Comparison of HPLC/ESI-FTICR MS versus MALDI-TOF/TOF MS for glycopeptide analysis of a highly glycosylated HIV envelope glycoprotein

Defining the structures and locations of the glycans attached on secreted proteins and virus envelope proteins is important in understanding how glycosylation affects their biological properties. Glycopeptide mass spectrometry (MS)-based analysis is a very powerful, emerging approach to characterize glycoproteins, in which glycosylation sites and the corresponding glycan structures are elucidated in a single MS experiment. However, to date there is not a consensus regarding which mass spectrometric platform provides the best glycosylation coverage information. Herein, we employ two of the most widely used MS approaches, online high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC/ESI-MS) and offline HPLC followed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), to determine which of the two approaches provides the best glycosylation coverage information of a complex glycoprotein, the group M consensus HIV-1 envelope, CON-S gp140DeltaCFI, which has 31 potential glycosylation sites. Our results highlight differences in the informational content obtained between the two methods such as the overall number of glycosylation sites detected, the numbers of N-linked glycans present at each site, and the type of confirmatory information obtained about the glycopeptide using MS/MS experiments. The two approaches are quite complementary, both in their coverage of glycopeptides and in the information they provide in MS/MS experiments. The information in this study contributes to the field of mass spectrometry by demonstrating the strengths and limitations of two widely used MS platforms in glycoprotein analysis.     

基质辅助激光解吸电离质谱和电喷雾电离质谱在辣根过氧化物酶糖肽结构分析中的应用

糖链结构的质谱解析是今后糖蛋白分析中的重要研究内容,其中完整糖肽的分析,由于可以同时获得糖基化位点和对应糖链的结构信息,更具有重要意义和研究前景。本工作对质谱软电离技术在完整糖肽分析中的应用进行了研究,其中包括了基质辅助激光解吸电离(matrix-assisted laser desorption ionization, MALDI)和电喷雾电离(electrospray ionization, ESI)技术。通过平行使用两种串联质谱(tandem mass spectrometry, MS/MS)分析策略: MALDI-MS/MS和ESI-MS/MS对目标糖蛋白——辣根过氧化物酶进行分析,并讨论了其互补性。结果表明,MALDI和ESI技术各有优劣,结合串联质谱分析,可获得糖肽的糖链结构信息;两条路线互补使用,在揭示蛋白质糖基化修饰(位点和结构)的研究中十分必要。     

Site-specific protein glycosylation analysis with glycan isomer differentiation

Glycosylation is one of the most common yet diverse post-translational modifications. Information on glycan heterogeneity and glycosite occupancy is increasingly recognized as crucial to understanding glycoprotein structure and function. Yet, no approach currently exists with which to holistically consider both the proteomic and glycomic aspects of a system. Here, we developed a novel method of comprehensive glycosite profiling using nanoflow liquid chromatography/mass spectrometry (nano-LC/MS) that shows glycan isomer-specific differentiation on specific sites. Glycoproteins were digested by controlled non-specific proteolysis in order to produce informative glycopeptides. High-resolution, isomer-sensitive chromatographic separation of the glycopeptides was achieved using microfluidic chip-based capillaries packed with graphitized carbon. Integrated LC/MS/MS not only confirmed glycopeptide composition but also differentiated glycan and peptide isomers and yielded structural information on both the glycan and peptide moieties. Our analysis identified at least 13 distinct glycans (including isomers) corresponding to five compositions at the single N-glycosylation site on bovine ribonuclease B, 59 distinct glycans at five N-glycosylation sites on bovine lactoferrin, 13 distinct glycans at one N-glycosylation site on four subclasses of human immunoglobulin G, and 20 distinct glycans at five O-glycosylation sites on bovine κ-casein. Porous graphitized carbon provided effective separation of glycopeptide isomers. The integration of nano-LC with MS and MS/MS of non-specifically cleaved glycopeptides allows quantitative, isomer-sensitive, and site-specific glycoprotein analysis.     

