ZIC-cHILIC Functionalized Magnetic Nanoparticle for Rapid and Sensitive Glycopeptide Enrichment from <1 µL Serum

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 &gt;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 (&lt;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 &gt;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.

Application of Microscopic Highly Hydrophilic Silica-Based Nanocomposites with High Surface Exposure in the Efficient Identification of Intact N-Glycopeptides

“…After treatment with 30NH 2 -SiO 2 , a total of 20 glycopeptides were identified, indicating the general applicability of this method. The detailed information on the above-captured glycopeptides enriched from fetuin digests is shown in Table S5 …”

“…6,7 Glycosylation is a critical branch of protein post-translational modifications that plays an important role in regulating protein function and abundant complex biological processes, such as molecular recognition, protein folding, and intracellular trafficking. 8,9 Aberrant glycosylation is associated with a variety of serious diseases such as cancers, and N-linked glycoproteins/glycopeptides have been found to be key disease biomarkers. 8 In-depth research on them is very attractive for the diagnosis and pathogenesis of related diseases.…”

“…Therefore, HILIC materials, zwitterionic HILIC materials (ZIC-HILIC), and ZIC-HILIC with the exposed choline group (ZIC-cHILIC) have shown good performance in the enrichment of glycopeptides. 8 For the improvement of the enrichment, specific organic groups were usually introduced on the surface of the SiO 2 -based materials as the active sites or modulating its hydrophilicity, including 3-aminopropyl, 9 sulfonic acid, 10 iminodiacetic acid (IDA), 11 zwitterionic cysteine (Cys), 12 glutathione, 13 arginine, poly(2-(methacryloyloxy)ethyl)dimethyl-(3-sul-fopropyl)ammonium hydroxide brushes, even the organic polymer, 14 and metal−organic framework materials. 15 For example, Lin et al prepared capillary trap columns of IDA-modified SiO 2 , and 207 glycopeptides were captured from human serum digests by on-column enrichment.…”

“…Among the separation and enrichment strategies that have been developed, hydrophilic interaction chromatography (HILIC) is an effective method for the enrichment of glycopeptides for its advantages of high selectivity, unbiasedness to the glycoproteome, and providing structural information about the sugar. Therefore, HILIC materials, zwitterionic HILIC materials (ZIC-HILIC), and ZIC-HILIC with the exposed choline group (ZIC-cHILIC) have shown good performance in the enrichment of glycopeptides . For the improvement of the enrichment, specific organic groups were usually introduced on the surface of the SiO 2 -based materials as the active sites or modulating its hydrophilicity, including 3-aminopropyl, sulfonic acid, iminodiacetic acid (IDA), zwitterionic cysteine (Cys), glutathione, arginine, poly­(2-(methacryloyloxy)­ethyl)­dimethyl-(3-sul-fopropyl)­ammonium hydroxide brushes, even the organic polymer, and metal–organic framework materials .…”

“…8,9 Aberrant glycosylation is associated with a variety of serious diseases such as cancers, and N-linked glycoproteins/glycopeptides have been found to be key disease biomarkers. 8 In-depth research on them is very attractive for the diagnosis and pathogenesis of related diseases. Currently, mass spectrometry (MS), with the characteristics of high sensitivity, convenient operation, and fast detection speed, has become an excellent and powerful technology for the comprehensive localization and identification of glycoproteins.…”