Abstract
Although mesoporous materials and magnetic materials are used to enrich glycopeptides, materials sharing both mesoporous structures and magnetic properties have not been reported for glycopeptide analyses. Here we prepared boronic acid-modified magnetic Fe3O4@mTiO2 microspheres by covalent binding of boronic acid molecules onto the surfaces of silanized Fe3O4@mTiO2 microspheres. The final particles (denoted as B-Fe3O4@mTiO2) showed a typical magnetic hysteresis curve, indicating superparamagnetic behavior; meanwhile, their mesoporous sizes did not change in spite of the reduction in surface area and pore volume. By using these particles together with conventional poly(methyl methacrylate) (PMMA) nanobeads, we then developed a synergistic approach for highly specific and efficient enrichment of N-glycopeptides/glycoproteins. Owing to the introduction of PMMA nanobeads that have strong adsorption towards nonglycopeptides, the number of N-glycopeptides detected and the signal-to-noise ratio in analyzing standard proteins mixture both increased appreciably. The recovery of N-glycopeptides by the synergistic method reached 92.1%, much improved than from B-Fe3O4@mTiO2 alone that was 75.3%. Finally, we tested this approach in the analysis of amniotic fluid, obtaining the maximum number and ratio of N-glycopeptides compared to the use of B-Fe3O4@mTiO2 alone and commercial SiMAG-boronic acid particles. This ensemble provides an interesting and efficient enrichment platform for glycoproteomics research.
Highlights
For the past years, methods for glyco-specific enrichment divide into several categories by means of different mechanisms, including lectin affinity[8, 9], size exclusion[10], hydrazide chemistry[11, 12], hydrophilic interaction[13, 14], www.nature.com/scientificreports/
The surfaces of those Fe3O4@mTiO2 particles became uneven, which was owing to the formation of crystalline TiO2 and generation of mesoporous structure
This was further demonstrated by Transmission electron microscopy (TEM) images (Fig. 2c,f,i), with a shell of TiO2 crystals with sizes falling within 20–30 nm surrounding the core for the Fe3O4@mTiO2 microspheres
Summary
Methods for glyco-specific enrichment divide into several categories by means of different mechanisms, including lectin affinity[8, 9], size exclusion[10], hydrazide chemistry[11, 12], hydrophilic interaction[13, 14], www.nature.com/scientificreports/. With phenylboronic acids typically employed, the boronic acid moiety can form cyclic ester with cis-diol group of glycoconjugates in an alkaline medium and at acidic pH the ester dissociates[19], making boronic acid a unique ligand for reversibly collecting and detaching glycopeptides This method isolates both N- and O-glycopeptides in an unbiased manner, complementing some limitations of other methods, such as biased glycol-enrichment with respect to lectin affinity and hydrazide chemistry, and insufficient selectivity and recovery related to hydrophilic interaction. Several types of materials have been developed to conjugate boronic acid groups for glycopeptide analyses, including agarose resin, mesoporous silica[15], polymer particles[16], magnetic particles[18,19,20,21,22,23,24], carbon nanotubes[25], and graphene oxide[26]. We tested the method in the glycopeptides analysis of human amniotic fluid samples with remarkable results
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