Mass spectrometry-based glycoprofiling of biopharmaceuticals by using an automated data processing tool: SimGlycan®

The therapeutic and immunological properties of biopharmaceuticals are governed by the glycoforms contained in them. Thus, bioinformatics tools capable of performing comprehensive characterization of glycans are significantly important to the biopharma industry. The primary structural elucidation of glycans using mass spectrometry is tricky and tedious in terms of spectral interpretation. In this study, the biosimilars of a therapeutic monoclonal antibody and an Fc-fusion protein with moderate and heavy glycosylation, respectively, were employed as representative biopharmaceuticals for released glycan analysis using liquid chromatography–tandem mass spectrometry instead of conventional mass spectrometry-based analysis. SimGlycan® is a software with proven ability to process tandem MS data for released glycans could identify eight additional glycoforms in Fc-fusion protein biosimilar, which were not detected during mass spectrometry analysis of released glycans or glyco-peptide mapping of the same molecule. Thus, liquid chromatography–tandem mass spectrometry analysis of released glycans not only complements conventional liquid chromatography–mass spectrometry-based glycan profiling but can also identify additional glycan structures that may otherwise be omitted during conventional liquid chromatography–tandem mass spectrometry based analysis of mAbs. The mass spectrometry data processing tools, such as PMI Byos™, SimGlycan^®, etc., can display pivotal analytical capabilities in automated liquid chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry-based glycan analysis workflows, especially for high-throughput structural characterization of glycoforms in biopharmaceuticals.     

Glycoform characterization of intact erythropoietin by capillary electrophoresis-electrospray-time of flight-mass spectrometry

Glycosylated proteins often show a large variation in their glycosylation pattern, complicating their structural characterization. In this paper, we present a method for the accurate mass determination of intact isomeric glycoproteins based on capillary electrophoresis-electrospray-time of flight-mass spectrometry. Human recombinant erythropoietin has been chosen as a showcase. The approach enables the on-line removal of nonglycosylated proteins, salts, and neutral and negatively charged species. More important, different glycosylation forms are separated both on the base of differences in the number of negatively charged sialic acid residues and the size of the glycans. Thus, 44 glycoforms and in total about 135 isoforms of recombinant human erythropoietin, taking also acetylation into account, could be distinguished for the reference material from the European Pharmacopeia. Distinct glycosylation differences for samples from different suppliers are clearly observed. Based on the accurate mass an overall composition of each single isoform is proposed, perfectly in agreement with data on glycan and glycopeptide analysis. This method is an ideal complement to the established techniques for glycopeptide and glycan analysis, not differentiating branching or linkage isoforms, but leading to an overall composition of the glycoprotein. The presented strategy is expected to improve significantly the ability to characterize and quantify isomeric glycoforms for a large variety of glycoproteins.     

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.     

Glycoprotein analysis using mass spectrometry: unraveling the layers of complexity

A glycoprotein exists as a heterogeneous mixture of forms due to differential glycosylation, each of which may confer different functionality and/or serve as a biochemical marker for disease. The complex structure of glycans make them a bioanalytical challenge requiring multiple mass spectrometry based approaches to gain different types of information. The following article will briefly describe recently utilized mass spectrometry methods to identify glycosylation sites and measure glycan composition, sequence, branching, and relative quantities. Potential metrological developments are discussed in light of current trends toward complete, reliable glycoanalytical characterization in a high-throughput manner.     

Glycan analysis: scope and limitations of different techniques—a case for integrated use of LC-MS(/MS) and NMR techniques

The structure of glycans from glycoproteins is highly relevant for their function. We tightly integrate liquid chromatography–mass spectrometry (LC-MS), MS/MS, and nuclear magnetic resonance (NMR) data to achieve a complete characterization of even isobaric glycans differing in only one linkage position or in the substitution in one branch. As example, we analyzed ten desialylated underivatized glycans from bovine fibrinogen. The molecules were separated on a PGC column, and LC-MS data allowed an assignment of the compositions of the glycans. MS/MS data of the same glycans allowed elucidation of sequence and to some extent of branching and linkage. All MS/MS fragmentation methods led to multiple dissociations, resulting in several cases in ambiguous data. The MS/MS data were interpreted both by scientists and automatically by software, and the differential results are compared. Additional data from a tight integration of LC-MS and NMR data resulted in a complete structural characterization of the glycans. The acquisition of simple 1D ¹H NMR data led—in combination with LC-MS and MS/MS data—to an unambiguous assignment of the isobaric glycans. Compounds that were not separated in the chromatography could easily be assigned structurally by applying the 3D cross-correlation (3DCC) technology to arrive at NMR spectra of the pure components—without actually separating them. By applying LC-MS, MS/MS, 1D ¹H NMR, and 3DCC together, one can assign glycan structures from glycoconjugates with high confidence affording only 200 pmol of glycan material.     

