Selective Enrichment Of N-linked Glycopeptides Research Articles

One-Step Synthesis of Hydrophilic Nano-Floral Inter-Polymeric Material for Highly Selective Enrichment of N-Linked Glycopeptides

A novel nano-floral inter-polymeric material (λ-Carr-Ag-SA) was developed to effectively capture N-linked glycopeptides from biological samples. A simple one-step reduction of silver ions was involved in the synthesis of the material in which λ-carrageenan and sodium alginate, with abundant hydrophilic groups, were used as the reducing agent and stabilizer. The performance of λ-Carr-Ag-SA was evaluated experimentally by enriching glycopeptides from horse radish peroxidase (HRP) and human serum. All the results demonstrated that λ-Carr-Ag-SA had a great selectivity, low detection limit and a large binding capacity toward glycopeptides. The successful application exhibits great potential of λ-Carr-Ag-SA for further glycoproteome analysis.

Gold nanoparticle-glutathione-functionalized porous graphene oxide-based hydrophilic beads for the selective enrichment of N-linked glycopeptides.

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 (2fmol), high enrichment selectivity (1:500), binding capacity (333.3mg/g), recovery yield (91.3 ± 2.1%), and repeatability (< 6.0% RSD) using matrix-assisted laser desorption/ionization time of flight mass spectrometrydetection 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 1μL 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.

One-step synthesis of hydrophilic microspheres for highly selective enrichment of N-linked glycopeptides

A polyacrylamide-based hydrophilic microsphere with a lot of hydroxyl groups on surface (PAM-OH HMS) was prepared in one step. The synthetic process was simple reverse suspension polymerization without any chemical derivation or grafting steps. The properties of obtained HMS were characterized by scanning electron microscopy (SEM), static water contact angle measurement, and FT-IR. The abundant hydroxyl groups on the surface make the material highly good hydrophilic and thus it was utilized for N-glycopeptides enrichment. The enrichment efficiency of PAM-OH HMSs was demonstrated by capturing N-linked glycopeptides from tryptic digest of human immunoglobulin G (IgG). The detection sensitivity for N-glycopeptides identification by MALDI-TOF MS was as low as 10 fmol for tryptic digest of standard human IgG. The selectivity of the HMS towards N-glycopeptides had almost no decrease when the molar ratio of BSA tryptic digest to IgG tryptic digest was increased from 10:1 to 100:1. Moreover, in the LC-MS/MS analysis of real biological sample, a total of 344 unique N-glycosites in 598 unique N-glycopeptides from 172 N-glycoproteins were identified from 2 μL human serum after deglycosylated by PNGase F, and 825 intact N-glycopeptides with different types of glycoform were detected when directly analyzed the N-glycopeptides enriched by PAM-OH HMS. To show the potential of our material in solving real biological issues, N-glycopeptides in the serum from hepatocelluar carcinoma (HCC) patient and health control were enriched and quantified. All the experiments demonstrated that this polyacrylamide-based hydrophilic microsphere shows a great potential to enrich the low-abundance N-glycopeptides for glycoproteome analysis of real complicated biological samples.

Facile synthesis of hydrophilic magnetic graphene nanocomposites via dopamine self-polymerization and Michael addition for selective enrichment of N-linked glycopeptides

The development of methods to effectively capture N-glycopeptides from the complex biological samples is crucial to N-glycoproteome profiling. Herein, the hydrophilic chitosan–functionalized magnetic graphene nanocomposites (denoted as Fe3O4-GO@PDA-Chitosan) were designed and synthesized via a simple two-step modification (dopamine self-polymerization and Michael addition). The Fe3O4-GO@PDA-Chitosan nanocomposites exhibited good performances with low detection limit (0.4 fmol·μL−1), good selectivity (mixture of bovine serum albumin and horseradish peroxidase tryptic digests at a molar ration of 10:1), good repeatability (4 times), high binding capacity (75 mg·g−1). Moreover, Fe3O4-GO@PDA-Chitosan nanocomposites were further utilized to selectively enrich glycopeptides from human renal mesangial cell (HRMC, 200 μg) tryptic digest, and 393 N-linked glycopeptides, representing 195 different glycoproteins and 458 glycosylation sites were identified. This study provides a feasible strategy for the surface functionalized novel materials for isolation and enrichment of N-glycopeptides.

