like easy operating, high enrichment specificity and intact glycopeptide retained. (amidoamine) dendrimer (PAMAM) was adopted for the synthesis of zwitterionically functionalized (ZICF) materials for glycopeptide enrichment. It is still pressing need to develop a glycopeptide enrichment material with high efficiency, good specificity and intact glycopeptides retained for in-depth glycoproteome research. We propose a chemical strategy for the zwitterionic functionalization (ZICF) of G5 PAMAM for glycopeptide enrichment With this method we successfully enriched and identified all the glycopeptides from standard glycoproteins with high selectivity and sensitivity. What’s more, we identified 44 unique glycopeptides with 48 glycosylation sites from 28 glycoproteins with only 0.1 μL human serum, demonstrating that this strategy can be a powerful tool in the study of protein glycosylation
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