Glycomics and glycoproteomics: Approaches to address isomeric separation of glycans and glycopeptides

Changes in the glycome of human proteins and cells are associated with the progression of multiple diseases such as Alzheimer's, diabetes mellitus, many types of cancer, and those caused by viruses. Consequently, several studies have shown essential modifications to the isomeric glycan moieties for diseases in different stages. However, the elucidation of extensive isomeric glycan profiles remains challenging because of the lack of analytical techniques with sufficient resolution power to separate all glycan and glycopeptide iso‐forms. Therefore, the development of sensitive and accurate approaches for the characterization of all the isomeric forms of glycans and glycopeptides is essential to tracking the progression of pathology in glycoprotein‐related diseases. This review describes the isomeric separation achievements reported in glycomics and glycoproteomics in the last decade. It focuses on the mass spectrometry–based analytical strategies, stationary phases, and derivatization techniques that have been developed to enhance the separation mechanisms in liquid chromatography systems and the detection capabilities of mass spectrometry systems.     

Analysis of glycans derived from glycoconjugates by capillary electrophoresis-mass spectrometry

The high structural variation of glycan derived from glycoconjugates, which substantially increases with the molecular size of a protein, contributes to the complexity of glycosylation patterns commonly associated with glycoconjugates. In the case of glycoproteins, such variation originates from the multiple glycosylation sites of proteins and the number of glycan structures associated with each site (microheterogeneity). The ability to comprehensively characterize highly complex mixture of glycans has been analytically stimulating and challenging. Although the most powerful MS and MS/MS techniques are capable of providing a wealth of structural information, they are still not able to readily identify isomeric glycan structures without high-order MS/MS (MS(n) ). The analysis of isomeric glycan structures has been attained using several separation methods, including high-pH anion-exchange chromatography, hydrophilic interaction chromatography and GC. However, CE and microfluidics CE (MCE) offer high separation efficiency and resolutions, allowing the separation of closely related glycan structures. Therefore, interfacing CE and MCE to MS is a powerful analytical approach, allowing potentially comprehensive and sensitive analysis of complex glycan samples. This review describes and discusses the utility of different CE and MCE approaches in the structural characterization of glycoproteins and the feasibility of interfacing these approaches to MS.     

Identification of isomeric N-glycan structures by mass spectrometry with 157 nm laser-induced photofragmentation

Characterization of structural isomers has become increasingly important and extremely challenging in glycobiology. This communication demonstrates the capability of ion-trap mass spectrometry in conjunction with 157 nm photofragmentation to identify different structural isomers of permethylated N-glycans derived from ovalbumin without chromatographic separation. The results are compared with collision-induced dissociation (CID) experiments. Photodissociation generates extensive cross-ring fragment ions as well as diagnostic glycosidic product ions that are not usually observed in CID MS/MS experiments. The detection of these product ions aids in characterizing indigenous glycan isomers. The ion trap facilitates MS(n) experiments on the diagnostic glycosidic fragments and cross-ring product ions generated through photofragmentation, thus allowing unambiguous assignment of all of the isomeric structures associated with the model glycoprotein used in this study. Photofragmentation is demonstrated to be a powerful technique for the structural characterization of glycans.     

Resolving and assigning N-linked glycan structural isomers from ovalbumin by IMS-MS

Ion mobility-mass spectrometry (IMS-MS) and molecular modeling techniques have been used to characterize ovalbumin N-linked glycans. Some glycans from this glycoprotein exist as multiple isomeric forms. The gas-phase separation makes it possible to resolve some isomers before MS analysis. Comparisons of experimental cross sections for selected glycan isomers with values that are calculated for iterative structures generated by molecular modeling techniques allow the assignment of sharp features to specific isomers. We focus here on an example glycan set, each having a m/z value of 1046.52 with formula [H₅N₄+2Na]²⁺, where H corresponds to a hexose, and N to a N-acetylglucosamine. This glycan appears to exist as three different isomeric forms that are assignable based on comparisons of measured and calculated cross sections. We estimate the relative ratios of the abundances of the three isomers to be in the range of ∼1.0:1.35:0.85 to ∼1.0:1.5:0.80. In total, IMS-MS analysis of ovalbumin N-linked glycans provides evidence for 19 different glycan structures corresponding to high-mannose and hybrid type carbohydrates with a total of 42 distinct features related to isomers and/or conformers.     