Amino Acid-Functionalized Two-Dimensional Hollow Cobalt Sulfide Nanoleaves for the Highly Selective Enrichment of N-Linked Glycopeptides.

Leaf-like hollow cobalt sulfides with a sulfur-gold-cysteine (S-Au-Cys) structure on the surface have been synthesized for efficient N-glycopeptide enrichment. A two-dimensional (2D) zeolitic imidazolate framework with cobalt (ZIF-L-Co, L for leaf) was used as a self-sacrificed template. After sulfidation, the S-Au-Cys architecture was created on the surface of the leaf-like hollow cobalt sulfide to obtain a material denoted ZIF-L-Co-S-Au-Cys. Enrichment of glycopeptides from trypsin digests of immunoglobulin G (IgG) standard samples and of IgG isolated from real human plasma samples was accomplished via hydrophilic interaction liquid chromatography processes using ZIF-L-Co-S-Au-Cys. The good sensitivity and selectivity ensure the effectiveness and robustness of ZIF-L-Co-S-Au-Cys for sample preconcentration, which is comparable to a commercial HILIC product. This work provides an efficient way to produce transition metal sulfides with a low-dimensional morphology and provides a novel concept for material design for exploitation in sample preparation, especially in glycoproteomics.

L-cysteine-modified metal-organic frameworks as multifunctional probes for efficient identification of N-linked glycopeptides and phosphopeptides in human crystalline lens

Highly selective enrichment of N-linked glycopeptides and phosphopeptides from complex biological samples is extremely important prior to mass spectrometry analysis due to their low abundance as well as numerous extrinsic interferences. In this work, l-cysteine (L-Cys)-modified multifunctional metal-organic frameworks denoted as Fe3O4@PDA@MIL-125@Au@L-Cys (mMIL-125@Au@L-Cys) were prepared by modifications step by step. By combining hydrophilic interaction chromatography (HILIC) with metal oxide affinity chromatography (MOAC), the as-prepared material was firstly utilized to identify N-linked glycopeptides and phosphopeptides from tryptic digests of horseradish peroxidase (HRP) and beta-casein (β-casein), respectively, with the help of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and exhibited outstanding sensitivity (0.1 fmol μL−1), great reusability (5 circles) and high selectivity (1: 100). Based on this, it was further applied into the enrichment of glycopeptides and phosphopeptides from tryptic digests of 100 μg human crystalline lens proteins. In the end, 81 N-linked glycopeptides corresponding to 35 glycoproteins and 175 phosphopeptides ascribed to 55 phosphorylated proteins were identified, respectively. The remarkable results were benefitted from the merits of improved hydrophilicity from L-Cys, strong affinity of TiO centers, numerous reaction sites on the large surface of MOFs and superparamagnetism from Fe3O4 cores. The design of mMIL-125@Au@L-Cys not only served as a multifunctional probe for efficient identification of N-linked glycopeptides and phosphopeptides in human crystalline lens, but also set a precedent for fabricating more MOFs with post-modifications for further proteomics research.

Preparation of iminodiacetic acid functionalized silica capillary trap column for on-column selective enrichment of N-linked glycopeptides

In this work, SiO2-IDA microspheres were obtained via a previous method by grafting iminodiacetic acid (IDA) groups onto silica microspheres, then the SiO2-IDA microspheres were packed in capillary columns to prepare SiO2-IDA capillary trap columns for the first time. Based on the trap columns, a novel on-column enrichment method toward glycopeptides was developed as a sample pretreatment step prior to matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) or nano-LC-MS/MS analysis. The preparation procedures of the trap columns are well reproducible. Thanks to outstanding hydrophilicity brought by the grafted zwitterionic IDA groups, the trap columns showed excellent on-column enrichment performance toward glycopeptides by hydrophilic interaction liquid chromatography (HILIC). Experimental results showed that the on-column enrichment method could reach a low detection limit (50 fmol HRP digest) and a high selectivity (molar ratio of HRP digest and BSA digest 1:100). Finally, it was applied to on-column enrichment of glycopeptides from digests of human serum followed by nano-LC-MS/MS analysis. In total, 207 glycopeptides assigned to 78 glycoproteins were identified from 2 μL human serum.

Maltose-Functionalized Hydrophilic Magnetic Nanoparticles with Polymer Brushes for Highly Selective Enrichment of N-Linked Glycopeptides.

