ABSTRACT
A three-dimensional structured porous graphene oxide-polyethylenimine bead (pGP) is synthesized for immobilizing gold nanoparticles and modifying glutathione molecules (denoted as pGP/AuG). The pGP/AuG has open pore structure, honeycomb-like channels, and excellent hydrophilicity. By taking advantages of the porous structure, abundant binding sites, and multivalent interactions between glycopeptides and both glutathione molecules and free amino groups, the pGP/AuG is adopted to the selective enrichment of N-linked glycopeptides with low limit of detection (2 fmol), high enrichment selectivity (1:500), binding capacity (333.3 mg/g), recovery yield (91.3 ± 2.1%), and repeatability (< 6.0% RSD) using matrix-assisted laser desorption/ionization time of flight mass spectrometry detection method. Furthermore, the practical applicability of pGP/AuG is evaluated, in which 209 N-glycosylated peptides corresponding to 128 N-glycosylated proteins are identified from 1 µL human serum in three independent analysis procedures, suggesting the great potential for application in glycoproteome fields.Graphical abstract Schematic presentation of preparation for porous graphene oxide-based hydrophilic beads (pGP/AuG) with honeycomb-like microstructure. The pGP/AuG was successfully used for enriching and identifying glycopeptides from actual biological sample.
Subject(s)
Glutathione/chemistry , Glycopeptides/isolation & purification , Graphite/chemistry , Metal Nanoparticles/chemistry , Animals , Cattle , Glycopeptides/analysis , Gold/chemistry , Humans , Hydrophobic and Hydrophilic Interactions , Immunoglobulin G/analysis , Immunoglobulin G/chemistry , Immunoglobulin G/isolation & purification , Limit of Detection , Peptide Fragments/analysis , Peptide Fragments/isolation & purification , Porosity , Proteolysis , Serum Albumin, Bovine/analysis , Serum Albumin, Bovine/chemistry , Serum Albumin, Bovine/isolation & purification , Solid Phase Extraction/methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
High-performance affinity materials are highly required in the sample preparation process in mass spectrometry-based glycoproteomics studies. In this research, a novel magnetic nanofiber-based zwitterionic hydrophilic material is prepared for glycopeptide enrichment and identification. The one-dimensional hydroxyapatite nanofiber (HN) acted as the supporting substance for immobilizing both Fe3O4 nanoparticles and Au nanoparticles, following the surface modification with a zwitterionic tripeptide l-glutathione (GSH) via the affinity interactions between the thiol group in GSH and both Au and Fe3O4 to form the magHN/Au-GSH nanofiber. Owing to the unique structural features, excellent hydrophilicity, abundant zwitterionic molecules, and strong magnetic responsiveness, the as-prepared magHN/Au-GSH nanofiber possesses satisfactory specificity for glycopeptide enrichment. As a result, the magHN/Au-GSH nanofiber demonstrated great detection sensitivity (2 fmol), satisfying enrichment recovery (89.65%), large binding capacity (100 mg g-1), and high enrichment selectivity (1 : 100) toward glycopeptides. Furthermore, 246 N-glycosylated peptides corresponding to 104 N-glycosylated proteins were identified from only 1 µL human serum, revealing the great potential of this affinity nanofiber for glycopeptide enrichment and glycoproteomics research.