基于固相萃取的微量糖蛋白糖基化修饰表征技术的建立

结合自制亲水固相萃取富集柱和生物质谱鉴定技术,实现了糖基化蛋白质核糖核酸酶B的糖含量测定、糖基化位点确认、聚糖富集及结构表征,以及不同糖型相对丰度分析。结果表明:其糖含量8.47%,糖基化位点为34位的Asn,糖链主要为5种高甘露糖型结构(Man5-9GlcNAc2)。所建立的HILIC富集技术,有利于针对微量生物样本,如生物工程药物糖蛋白及重要功能糖蛋白,开展位点特异性糖链结构解析,为糖蛋白质的药效或功能研究提供线索。     

Mass spectrometry characterization for chemoenzymatic glycoprotein synthesis

The current project describes the chemoenzymatic modification of bovine ribonuclease B (RNase B) to contain a single glycosylation site with a known glycan. A reactive disaccharide oxazoline derivative was synthesized and stereospecifically added to deglycosylated RNase B through endo-β-N-acetylglucosaminidase M catalyzed chemoenzymatic transglycosylation. Oxazoline formation conditions were optimized using mass spectrometry, and the product verified based on its collision-induced dissociation (CID) mass spectrum. Enzymatic removal of native glycans as well as formation of the desired homogeneous product was also monitored using mass spectrometry. LC-MS(n) using four sequential rounds of CID was used to verify that the original glycosylation site had been reorganized to contain the new glycan. The techniques described herein are not limited to this analyte or glycan and should be amenable to the synthesis of numerous homogeneous glycoconjugates with judicious choice of enzyme/substrate combinations. The combined use of chemoenzymatic synthesis and mass spectrometry-based characterization shows promise for the development of homogeneous glycoprotein reference materials. A well-defined glycoprotein standard containing a single glycan of known composition, linkage and stereochemistry would be of great value for the comparison and evaluation of glycoprotein analysis techniques.     

糖蛋白结构质谱解析的样品前处理

基于高分辨率、高精度、高灵敏度及良好表现力的多级质谱,以标准糖蛋白辣根过     

伴刀豆球蛋白亲和色谱柱的制备及其在糖蛋白核糖核酸酶B结构分析中的应用

通过对硅胶基质进行化学改性键合伴刀豆球蛋白(Con A),制备了对糖蛋白具有特异亲和作用的亲和色谱固定相;该固定相非特异性吸附弱,对于糖蛋白和糖肽的分离效果良好。对亲和色谱的分离条件进行了优化,以标准糖蛋白核糖核酸酶B(RNase B)为模型,对其进行了纯化;用糖苷酶切除糖链,并对切除糖链前后的RNase B用胰蛋白酶酶解;用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)对亲和色谱分离得到的糖蛋白、糖链及糖肽进行了分析,确定了RNase B的一级结构、糖含量、糖基化位点及糖连接方式。该方法快速准确,适于糖蛋白和糖肽的分离表征。将其应用于血清中糖蛋白及酶解后血清中糖肽的分离富集,取得了很好的效果。     

Studying the Glycan Moiety of RNase B by Means of Raman and Raman Optical Activity

Raman and Raman optical activity (ROA) spectroscopy are used to study the solution‐phase structure of the glycan moiety of the protein ribonuclease B (RNase B). Spectral data of the intact glycan moiety of RNase B is obtained by subtracting high‐quality spectral data of RNase A, the non‐glycosylated form of the RNase, from the spectra of the glycoprotein. The remaining difference spectra are compared to spectra generated from Raman and ROA data of the constituent disaccharides of the RNase glycan, achieving convincing spectral overlap. The results show that ROA spectroscopy is able to extract detailed spectral data of the glycan moieties of proteins, provided that the non‐glycosylated isoform is available. Furthermore, good comparison between the full glycan spectrum and the regenerated spectra based on the disaccharide data lends great promise to ROA as a tool for the solution‐phase structural analysis of this structurally elusive class of biomolecules.     