Efficient enrichment glycoproteins/glycopeptides from complex biological solutions are very important in the biomedical sciences, in particular biomarker research. In this work, the high hydrophilic polyethylenimine conjugated polymaltose polymer brushes functionalized magnetic Fe3O4 nanoparticles (NPs) denoted as Fe3O4–PEI–pMaltose were designed and synthesized via a simple two-step modification. The obtained superhydrophilic Fe3O4–PEI–pMaltose NPs displayed outstanding advantages in the enrichment of N-linked glycopeptides, including high selectivity (1:100, mass ratios of HRP and bovine serum albumin (BSA) digest), low detection limit (10 fmol), large binding capacity (200 mg/g), and high enrichment recovery (above 85%). The above-mentioned excellent performance of novel Fe3O4–PEI–pMaltose NPs was attributed to graft of maltose polymer brushes and efficient assembly strategy. Moreover, Fe3O4–PEI–pMaltose NPs were further utilized to selectively enrich glycopeptides from human renal mesangial cell (HRMC, 200 μg) tryptic digest, and 449 N-linked glycopeptides, representing 323 different glycoproteins and 476 glycosylation sites, were identified. It was expected that the as-synthesized Fe3O4–PEI–pMaltose NPs, possessing excellent performance (high binding capacity, good selectivity, low detection limit, high enrichment recovery, and easy magnetic separation) coupled to a facile preparation procedure, have a huge potential in N-glycosylation proteome analysis of complex biological samples.

Polymeric hydrophilic ionic liquids used to modify magnetic nanoparticles for the highly selective enrichment of N-linked glycopeptides

The low abundance of glycopeptides in biological samples makes it necessary to enrich them before further analysis. In this study, the polymeric hydrophilic ionic liquid-modified magnetic (Fe3O4@MPS@PMAC) nanoparticles were synthesized via a one-step reflux-precipitation polymerization. Owing to the excellent hydrophilicity and strong electrostatic interaction toward glycopeptides of the polymerized hydrophilic ionic liquid, [2-(methacryloyloxy) ethyl] trimethylammonium chloride (MAC), the synthesized Fe3O4@MPS@PMAC nanoparticles exhibited outstanding performance in glycopeptide enrichment with high detection sensitivity (10 fmol), large binding capacity (100 μg mg−1) and satisfied enrichment recovery (approximately 82%). Furthermore, the newly developed Fe3O4@MPS@PMAC nanoparticles were applied for the glycopeptide enrichment of HeLa exosome proteins. A total of 1274 glycopeptides from 536 glycoproteins were identified in three replicate analyses of 50 μg of HeLa exosome proteins. These results demonstrate the potential of Fe3O4@MPS@PMAC nanoparticles for both glycoproteomic analysis and exosome research.

One-step synthesis of carboxyl-functionalized metal-organic framework with binary ligands for highly selective enrichment of N-linked glycopeptides

Highly efficient and selective enrichment of glycopeptides from complex biosamples is vital prior to mass spectrometry analysis. In this work, a hydrophilic metal organic framework with free carboxylic groups (denoted as UiO-66-COOH) was synthesized in one step with binary ligands. Compared with pure UiO-66 with one ligand, UiO-66-COOH containing binary ligands demonstrated greater hydrophilicity and performed much better in selective enrichment of glycopeptides. The as-prepared material demonstrated excellent performance for selective enrichment of glycopeptides from tryptic digests of standard glycoproteins and practical biological samples.

Synthesis of magnetic zwitterionic–hydrophilic material for the selective enrichment of N-linked glycopeptides

In consideration of the close connection between glycopeptides and human diseases, the efficient method to separate and enrich glycopeptides from complex biological samples is urgently required. In the work, we developed a magnetic zwitterionic-hydrophilic material for highly effective separation and analysis of glycopeptides from complex samples. The Fe3O4 particles were covered with a thick layer of polymer by one-step reflux-precipitation polymerization (RPP), subsequently decorated by Au nanoparticles (Au NPs) through in situ reduction and finally modified with zwitterionic groups. The abundant zwitterionic sites facilitate the selective enrichment of glycopeptides. Besides, the prepared Fe3O4@PGMA@Au-l-cys showed high detection sensitivity (5 fmol IgG digest), approving enrichment capacity (75mgg−1), satisfactory enrichment recovery (89.8%), and great performance in the analysis and profiling of low-abundance N-linked glycopeptides. Furthermore, the prepared material was employed in the enrichment of glycopeptides in intricate biological samples, and 774 unique N-glycosylation sites from 411 N-glycosylated proteins were reliably identified in three replicate analyses of a 75μg protein sample extracted from mouse liver, suggesting wide application prospect in glycoproteomics.

