Abstract
Due to their unique glycan composition and linkage, protein glycosylation plays significant roles in cellular function and is associated with various diseases. For comprehensive characterization of their extreme structural complexity occurring in >50% of human proteins, time-consuming multi-step enrichment of glycopeptides is required. Here we report zwitterionic n-dodecylphosphocholine-functionalized magnetic nanoparticles (ZIC-cHILIC@MNPs) as a highly efficient affinity nanoprobe for large-scale enrichment of glycopeptides. We demonstrate that ZIC-cHILIC@MNPs possess excellent affinity, with 80–91% specificity for glycopeptide enrichment, especially for sialylated glycopeptide (90%) from biofluid specimens. This strategy provides rapidity (~10 min) and high sensitivity (<1 μL serum) for the whole enrichment process in patient serum, likely due to the rapid separation using magnetic nanoparticles, fast reaction, and high performance of the affinity nanoprobe at nanoscale. Using this strategy, we achieved personalized profiles of patients with hepatitis B virus (HBV, n = 3) and hepatocellular carcinoma (HCC, n = 3) at the depth of >3000 glycopeptides, especially for the large-scale identification of under-explored sialylated glycopeptides. The glycoproteomics atlas also revealed the differential pattern of sialylated glycopeptides between HBV and HCC groups. The ZIC-cHILIC@MNPs could be a generic tool for advancing the glycoproteome analysis, and contribute to the screening of glycoprotein biomarkers.
Highlights
Protein glycosylation is a ubiquitous post-translational modification that is estimated to occur in over 50% of the human proteome [1]
The ZIC-cHILIC@Magnetic nanoparticles (MNPs) were prepared by incubation of core MNPs and ndodecylphosphocholine through comprehensive van der Waals interaction, giving ZICcHILIC@MNPs a zwitterionic feature where the positive charge of the choline group is exposed on the outer layer and the negative charge of phosphoryl group is on the inner part of the nanoparticle
To characterize the chemical bonding of different materials, the functional group of core MNPs and ZIC-cHILIC@MNPs were analyzed by Fourier-Transform Infrared
Summary
Protein glycosylation is a ubiquitous post-translational modification that is estimated to occur in over 50% of the human proteome [1]. Glycopeptides are significantly more hydrophilic and have several hydroxyl groups from the glycan that can participate in H-bonding They are preferentially retained over non-glycopeptides in HILIC [11]. Wohlgemut et al utilized ZIC-HILIC on a monolithic nano-LC column for glycopeptide enrichment, demonstrating sensitive results at single glycoprotein level, including 27 and 62 glycopeptides enriched in IgG1 and α-acid glycoprotein, respectively [23]. Yeh et al fabricated magnetic bead-based ZIC-HILIC material for glycopeptide enrichment. Taking advantage andand sensitive enrichment of magnetic nanoparticles (MNPs) for glycopeptides, in this study, we designed and fabricated. The ZIC-cHILIC@MNPs were expected mechanism with zwitterionic features to improve the selectivity towardsthe hydrophilic polar to combine HILIC mechanism with zwitterionic features to improve selectivity tomolecules (i.e., glycan moiety of glycopeptides). Our results demonstrated that ZIC-cHILIC@MNPs provide a high enrichment specificity of 81–90% for sialylated glycopeptides in sera
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