Absolute Quantitation of Glycosylation Site Occupancy Using Isotopically Labeled Standards and LC-MS

N-linked glycans are required to maintain appropriate biological functions on proteins. Underglycosylation leads to many diseases in plants and animals; therefore, characterizing the extent of glycosylation on proteins is an important step in understanding, diagnosing, and treating diseases. To determine the glycosylation site occupancy, protein N-glycosidase F (PNGase F) is typically used to detach the glycan from the protein, during which the formerly glycosylated asparagine undergoes deamidation to become an aspartic acid. By comparing the abundance of the resulting peptide containing aspartic acid against the one containing non-glycosylated asparagine, the glycosylation site occupancy can be evaluated. However, this approach can give inaccurate results when spontaneous chemical deamidation of the non-glycosylated asparagine occurs. To overcome this limitation, we developed a new method to measure the glycosylation site occupancy that does not rely on converting glycosylated peptides to their deglycosylated forms. Specifically, the overall protein concentration and the non-glycosylated portion of the protein are quantified simultaneously by using heavy isotope-labeled internal standards coupled with LC-MS analysis, and the extent of site occupancy is accurately determined. The efficacy of the method was demonstrated by quantifying the occupancy of a glycosylation site on bovine fetuin. The developed method is the first work that measures the glycosylation site occupancy without using PNGase F, and it can be done in parallel with glycopeptide analysis because the glycan remains intact throughout the workflow.

Using optimized collision energies and high resolution,high accuracy fragment ion selection to improve glycopeptide detection by precursor ion scanning

Glycosylation is the most widespread protein modification and is known to modulate signal transduction and several biologically important interactions. In order to understand and evaluate the biological role of glycosylation it is important to identify the glycosylated protein and localize the site glycosylation under particular biological conditions. To identify glycosylated peptides from simple mixtures, i.e., in-gel digests from single SDS PAGE bands we performed high resolution, high accuracy precursor ion scanning using a quadrupole TOF instrument equipped with the Q(2) pulsing function. The high resolving power of the quadrupole TOF instrument results in the selective detection of glycan specific fragment ions minimizing the interference of peptide derived fragment ions with the same nominal mass. Precursor ion scanning has been previously described for these glycan derived ions. However the use of this method has been limited by the low specificity of the method. The analysis using precursor ion scanning can be applied to any peptide mixture from a protein digest without having previous knowledge of the glycosylation of the protein. In addition to the low femtomole (nanomolar) detection limits, this method has the advantage that no prior derivatization or enzymatic treatment of the peptide mixtures is required.     

Improvement of mass spectrometry analysis of glycoproteins by MALDI-MS using 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid

Protein glycosylation analysis is important for elucidating protein function and molecular mechanisms in various biological processes. We previously developed a glycan analysis method using a 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid liquid matrix (3-AQ/CHCA LM) and applied it to the quantitative glycan profiling of glycoproteins. However, information concerning glycosylation sites is lost; glycopeptide analysis is therefore required to identify the glycosylation sites in glycoproteins. Human epidermal growth factor receptor 2 (HER2) is a glycoprotein that plays a role in the regulation of cell proliferation, differentiation, and migration. Several reports have described the structure of HER2, but the structures of N-glycans attached to this protein remain to be fully elucidated. In this study, 3-AQ/CHCA LM was applied to tryptic digests of HER2 to reveal its N-glycosylation state and to evaluate the utility of this LM in characterizing glycopeptides. Peptide sequence coverage was considerably improved compared to analysis of HER2 using α-cyano-4-hydroxycinnamic acid or 2,5-dihydroxybenzoic acid. Most of the peaks observed using only this LM were localized at the inner or outer regions of sample spots. Furthermore, five of the peptide peaks that were enriched within the inner region were confirmed to be glycosylated by MS/MS analysis. Three glycosylation sites were identified and their glycan structures were elucidated. The reduction in sample complexity by on-target separation allowed for higher sequence coverage, resulting in effective detection and characterization of glycopeptides. In conclusion, these results demonstrate that MS-based glycoprotein analysis using 3-AQ/CHCA is an effective method to identify glycosylation sites in proteins and to elucidate the glycan structures of glycoproteins in complex samples.     

Characterization of the N-linked glycosylation site of recombinant pectate lyase

Recombinant pectate lyase from Aspergillus niger was overexpressed in Aspergillus nidulans. The two recombinant proteins produced differed in molecular mass by 1200 Da, which suggested that the larger molecular weight protein was glycosylated. The deduced amino acid sequence was searched for potential N-linked glycosylation sites, and one potential site was identified at residue 64. The proteins were analyzed for their ability to bind various lectins as an assay for the presence of carbohydrates. The proteins were then digested with trypsin to facilitate the isolation of the potential glycosylation site. The resulting digestion products were subsequently analyzed by liquid chromatography/mass spectrometry using in-source collision induced dissociation to detect glycopeptides. Once the glycopeptide had been identified, treatment with an endoglycosidase both verified the location of glycosylation and identified the mass of the glycan. The Complex Carbohydrate Structural Database was searched for possible N-linked structures with the same mass, and the suggested primary sequence was confirmed by an exoglycosidase digestion. The data demonstrated that the larger recombinant protein contained a high mannose N-linked structure (Man(5)GlcNAc(2)) attached to N-64, while this site was not occupied in the smaller protein.     

