Selective enrichment of N‐linked glycopeptides and glycans by using a dextran‐modified hydrophilic material

Glycosylation analysis of proteins from biological sources utilizing mass spectrometry based approaches is challenging due to the relatively low abundance of glycopeptides, the structural diversity of glycans, and the coexisting matrices. In this study, a customized dextran‐bonded silica‐based stationary phase was introduced for selective enrichment of glycopeptides and glycans from complex biological samples. This material has exhibited superior selectivity and broader glycosylation site coverage over commercial Sepharose in glycoproteomic evaluation. Additionally, the glycomic analysis of fetuin, α₁‐acid glycoprotein, and human serum N‐glycome also indicated the relatively higher sensitivity, selectivity, and glycoform coverage of dextran‐bonded silica than that of Sepharose and porous graphitized carbon. Therefore, the dextran‐bonded silica is expected to make contributions in the fields of glycoproteomics and glycomics.     

两性离子双功能化超亲水金属有机框架纳米复合材料的制备及其用于糖肽的选择性富集

蛋白糖基化是生物体中普遍发生且重要的生物学过程,其参与多种分子生物学的功能和途径,是临床诊断重要的生物标志物.但是,糖肽因其丰度低、离子化效率低、糖链异质性等难点,使糖蛋白分析一直面临巨大的挑战.因此,研究合成了一种新型的两性离子双功能化纳米金(AuGC)修饰的超亲水性沸石咪唑骨架(ZIF-8)纳米复合材料(AuGC/...     

Facile preparation of hydrophilic glutathione modified magnetic nanomaterials for specific enrichment of glycopeptides

Glutathione modified magnetic nanoparticles (Fe₃O₄@Au-GSH) were synthesized through a simple process and exploited to enrich glycopeptides from complex samples.     

Facile preparation of hydrophilic glutathione modified magnetic nanomaterials for specific enrichment of glycopeptides

Investigations of glycosylated proteins or peptides and their related biological pathways provide new possibilities for illuminating the physiological and pathological mechanisms of glycosylation modification. However, open-ended and in-depth analysis of glycoproteomics is usually subjected to the low-abundance of glycopeptides, heterogeneous glycans, and a variety of interference molecules. In order to alleviate the influence of these obstacles, effective preconcentration of glycopeptides are indispensable. Here, we employed a hydrophilic interaction liquid chromatography (HILIC)-based method to universally capture glycopeptides. Glutathione modified magnetic nanoparticles (Fe₃O₄@Au-GSH) were synthesized through a simple process and exploited to enrich glycopeptides from complex samples. The prepared materials showed excellent ability to trap glycopeptides from standard glycoproteins digests, low detection limit (10 fmol/μL), and good selectivity (HRP:BSA = 1:100). These results indicated that glutathione-based magnetic nanoparticles synthesized in this work had great potential for glycopeptides enrichment.     

Synthesis of zwitterionic polymer brushes hybrid silica nanoparticles via controlled polymerization for highly efficient enrichment of glycopeptides

Zwitterionic hydrophilic interaction chromatography (ZIC-HILIC) materials have been increasingly attractive in glycopeptide enrichment. However, the traditional ZIC-HILIC materials are modified with monolayer zwitterionic molecules on the surface, therefore, the hydrophilicity, detection sensitivity and loading capacity are limited. In this work, we synthesized novel silica nanoparticles with uniform poly(2-(methacryloyloxy)ethyl)dimethyl-(3-sul-fopropyl)ammonium hydroxide (PMSA) brushes grafted onto the surface via reversible addition-fragmentation chain transfer (RAFT) polymerization (denoted as SiO₂-RAFT@PMSA). The resulting SiO₂-RAFT@PMSA nanoparticles demonstrated low detection limit (10 fmol) and high recovery yield (over 88%) for glycopeptide enrichment from tryptic digest of human IgG. The SiO₂-RAFT@PMSA nanoparticles were further applied for the analysis of mouse liver glycoproteome, a total number of 303 unique N-glycosylation sites corresponding to 185 glycoproteins was reliably profiled in three replicate nano-LC–MS/MS runs. Significantly, more glycopeptides were identified than those of nanoparticles, monolayer MSA molecules modified SiO₂@single-MSA and nonuniform multi-layer PMSA brushes coated SiO₂@PMSA, as well as commercial ZIC@HILIC beads and Click Maltose beads. The excellent performance of SiO₂-RAFT@PMSA nanoparticles results from the non-fouling property, a large quantity of functional molecules and suitable link arms provided by uniform PMSA brushes, as well as efficient interaction between glycopeptides and uniform PMSA brushes. It is concluded that the synthesized SiO₂-RAFT@PMSA nanoparticles exhibit great potential in glycoproteome analysis. Moreover, this strategy to modify nanopaticles with uniform polymer brushes via RAFT polymerization can also be explored to design other types of materials for bioseparation application.     

