Hydrophilic Phytic Acid-Coated Magnetic Graphene for Titanium(IV) Immobilization as a Novel Hydrophilic Interaction Liquid Chromatography–Immobilized Metal Affinity Chromatography Platform for Glyco- and Phosphopeptide Enrichment with Controllable Selectivity

Abstract

In this work, multifunctional Ti-immobilized phytic acid-modified magnetic graphene (denoted as MagG@PEI@PA-Ti) nanocomposites were fabricated through a facile route for simultaneous/respective enrichment of N-glyco- and phosphopeptides. Phytic acid (PA), with six phosphate groups, possesses excellent hydrophilicity and metal ion coordination ability, which endowed the MagG@PEI@PA-Ti with combined properties of immobilized metal ion affinity chromatography (IMAC)- and hydrophilic interaction liquid chromatography (HILIC)-based materials. On the basis of the different binding ability of N-glyco- and phosphopeptides on MagG@PEI@PA-Ti, the MagG@PEI@PA-Ti nanocomposites could enrich N-glyco- and phosphopeptides simultaneously or respectively by using different enrichment conditions, achieving controllable selective enrichment of N-glyco- and phosphopeptides. The proposed nanocomposites demonstrated an outstanding performance for selective enrichment of N-glycopeptides (selectivity, 1:1000 molar ratios of IgG/BSA; sensitivity, 0.5 fmol/μL IgG; loading capacity, 300 mg g; recovery, >90%) and phosphopeptides (selectivity, 1:5000 molar ratios of α-casein/BSA; sensitivity, 0.1 fmol/μL α-casein; loading capacity, 100 mg g; recovery, >90%). Taking advantage of these merits, a total of 393 N-glycopeptides derived from 259 glycoproteins and 574 phosphopeptides derived from 341 phosphoproteins were identified from 200 μg of HeLa cell extracts through a single-step enrichment using MagG@PEI@PA-Ti.

Site-specific N-glycosylation of HeLa cell glycoproteins

“…Using this method 268 unique N -glycosylation sites corresponding to 106 glycoproteins have been identified so far 11 . In two recent articles, glycopeptides were enriched from the HeLa cell lysate 7,17 followed by cleaving the sugars from the glycopeptides. Subsequent mass spectrometry analysis was used to locate the site of glycosylation but the attached sugar structures were not studied.…”

Phosphopeptide enrichment for phosphoproteomic analysis - A tutorial and review of novel materials

“…(1) The application of graphene with large surface areas and the introduction of a phytic 577 acid (PA) molecule provide a benefit due to the presence of six phosphate groups with 578 metal ion coordination ability resulting in more affinity sites for IMAC. Moreover, the excellent hydrophilicity of PA can further reduce non-specific adsorption [51]. Example formats include MagG@PEI@PA-Ti 4+ [51] (Fig 2a) which yielded a total of 574 p-peptides from 341 phosphoproteins were detected from 200 μg of HeLa cells using MagG@ @PA-Ti 4+ [51].…”

Nanomaterials in Proteomics

“…The result demonstrated the CS@PGMA@IDA‐Ti 4+ possessed simultaneous capture ability toward glyco‐ and phosphopeptides to some extent, however, the ability needed to be improved compared with the researches of nanomaterials in single PTM . More recently, the reported Ti 4+ ‐immobilized magnetic graphene (dubbed MagG@PEI@PA‐Ti 4+ ) also presented the similar problem of inadequate capability by comparison . As a result, the nanomaterials with sufficient ability to simultaneously capture glycopeptides and phosphopeptides are in the demand to be developed energetically.…”