Convergent synthesis of homogeneous Glc1Man9GlcNAc2-protein and derivatives as ligands of molecular chaperones in protein quality control

A detailed understanding of the molecular mechanism of chaperone-assisted protein quality control is often hampered by the lack of well-defined homogeneous glycoprotein probes. We describe here a highly convergent chemoenzymatic synthesis of the monoglucosylated glycoforms of bovine ribonuclease (RNase) as specific ligands of lectin-like chaperones calnexin (CNX) and calreticulin (CRT) that are known to recognize the monoglucosylated high-mannose oligosaccharide component of glycoproteins in protein folding. The synthesis of a selectively modified glycoform Gal(1)Glc(1)Man(9)GlcNAc(2)-RNase was accomplished by chemical synthesis of a large N-glycan oxazoline and its subsequent enzymatic ligation to GlcNAc-RNase under the catalysis of a glycosynthase. Selective removal of the terminal galactose by a β-galactosidase gave the Glc(1)Man(9)GlcNAc(2)-RNase glycoform in excellent yield. CD spectroscopic analysis and RNA-hydrolyzing assay indicated that the synthetic RNase glycoforms maintained essentially the same global conformations and were fully active as the natural bovine ribonuclease B. SPR binding studies revealed that the Glc(1)Man(9)GlcNAc(2)-RNase had high affinity to lectin CRT, while the synthetic Man(9)GlcNAc(2)-RNase glycoform and natural RNase B did not show CRT-binding activity. These results confirmed the essential role of the glucose moiety in the chaperone molecular recognition. Interestingly, the galactose-masked glycoform Gal(1)Glc(1)Man(9)GlcNAc(2)-RNase also showed significant affinity to lectin CRT, suggesting that a galactose β-1,4-linked to the key glucose moiety does not significantly block the lectin binding. These synthetic homogeneous glycoprotein probes should be valuable for a detailed mechanistic study on how molecular chaperones work in concert to distinguish between misfolded and folded glycoproteins in the protein quality control cycle.     

The glycan patterns at the individual glycosylation sites in orosomucoid from allergic reaction patients

Summary Little is known about the alterations that have occurred at the individual glycosylation sites in allergy patients or how these glycosylation patterns may change after anti-allergy treatments. Using reverse-phase HPLC, we have quantitated the glycoforms present at the individual glycosylation sites on orosomucoid isolated from the sera of allergic reaction patients and an allergic reaction patient treated with the antihistamine Terfenadine. The glycan structures isolated from the five glycosylation sites for the individual taking Terfenadine were all within normal ranges. It is suggested that if the changes in glycosylation in OMD in the allergic state are functionally driven, then it should be possible to correlate biological activities with quantitative changes at the individual glycosylation sites, and hence further define the role of OMD in allergy and inflammation.     

Enzymatic removal of N‐glycans by PNGase F coated magnetic microparticles

Investigation of protein glycosylation is an important area in biomarker discovery and biopharmaceutical research. Alterations in protein N‐glycosylation can be an indication of changes in pathological conditions in the medical field or production parameters of biotherapeutics. Rapid development of these disciplines calls for fast, high‐throughput, and reproducible methods to analyze protein N‐glycosylation. Currently used methods require either long deglycosylation times or large excess of enzymes. In this paper, we report on the use of PNGase F immobilization onto the surface of magnetic microparticles and their use in rapid and efficient removal of N‐glycans from glycoproteins. The use of immobilized PNGase F also allowed reusability of the enzyme‐coated beads as the magnetic microparticles can be readily partitioned from the sample by a magnet after each deglycosylation reaction. The efficiency and activity of the PNGase F coated magnetic beads was compared with in‐solution enzyme reactions using standard glycoproteins possessing the major N‐glycan types of neutral, high mannose, and highly sialylated carbohydrates. The PNGase F coated magnetic beads offered comparable deglycosylation level to the conventional in‐solution based method in 10‐min reaction times for the model glycoproteins of immunoglobulin G (mostly neutral carbohydrates), ribonuclease B (high mannose type sugars), and fetuin (highly sialylated oligosaccharides) with the special features of easy removal of the enzyme from the reaction mixture and reusability.     