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 glycopeptides based on copper tetra(N‐carbonylacrylic) aminephthalocyanine and iminodiacetic acid functionalized polymer monolith

Efficient separation and enrichment of low‐abundance glycopeptides from complex biological samples is the key to the discovery of disease biomarkers. In this work, a new material was prepared by coating copper tetra(N‐carbonylacrylic) aminephthalocyanine and iminodiacetic acid onto poly(glycidyl methacrylate‐pentaerythritol triacrylate) monolith. The monolith was applied to polymer monolithic microextraction for specific capture of glycopeptides coupled with matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. The developed monolith exhibited satisfactory efficiency for glycopeptide enrichment with high selectivity and detection sensitivity. When the tryptic digest of immunoglobulin G was used as the sample, total 24 glycopeptides were identified and the detection limit was determined as 5 fmol. When the approach was applied to the analysis of glycopeptides in the mixture of bovine serum albumin and immunoglobulin G (100:1, m/m) digests, 16 glycopeptides could still be observed. Moreover, the monolith was successfully applied to the selective enrichment of glycopeptides from human serum digests, exhibiting great practicability in identifying low‐abundance glycopeptides in complex biological samples.     

Polyelectrolyte complex nanoparticles from N‐carboxyethylchitosan and polycationic double hydrophilic diblock copolymers

For the first time, the polyelectrolyte complex (PEC) formation tool was used for preparation of core‐shell nanoparticles form the natural polyampholyte N‐carboxyethylchitosan (CECh) and weak polycationic (protonated) polyoxyethylene‐b‐poly[2‐(dimethyl‐amino)ethyl methacrylate] (POE‐b‐PDMAEMA) diblock copolymers. The performed dynamic light scattering analyses revealed that nanoparticles with a PEC core and a POE shell could be formed at mixing ratio between the oppositely charged groups equal to 1/1 depending on CECh molar mass, polymerization degree of PDMAEMA block and ionic strength. The results were confirmed by the performed AFM and cryo‐TEM analyses. When high molar mass CECh was used, core‐shell nanoparticles were obtained with the diblock copolymer of the shortest PDMAEMA block at ionic strength (I) of 0.01. At ionic strength value close to the physiological one (I = 0.1) secondary aggregation occurred. Spherical nanoparticles at I = 0.1 were obtained upon lowering the CECh molar mass. Depending on the polymer partners and medium parameters the size of the obtained particles varied from 60 to 600 nm. The X‐ray photoelectron spectra evidenced the hydrophilic POE‐block shell—coacervate CECh/PDMAEMA‐block core structure. The nanoparticles are stable in a rather narrow pH range around 7.0, thus revealing the high pH‐sensitivity of the obtained core‐shell particles. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 2105–2117, 2009     

半胱氨酸基麦芽糖修饰亲水硅胶的制备及其对糖肽的富集

糖蛋白与糖肽在复杂生物体系中丰度一般较低,为了在糖蛋白质组学研究中深入和全面分析鉴定糖基化位点与糖链,通常需要进行富集前处理操作。该文设计并合成了一种半胱氨酸基麦芽糖修饰亲水硅胶分离介质(Cys-Mal@SiO_2),将其装填入固相萃取柱中制备新型亲水固相萃取柱。在以人免疫球蛋白G酶解液为样品进行富集鉴定时,Cys-Mal@SiO_2鉴定糖肽的质谱信号强度和信噪比比半胱氨酸修饰硅胶(Cys@SiO_2)、麦芽糖修饰硅胶(Mal@SiO_2)和商品化ZIC-HILIC更高。在复杂的鼠肝蛋白质提取物的糖蛋白质组学分析中,Cys-Mal@SiO_2共鉴定出1 551条糖肽,属于466个糖蛋白的906个N-糖基化位点,比Cys@SiO_2和Mal@SiO_2鉴定糖肽数、蛋白质数和N-糖基化位点数分别多211、67、127个和289、76、193个。将Cys-Mal@SiO_2成功应用于低丰度糖肽的选择性富集与鉴定,在糖蛋白质组学研究中体现出良好的应用潜力。     

Design and fabrication of highly hydrophilic magnetic material by anchoring l-cysteine onto chitosan for efficient enrichment of glycopeptides

Hydrophilic interaction liquid chromatography(HILIC) has been recognized as an effective strategy for glycopeptide enrichment. Hydrophilic materials pave the way to solve the limit of low enrichment capacity and poor selectivity. The present study is the first attempt to combine chitosan(CS) and L-cysteine(L-Cys) to design a novel hydrophilic material focusing on glycopeptide enrichment. CS containing a large number of hydrophilic amino and hydroxyl groups has unique chemical properties, which m...     