Characterization of Minor N-linked Glycans on Antibodies Using Endo H Release and MALDI–Mass Spectrometry

Abstract

A MALDI mass spectrometry method using Bruker Daltonic's LIFT technology for MS/MS analysis has been developed for profiling and characterizing low abundant N-glycans from recombinant immunoglobulin G (IgG) antibodies. In this method, Endoglycosidase H (Endo H) released N-glycans are derivatized at their reducing end with 2-aminobenzamide (2-AB) and separated by normal phase chromatography. Endo H hydrolyses the bond between the two GlcNAc residues of the trimannosyl core of high mannose and hybrid N-linked glycans, leaving the core GlcNAc attached to the protein. High mannose and hybrid type N-glycans are released from the glycoprotein whereas the more abundant, complex biantennary type oligosaccharide structures are unaffected. Analysis of Endo H treated glycan moieties by MALDI mass spectrometry identified several minor species of high mannose and hybrid type glycans. Subsequent MALDI TOF MS/MS analysis of the resulting products yielded information about structural features of the high mannose and hybrid type glycans. This study involving Endo H treatment followed by MALDI mass spectrometry coupled with LIFT technology for MS/MS analysis offers a specific and sensitive technique for visualizing, and characterizing minor glycan species.     

A rapid sample preparation method for mass spectrometric characterization of N-linked glycans

A rapid method for analysis of glycans of glycoproteins is presented. This method comprised deglycosylation, sample cleanup and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of glycans. The enzymatic deglycosylation of N-linked glycoproteins was enhanced in terms of speed and reproducibility using an enzyme-friendly surfactant. The released glycans were desalted using a micro-scale solid phase extraction (SPE) device packed with a hydrophilic interaction chromatography (HILIC) sorbent. Hydrophilic glycans were well retained by SPE, while salts and surfactants were removed from the sample. The glycans were eluted using 25-50 microL of solvent and analyzed directly without derivatization using MALDI-MS. MALDI quadrupole time-of-flight (Q-Tof) instrumentation was utilized for glycan profiling and structure characterization by tandem mass spectrometry (MS/MS). The presented method allows sensitive analysis of glycans benefiting from optimized deglycosylation reactions and efficient sample cleanup.     

Hydrophilic interaction 10 microm I.D. porous layer open tubular columns for ultratrace glycan analysis by liquid chromatography-mass spectrometry

The sensitivity of glycan analysis using nano-liquid chromatography interfaced with electrospray ionization mass spectrometry (ESI-MS) increases with the decrease of the mobile phase flow rate, accompanied by reduced ion suppression. In this study, we describe the preparation and performance of high efficiency 10 microm I.D. amine-bonded poly(vinylbenzyl chloride-divinylbenzene) hydrophilic interaction (HILIC) porous layer open tubular (PLOT) columns operated at 20 nL/min for the separation and analysis of glycan mixtures. HILIC-PLOT columns with a uniform porous polymer layer were reproducibly prepared ( approximately 4% RSD in retention time from column-to-column) via in situ polymerization, followed by one step modification with ethylenediamine. When coupled on-line with negative ESI-MS, low detection limits (0.3fmol) for a 3-sialyl-tetrasaccharide were achieved using a 2.5mx10 microm I.D. HILIC-PLOT column. A dextran ladder standard was used to evaluate the performance of the column, and high efficiency separation was achieved with detection of the dextrans up to G22 from approximately 50 fmol amounts injected. As an example of the high sensitivity of the column, MS(6) characterization of glycan structures was possible from the injection of 10 fmol of a neutral and sialylated glycan. As another example of high sensitivity LC-MS analysis of 3 ng of a PNGase F digest of ovalbumin allowed 28 N-linked glycans to be confidently identified from a single analysis. High quality MS/MS spectra for each ovalbumin glycan were acquired and manually interpreted for structure analysis. The HILIC-PLOT column is a very promising approach for LC-MS analysis of glycans at the ultratrace level.     