Amine Chemistry Method for Selective Enrichment of N-Linked Glycopeptides for Glycoproteomics Analysis.

An amine chemistry method was developed for the extraction of N-glycopeptides using amine-functionalized beads for glycoproteomics analysis. Two reductive amination reactions between primary amine and aldehyde were employed in this approach. The first one was to block the primary amines in the peptides by addition of formaldehyde and sodium cyanoborohydride into the peptide sample, and the second one was to couple the glycopeptides onto solid phase beads by incubating the glycopeptides containing aldehyde groups (oxidized by periodate) with the amine-functionalized beads in the presence of sodium cyanoborohydride. It was demonstrated that the blocking of primary amines in the peptides by the first reductive amination reaction prior to the periodate oxidation made the amine chemistry method very efficient and sensitive. This new method was validated by analysis of glycoprotein standards as well as proteome samples. It was found that this new method led to significant increase in the identification of N-glycosites compared with the conventional hydrazide chemistry method.

An ultra hydrophilic dendrimer-modified magnetic graphene with a polydopamine coating for the selective enrichment of glycopeptides.

In recent MS-based glycoproteomics, the selective enrichment of glycopeptides from complex biological samples is essential. In this work, for the first time, a novel ultra hydrophilic dendrimer-modified magnetic graphene@polydopamine@poly(amidoamine) (magG@PDA@PAMAM) was synthesized via three simple and rapid steps. The magnetic composites combined large surface of graphene, strong magnetic responsiveness of Fe3O4 with double hydrophilic abilities of PDA and PAMAM. Especially PAMAM had long dendritic chains and abundant amino groups. When it was grafted onto magG@PDA, it strongly enhanced the hydrophilic properties of the magnetic composites. The PAMAM-functionalized magnetic composites were employed in the enrichment of glycopeptides, 15 glycopeptides from horseradish peroxidase (HRP) digestion were identified and the limit of detection was as low as 1 fmol μL-1. Also, it showed a good selectivity when the background nonglycopeptides had a concentration 100 fold higher than the target glycopeptides. All the results proved that magG@PDA@PAMAM has great potential in the glycoproteome analysis of complex biological samples.

Highly specific enrichment of N-linked glycopeptides based on hydrazide functionalized soluble nanopolymers

In this work, for the first time, hydrazide functionalized PAMAM was designed and synthesized for efficient and selective enrichment of N-linked glycopeptides from complex biological samples using FASP (filter-aided sample preparation) mode.

Click aspartic acid as H ILIC SPE material for selective enrichment of N-linked glycopeptides

A novel hydrophilic interaction chromatography (HILIC) material was developed via clicking aspartic acid onto silica gel (termed as Click AA). The material demonstrated highly hydrophilic property and was used as solid phase extraction sorbent for selective enrichment of glycopeptides. By optimization of extraction conditions, Click AA exhibited high selectivity to glycopeptides with various peptide length and different types of glycans, which was much superior to similar commercial products. The application of Click AA to simulated proteomic samples further proved its specialty toward glycopeptides.

Synthesis of branched PEG brushes hybrid hydrophilic magnetic nanoparticles for the selective enrichment of N-linked glycopeptides

Hybrid Fe(3)O(4)@SiO(2)@PEG-Maltose MNPs were synthesized by SI-ATRP of branched PEG brushes on the surface and subsequent functionalization with hydrophilic maltose group, and the multifunctional materials were utilized for selective enrichment of N-linked glycopeptides from biological samples with high specifity, high sensitivity, and large binding capacity.

Selective enrichment of N-linked glycopeptides by using a highly hydrophilic matrix synthesized via click chemistry

A highly hydrophilic matrix named Click CA was developed via click chemistry for the selective enrichment of N-linked glycopeptides. This matrix exhibited superior selectivity and coverage for N-linked glycopeptides.