Performance of zwitterionic hydrophilic interaction LC for the determination of iodinated X‐ray contrast agents

This study compares the separation performance of a group of iodinated X‐ray contrast media on four different columns. The first three were two stationary phases (SPs) modified with C₁₈ and a polar‐embedded SP (polar amide group bonded to an alkyl chain), all of which worked under RP‐LC mode. The fourth was a zwitterionic sulphoalkylbetaine SP, working under the hydrophilic interaction LC (HILIC) mode. After the optimisation of the different parameters, the zwitterionic column displayed the best separation, which also overcomes the problems encountered when these analytes were separated under RP‐LC. Moreover, when HILIC is coupled to MS/MS, sensitivity is enhanced. However, when sewage samples were analysed by SPE followed by the optimal HILIC–MS/MS, the sensitivity of the method was affected due to the high matrix effect, which had to be solved by dilution of the extract. Finally, the method was preliminarily validated with sewage and the figures of merit were comparable to those of the SPE–RP‐LC–MS/MS.     

Gold nanoparticles immobilized hydrophilic monoliths with variable functional modification for highly selective enrichment and on-line deglycosylation of glycopeptides

The poly (glycidyl methacrylate-co-poly (ethylene glycol) diacrylate) monoliths modified with gold nanoparticles, with advantages of enhanced reactive sites, good hydrophilicity and facile modification, were prepared as the matrix, followed by variable functionalization with cysteine and PNGase F for glycopeptide enrichment and on-line deglycosylation respectively. By the cysteine functionalized monolithic column, glycopeptides could be efficiently and selectively enriched with good reproducibility based on hydrophilic interaction chromatography (HILIC). Furthermore, the enrichment was specially achieved in weak alkaline environment, with 10 mM NH₄HCO₃ as the elution buffer, compatible with deglycosylation conditions. Therefore, the glycopeptides could be on-line deglycosylated with high efficiency and throughput by directly coupling the PNGase F functionalized monolithic column with the enrichment column during elution without the requirement of buffer exchange and pH adjustment. By such a method, within only 70-min pretreatment, 196 N-linked glycopeptides, corresponding to 122 glycoproteins, could be identified from 5 μg of human plasma with 14 high-abundant proteins removed, and the N-linked glycopeptides occupied 81% of all identified peptides, achieving to the best of our knowledge, the highest selectivity of HILIC-based methods. All the results demonstrated the high efficiency, selectivity and throughput of our proposed strategy for the large scale glycoproteome analysis.     

N‐ and O‐linked glycosylation site profiling of the human basic salivary proline‐rich protein 3M

In the present study, we show that the heterogeneous mixture of glycoforms of the basic salivary proline‐rich protein 3M, encoded by PRB3‐M locus, is a major component of the acidic soluble fraction of human whole saliva in the first years of life. Reversed‐phase high‐performance liquid chromatography with high‐resolution electrospray ionization mass spectrometry analysis of the intact proteoforms before and after N‐deglycosylation with Peptide‐N‐Glycosidase F and tandem mass spectrometry sequencing of peptides obtained after Endoproteinase GluC digestion allowed the structural characterization of the peptide backbone and identification of N‐ and O‐glycosylation sites. The heterogeneous mixture of the proteoforms derives from the combination of 8 different neutral and sialylated glycans O‐linked to Threonine 50, and 33 different glycans N‐linked to Asparagine residues at positions 66, 87, 108, 129, 150, 171, 192, and 213.     

Facile synthesis of 4‐mercaptophenylboronic acid functionalized gold nanoparticles for selective enrichment of glycopeptides

