Abstract
The low abundance of glycopeptides in biological samples makes it necessary to enrich them before further analysis. In this study, the polymeric hydrophilic ionic liquid-modified magnetic (Fe3O4@MPS@PMAC) nanoparticles were synthesized via a one-step reflux-precipitation polymerization. Owing to the excellent hydrophilicity and strong electrostatic interaction toward glycopeptides of the polymerized hydrophilic ionic liquid, [2-(methacryloyloxy) ethyl] trimethylammonium chloride (MAC), the synthesized Fe3O4@MPS@PMAC nanoparticles exhibited outstanding performance in glycopeptide enrichment with high detection sensitivity (10 fmol), large binding capacity (100 μg mg−1) and satisfied enrichment recovery (approximately 82%). Furthermore, the newly developed Fe3O4@MPS@PMAC nanoparticles were applied for the glycopeptide enrichment of HeLa exosome proteins. A total of 1274 glycopeptides from 536 glycoproteins were identified in three replicate analyses of 50 μg of HeLa exosome proteins. These results demonstrate the potential of Fe3O4@MPS@PMAC nanoparticles for both glycoproteomic analysis and exosome research.
Highlights
Protein glycosylation is one of the most important post-translational modifications, which is closely related to a variety of biological processes such as cell division, signal transduction, protein-protein interactions and tumor immunology[1,2,3,4,5]
Several novel materials and methods have been reported for glycopeptide enrichment including boronic acid chemistry[11,12,13], hydrazide chemistry[14,15,16], lectin affinity chromatography[17, 18] and hydrophilic interaction chromatography (HILIC)[19,20,21]
The lectin affinity chromatography could be utilized for glycopeptide and glycoprotein enrichment by the affinity interactions between lectin and glycans, while a single lectin could not be used for a global enrichment of multiple types of glycopeptides
Summary
To evaluate the enrichment selectivity of Fe3O4@MPS@PMAC, the mixture of the tryptic digests of BSA and human IgG were incubated with the nanoparticles. The number of identified glycopeptide decreased to 2 when the mass ratio of human IgG:BSA was further reduced to 1:100 (Fig. 6f) These results demonstrated the high glycopeptide enrichment selectivity of the synthesized Fe3O4@MPS@PMAC. The binding capacity of the nanoparticles for glycopeptides was tested by adding different amounts of Fe3O4@ MPS@PMAC (5–50 μg) to 3 μg of tryptic digests of human IgG. 996 N-glycopeptides from 444 glycoproteins were identified by using the commercial material (Figs S4 and S5) This result demonstrated the superiority of Fe3O4@MPS@PMAC for glycoprotein analysis to that of the currently used enrichment material. This work provides a new avenue for glycoproteome research and broadens the research possibilities for the study of exosomes
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