HILIC analysis of fluorescence-labeled N-glycans from recombinant biopharmaceuticals

In contrast with conventional drugs, biopharmaceuticals are highly complex molecules with remarkable heterogeneity. Protein glycosylation is an inherent source of this heterogeneity and also affects the safety, efficacy, and serum half-life of therapeutic glycoproteins. Therefore analysis of the glycan pattern is an important issue for characterization and quality control in the biopharmaceutical industry. In this publication we describe a complete workflow for the analysis of protein N-glycans. The sample-preparation procedure, consisting of the release of the N-glycans by PNGase-F, followed by fluorescence labeling with 2-aminobenzamide and removal of excess label, was optimized to avoid alteration of the glycan sample. Subsequently, labeled glycans were analyzed by hydrophilic-interaction liquid chromatography (HILIC) with fluorescence detection. The developed method was validated for analysis of antibody N-glycans. To demonstrate the accuracy of the method an antibody sample was additionally analyzed by an orthogonal method. The antibody was digested with lysyl endopeptidase and the (glyco-)peptides were analyzed by RP-HPLC–MS. The consistency of the results between these two methods demonstrates the reliability of the glycan analysis method introduced herein.     

Quantitative analysis of N-glycans from human alfa-acid-glycoprotein using stable isotope labeling and zwitterionic hydrophilic interaction capillary liquid chromatography electrospray mass spectrometry as tool for pancreatic disease diagnosis

In this work we demonstrate the potential of glycan reductive isotope labeling (GRIL) using [¹²C]- and [¹³C]-coded aniline and zwitterionic hydrophilic interaction capillary liquid chromatography electrospray mass spectrometry (μZIC-HILIC-ESI-MS) for relative quantitation of glycosylation variants in selected glycoproteins present in samples from cancer patients. Human α₁-acid-glycoprotein (hAGP) is an acute phase serum glycoprotein whose glycosylation has been described to be altered in cancer and chronic inflammation. However, it is not clear yet whether some particular glycans in hAGP can be used as biomarker for differentiating between these two pathologies. In this work, hAGP was isolated by immunoaffinity chromatography (IAC) from serum samples of healthy individuals and from those suffering chronic pancreatitis and different stages of pancreatic cancer, respectively. After de-N-glycosylation, relative quantitation of the hAGP glycans was carried out using stable isotope labeling and μZIC-HILIC-ESI-MS analysis. First, protein denaturing conditions prior to PNGase F digestion were optimized to achieve quantitative digestion yields, and the reproducibility of the established methodology was evaluated with standard hAGP. Then, the proposed method was applied to the analysis of the clinical samples (control vs. pathological). Pancreatic cancer samples clearly showed an increase in the abundance of fucosylated glycans as the stage of the disease increases and this was unlike to samples from chronic pancreatitis. The results gained here indicate the mentioned glycan in hAGP as a candidate structure worth to be corroborated by an extended study including more clinical cases; especially those with chronic pancreatitis and initial stages of pancreatic cancer. Importantly, the results demonstrate that the presented methodology combining an enrichment of a target protein by IAC with isotope coded relative quantitation of N-glycans can be successfully used for targeted glycomics studies. The methodology is assumed being suitable as well for other such studies aimed at finding novel cancer associated glycoprotein biomarkers.     

N-glycosylation proteome of endoplasmic reticulum in mouse liver by ConA affinity chromatography coupled with LTQ-FT mass spectrometry

Glycosylation is the most versatile and one of the most significant protein post-translational modifications. It is generally classified into three categories according to the amino acid to which the glycan is attached: N-glycosylation, O-glycosylation and C-glycosylation. Synthesis of N-glycoproteins occurs in the rough endoplasmic reticulum (rER), and all N-glycoproteins synthesized in rER have uniform glycan endings with mannose (Man) and glucose (Glc). A systematic strategy was developed to comprehensiv...     