In the work, 4‐mercaptophenylboronic acid (4‐MPBA)‐functionalized gold nanoparticles were synthesized via a facile approach. At first, gold nanoparticles (about 50 nm) were prepared by a simple and convenient hydrothermal method based on a polyol process. Then, gold nanoparticles were modified with 4‐MPBA by the well‐known reaction of Au with the thiol groups. The MPBA‐functionalized gold nanoparticles were characterized by Fourier transform infrared spectra and UV/Vis adsorption spectra. Due to the fact that the boronic acid group on the surface of 4‐MPBA‐modified gold particles can form tight yet reversible covalent bonds with glycopeptides containing cis‐1,2‐diols groups, the MPBA‐modified gold nanoparticles were successfully applied to selective enrichment of glycopeptides. Isolation and enrichment of glycopeptides in a standard protein (asialofetuin and horseradish peroxidase) digestion and a complex sample were performed using MPBA‐modified gold nanoparticles, followed by matrix‐assisted laser desorption/ionization quadruple ion trap time‐of‐flight (MALDI‐QIT‐TOF) mass spectrometric analysis. The experimental results demonstrated that MPBA‐modified gold nanoparticles synthesized by the facile approach have the powerful potential for selective enrichment of glyciopeptides, and can be an alternative tool in glycoproteomics. Copyright © 2009 John Wiley & Sons, Ltd.     

Reversed-phase depletion coupled with hydrophilic affinity enrichment for the selective isolation of N-linked glycopeptides by using Click OEG-CD matrix

Selective enrichment of glycopeptides is of great importance for protein glycosylation analysis using mass spectrometry since the signals of glycopeptides could be severely suppressed by the coexisting non-glycosylated peptides in the protein digest. In the present work, a strategy for N-linked glycopeptide enrichment through reversed-phase depletion coupled with hydrophilic affinity enrichment by applying the customized matrix named Click OEG-CD is developed. Compared with single hydrophilic interaction liquid chromatography (HILIC) mode, the strategy exhibited remarkably higher selectivity for N-linked glycopeptides. As many as 22, 18, and eight glycopeptides were detected in the glycopeptide fraction enriched with the strategy from the digests of human immunoglobulin G, horseradish peroxidase and bovine ribonuclease B, respectively. In addition, the strategy also showed high glycosylation microheterogeneity coverage for the enrichment of human α₁-acid glycoprotein glycopeptides. More than 170 glycopeptides covering all the glycosylation sites were detected in the enriched fraction. The revered-phase liquid chromatography depletion coupled with HILIC enrichment strategy by using Click OEG-CD matrix is expected to show more potential in further applications in glycosylation analysis.     

Click novel glycosyl amino acid hydrophilic interaction chromatography stationary phase and its application in enrichment of glycopeptides

A novel glycosyl amino acid hydrophilic interaction chromatography (HILIC) stationary phase was prepared via click chemistry. The key intermediate N₃-glycosyl d-phenylglycine was prepared by a three steps procedure, including selective condensation of amino glucose with N-succinimidyl ester of Boc-d-phenylglycine, deprotection and transformation of amino group to azido group. The structure of all the intermediates and functionalized silica beads were confirmed by ¹H NMR, IR, elemental analysis and ¹³C CP-MAS. The chromatography test showed that this new type of separation material possessed good HILIC properties and glycopeptide enrichment characteristics. Nucleosides and bases could be separated in a simple eluent composition (only acetonitrile in combined with water), and with the same condition, these model compounds could not be separated on the commercial HILIC column (Atlantis). Click glycosyl amino acid thus prepared also showed longer retention and better separation ability in the separation of polar organic acids.     

Optimization of N‐methyl‐N‐[tert‐butyldimethylsilyl]trifluoroacetamide as a derivatization agent for determining isotopic enrichment of glycerol in very‐low density lipoproteins

Stable isotope kinetic studies play an important role in the study of very‐low density lipoprotein (VLDL) metabolism, including basic and clinical research. Today, [1,1,2,3,3‐²H₅]glycerol is the most cost‐effective alternative to measure glycerol and triglyceride kinetics. Recycling of glycerol from glycolysis and gluconeogenesis may lead to incompletely labelled tracer molecules. Many existing methods for the measurement of glycerol isotopic enrichment involve the production of glycerol derivatives that result in fragmentation of the glycerol molecule after ionization. It would be favourable to measure the intact tracer molecule since incompletely labelled tracer molecules may be measured as fully labelled. The number of methods available to measure the intact tracer in biological samples is limited. The aim of this project was to develop a gas chromatography/mass spectrometry (GC/MS) method for glycerol enrichment that measures the intact glycerol backbone and is suitable for electron ionization (EI), which is widely available. A previously published method for N‐methyl‐N‐[tert‐butyldimethylsilyl]trifluoroacetamide (MTBSTFA) derivatization was significantly improved; we produced a stable derivative and increased recovery 27‐fold in standards. We used the optimized MTBSTFA method in VLDL‐triglyceride and found that further modification was required to take matrix effects into account. We now have a robust method to measure glycerol isotopic enrichment by GC/EI‐MS that can be used to rule out the known problem of tracer recycling in studies of VLDL kinetics. Copyright © 2010 John Wiley & Sons, Ltd.