Facile and easily popularized synthesis of l-cysteine-functionalized magnetic nanoparticles based on one-step functionalization for highly efficient enrichment of glycopeptides

Abstract

Protein glycosylation is one of the most important post-translational modifications. Also, efficient enrichment and separation of glycopeptides from complex samples are crucial for the thorough analysis of glycosylation. Developing novel hydrophilic materials with facile and easily popularized synthesis is an urgent need in large-scale glycoproteomics research. Herein, for the first time, a one-step functionalization strategy based on metal-organic coordination was proposed and FeO nanoparticles were directly functionalized with zwitterionic hydrophilic L-cysteine (L-Cys), greatly simplifying the synthetic procedure. The easily synthesized FeO/L-Cys possessed excellent hydrophilicity and brief composition, contributing to affinity for glycopeptides and reduction in nonspecific interaction. Thus, FeO/L-Cys nanoparticles showed outstanding sensitivity (25 amol/μL), high selectivity (mixture of bovine serum albumin and horseradish peroxidase tryptic digests at a mass ratio of 100:1), good reusability (five repeated times), and stability (room temperature storage of 1 month). Encouragingly, in the glycosylation analysis of human serum, a total of 376 glycopeptides with 393 N-glycosylation sites corresponding to 118 glycoproteins were identified after enrichment with FeO/L-Cys, which was superior to ever reported L-Cys modified magnetic materials. Furthermore, applying the one-step functionalization strategy, cysteamine and glutathione respectively direct-functionalized FeO nanoparticles were successfully synthesized and also achieved efficient glycopeptide enrichment in human serum. The results indicated that we have presented an efficient and easily popularized strategy in glycoproteomics as well as in the synthesis of novel materials. Graphical abstract FeO/L-Cys nanoparticles based on one-step functionalization for highly efficient enrichment of glycopeptides.

A review on recent advances in the enrichment of glycopeptides and glycoproteins by liquid chromatographic methods: 2016–Present

“…L‐Cys, an amino acid, is a zwitterion molecule that possesses both positive and negative (NH 2 and COOH) groups within its structure. Seven out of eight research articles that employed L‐Cys as ligand had used Fe 3 O 4 NPs grafted with either a series of polymer layers or SiO 2 layer or a polymer layer‐SiO 2 layer combination as the stationary phase [49–53, 60, 79]. Only in one study [53], L‐Cys was directly immobilized on Au NPs via Au‐S bond.…”

“…Feng et al. [53] had developed a simple and efficient approach to directly immobilize l ‐Cys on Fe 3 O 4 NPs (L‐Cys@Fe 3 O 4 NPs). This approach had resulted in the enrichment of three HRP glycopeptides at 25 amol/μL within 1 min.…”

“…Feng et al. had extended their single‐step strategy to prepare cysteamine@Fe 3 O 4 NPs that enriched six glycoproteins [53]. Porous p ‐TpBDH‐OH nanomaterial with enhanced hydrophilicity was synthesized by Ding et al.…”

“…Seven out of eight research articles that employed L-Cys as ligand had used Fe 3 O 4 NPs grafted with either a series of polymer layers or SiO 2 layer or a polymer layer-SiO 2 layer combination as the stationary phase [49-53, 60, 79]. Only in one study [53], L-Cys was directly immobilized on Au NPs via Au-S bond. Sun et al [95] had used L-Cys@Au@poly(GMA-co-EDMA) resin pipette tip to enrich 26 IgG and 6 HRP glycopeptides from 0.5 pmol/μL of the respective protein digests.…”

“…Feng et al [53] enriched 117 glycoproteins from human serum using GSH@Fe 3 O 4 NPs. Qi et al [54] incorporated the gold layer on Fe 3 O 4 NPs prior to GSH immobilization (GSH@Au@Fe 3 O 4 NPs), which resulted in the enrichment of 22 glycopeptides from human serum.…”