Qualitative and Quantitative Assessment on the Use of Magnetic Nanoparticles for Glycopeptide Enrichment

Abstract

Glycoproteomics represent the field of study of the dynamic changes occurring among glycoconjugates within the cellular compartments. Changes in glycosylation have been linked to various diseases, including metastatic carcinomas in which the 9 carbon sialic acid moiety has been shown to play a prominent role. The common method used to study these aberrant changes most often includes a mass spectrometer at some stage in the workflow. However, serum samples contain many proteins which inhibit the analysis of these glycosylation changes, and ergo, enrichment steps are employed as a measure to help alleviate this ailment. Routinely, this is accomplished using lectins, either alone or in combination, to retrieve proteins with specific sugar linkages within the serum sample. This methodology, although known to be very specific, requires many washing steps, making it a cumbersome addition to a high throughput workflow. Presented here is an alternative protocol using custom-made amine functionalized magnetic nanoparticles (MNP) which are nearly 4× smaller than those used before for similar purposes. The developed protocol is based on both hydrophilic interaction and weak anion exchange principles, allowing it to target glycopeptides but, more specifically, those which contain sialylation. For quantification purposes, tandem mass tags from Thermo Scientific were utilized to compare the enrichment efficiencies between the magnetic nanoparticle method and a commercially available glycopeptide enrichment kit offered through EMD Millipore. The MNP method is fast (~10 min) and simple and can quantitatively and qualitatively enrich sialylated glycopeptides more than the commercially available kit.

Carbohydrates on Proteins: Site-Specific Glycosylation Analysis by Mass Spectrometry

“…Elution of glycopeptides can be achieved by increasing the content of water in the mobile phase. Based on hydrophilic interactions and weak anion exchange principles, a recent application uses amine-functionalized magnetic nanoparticles with greater surface area to selectively enrich sialylated glycopeptides (38). Zwitterionic-HILIC (ZIC-HILIC), a variation of HILIC that uses zwitterions as stationary phases, has better separation efficiency for glycopeptides because additional ion-pairing effects exist in the separation process (39,40).…”

Mass spectrometric analysis of products of metabolic glycan engineering with azido-modification of sialic acids

“…Methods based on hydrazide chemistry have been reported, exploiting oxidized cis-diol interactions [14,15], and other protocols focus on the capture of glycans through boronic acid interactions [16] or aniline groups functionalized to magnetic nanoparticles [17,18]. Hydrophilic interaction liquid chromatography (HILIC) approaches have shown to be simple, fast, and efficient relative to reversed-phase approaches for glycopeptide enrichment [19][20][21][22]. Much emphasis has been placed around the design of niche Bglyco-modified^columns usable under HILIC conditions including Bclick saccharides^onto polymer beads [23], silica beads [24,25], and magnetic nanoparticles [26].…”

Synthesis and evaluation of carboxymethyl chitosan for glycopeptide enrichment

“…Although the loading capacity of CMCH was not optimized, the amount of digest loaded, 25 mg, was well within the recommended maximum load for EMD ( [36], as well as optimization of isolation of glycopeptides from magnetic nanoparticles [34], and also from optimization of glycopeptide enrichment using cellulose [16], it was found that a ratio of approximately 7:3 is required for elution. If RP is used, approximately 30% ACN in water is needed to elute the bulk of glycopeptides, whereas by HILIC, glycopeptides will elute with 30% H 2 O in ACN.…”

“…However, as the CMCH beads were utilized according to HILIC protocols developed in our laboratory [16,34] and as they dramatically enrich glycopeptides, HILIC interactions are deemed to play an important role in glycopeptide enrichment. As for the WAX process, the amine on chitosan is proposed to interact with negatively charged sialylated species, as it can explain the more efficient enrichment of sialylated glycopeptides than seen using the EMD process.…”

“…It is only when fragmentation occurs [39] that a mass difference in reporter ions can be observed, whereby the ratio between these reporter ions can be directly correlated to the enrichment efficiencies of the two methodologies. This workflow for glycopeptide enrichment has been previously demonstrated by our group using amine functionalized magnetic nanoparticles (MNP) [34]. From this work, and other published glycomic studies, a standard library of glycoproteins has been generated for reference [16,40e42].…”