Glycopeptide analysis by mass spectrometry

Glycosylation is one of the most important post-translational modifications found in nature. Identifying and characterizing glycans is an important step in correlating glycosylation structure to the glycan's function, both in normal glycoproteins and those that are modified in a disease state. Glycans on a protein can be characterized by a variety of methods. This review focuses on the mass spectral analysis of glycopeptides, after subjecting the glycoprotein to proteolysis. This analytical approach is useful in characterizing glycan heterogeneity and correlating glycan compositions to their attachment sites on the protein. The information obtained from this approach can serve as the foundation for understanding how glycan compositions affect protein function, in both normal and aberrant glycoproteins.     

Visualization of Protein‐Specific Glycosylation inside Living Cells

Protein glycosylation is a ubiquitous post‐translational modification that is involved in the regulation of many aspects of protein function. In order to uncover the biological roles of this modification, imaging the glycosylation state of specific proteins within living cells would be of fundamental importance. To date, however, this has not been achieved. Herein, we demonstrate protein‐specific detection of the glycosylation of the intracellular proteins OGT, Foxo1, p53, and Akt1 in living cells. Our generally applicable approach relies on Diels–Alder chemistry to fluorescently label intracellular carbohydrates through metabolic engineering. The target proteins are tagged with enhanced green fluorescent protein (EGFP). Förster resonance energy transfer (FRET) between the EGFP and the glycan‐anchored fluorophore is detected with high contrast even in presence of a large excess of acceptor fluorophores by fluorescence lifetime imaging microscopy (FLIM).     

Aptamers targeting protein-specific glycosylation in tumor biomarkers: general selection,characterization and structural modeling

Detecting specific protein glycoforms is attracting particular attention due to its potential to improve the performance of current cancer biomarkers. Although natural receptors such as lectins and antibodies have served as powerful tools for the detection of protein-bound glycans, the development of effective receptors able to integrate in the recognition both the glycan and peptide moieties is still challenging. Here we report a method for selecting aptamers toward the glycosylation site of a protein. It allows identification of an aptamer that binds with nM affinity to prostate-specific antigen, discriminating it from proteins with a similar glycosylation pattern. We also computationally predict the structure of the selected aptamer and characterize its complex with the glycoprotein by docking and molecular dynamics calculations, further supporting the binary recognition event. This study opens a new route for the identification of aptamers for the binary recognition of glycoproteins, useful for diagnostic and therapeutic applications.

Binary recognition of the glycoprotein prostate specific antigen by aptamers: a tool for detecting aberrant glycosylation associated with cancer.     

Glycan Reader: automated sugar identification and simulation preparation for carbohydrates and glycoproteins

Understanding how glycosylation affects protein structure, dynamics, and function is an emerging and challenging problem in biology. As a first step toward glycan modeling in the context of structural glycobiology, we have developed Glycan Reader and integrated it into the CHARMM-GUI, http://www.charmm-gui.org/input/glycan. Glycan Reader greatly simplifies the reading of PDB structure files containing glycans through (i) detection of carbohydrate molecules, (ii) automatic annotation of carbohydrates based on their three-dimensional structures, (iii) recognition of glycosidic linkages between carbohydrates as well as N-/O-glycosidic linkages to proteins, and (iv) generation of inputs for the biomolecular simulation program CHARMM with the proper glycosidic linkage setup. In addition, Glycan Reader is linked to other functional modules in CHARMM-GUI, allowing users to easily generate carbohydrate or glycoprotein molecular simulation systems in solution or membrane environments and visualize the electrostatic potential on glycoprotein surfaces. These tools are useful for studying the impact of glycosylation on protein structure and dynamics.     

Glycosylation Enhances Peptide Hydrophobic Collapse by Impairing Solvation

Post‐translational N‐glycosylation of proteins is ubiquitous in eukaryotic cells, and has been shown to influence the thermodynamics of protein collapse and folding. However, the mechanism for this influence is not well understood. All‐atom molecular dynamics simulations are carried out to study the collapse of a peptide linked to a single N‐glycan. The glycan is shown to perturb the local water hydrogen‐bonding network, rendering it less able to solvate the peptide and thus enhancing the hydrophobic contribution to the free energy of collapse. The enhancement of the hydrophobic collapse compensates for the weakened entropic coiling due to the bulky glycan chain and leads to a stronger burial of hydrophobic surface, presumably enhancing folding. This conclusion is reinforced by comparison with coarse‐grained simulations, which contain no explicit solvent and correspondingly exhibit no significant thermodynamic changes on glycosylation.