Sequential enrichment of sulfated glycans by strong anion-exchange chromatography prior to mass spectrometric measurements

Structural characterization of sulfated glycans through mass spectrometry (MS) has been often limited by their low abundance in biological materials and inefficient ionization in the positive-ion mode. Here, we describe a microscale method for sequentially enriching sulfated glycans according to their degree of sulfation. This method is based on modifying the binding ability of strong anion-exchange material through the use of different sodium acetate concentrations, thus enabling fairly selective binding and a subsequent elution of different glycans according to their degree of sulfation. Before this enrichment, the negative charge on the sialic acid, which is commonly associated with such glycans, was eliminated through permethylation that is used to enhance the positive-ion mode matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-MS) signal for all glycans. This enrichment approach minimizes competitive ionization between sulfated and neutral glycans, as well as that between sulfated species with a different degree of sulfation. The described method was initially optimized using sulfated oligosaccharide standards, while its potential has been verified for the sulfated N-glycans originated from the bovine thyroid-stimulating hormone (bTSH), a glycoprotein possessing mono- and disulfated N-glycans. This enhancement of the MALDI-MS signal facilitates analysis of some otherwise undetected components.     

Rapid and efficient glycoprotein identification through microwave-assisted enzymatic digestion

Identification of protein glycosylation sites is analytically challenging due to the diverse glycan structures associated with a glycoprotein. Mass spectrometry (MS)-based identification and characterization of glycoproteins has been achieved predominantly with the bottom-up approach, which typically involves the enzymatic cleavage of proteins to peptides prior to LC/MS or LC/MS/MS analysis. However, the process can be challenging due to the structural variations and steric hindrance imposed by the attached glycans. Alternatives to conventional heating protocols, that increase the rate of enzymatic cleavage of glycoproteins, may aid in addressing these challenges. An enzymatic digestion of a glycoprotein can be accelerated and made more efficient through microwave-assisted digestion. In this paper, a systematic study was conducted to explore the efficiency of microwave-assisted enzymatic (trypsin) digestion (MAED) of glycoproteins as compared with the conventional method. In addition, the optimum experimental parameters for the digestion such as temperature, reaction time, and microwave radiation power were investigated. It was determined that efficient tryptic digestion of glycoproteins was attained in 15 min, allowing comparable if not better sequence coverage through LC/MS/MS analysis. Optimum tryptic cleavage was achieved at 45°C irrespective of the size and complexity of the glycoprotein. Moreover, MAED allowed the detection and identification of more peptides and subsequently higher sequence coverage for all model glycoprotein. MAED also did not appear to prompt a loss or partial cleavage of the glycan moieties attached to the peptide backbones.     

LC-MS/MS Peptide Mapping with Automated Data Processing for Routine Profiling of N-Glycans in Immunoglobulins

Protein N-Glycan analysis is traditionally performed by high pH anion exchange chromatography (HPAEC), reversed phase liquid chromatography (RPLC), or hydrophilic interaction liquid chromatography (HILIC) on fluorescence-labeled glycans enzymatically released from the glycoprotein. These methods require time-consuming sample preparations and do not provide site-specific glycosylation information. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) peptide mapping is frequently used for protein structural characterization and, as a bonus, can potentially provide glycan profile on each individual glycosylation site. In this work, a recently developed glycopeptide fragmentation model was used for automated identification, based on their MS/MS, of N-glycopeptides from proteolytic digestion of monoclonal antibodies (mAbs). Experimental conditions were optimized to achieve accurate profiling of glycoforms. Glycan profiles obtained from LC-MS/MS peptide mapping were compared with those obtained from HPAEC, RPLC, and HILIC analyses of released glycans for several mAb molecules. Accuracy, reproducibility, and linearity of the LC-MS/MS peptide mapping method for glycan profiling were evaluated. The LC-MS/MS peptide mapping method with fully automated data analysis requires less sample preparation, provides site-specific information, and may serve as an alternative method for routine profiling of N-glycans on immunoglobulins as well as other glycoproteins with simple N-glycans.

Structural analysis of sulfated glycans by sequential double-permethylation using methyl iodide and deuteromethyl iodide

MALDI mass spectrometric characterization of sulfated glycans is often challenging due to their low ionization response in the positive ion mode. Here we demonstrate a new analytical approach, allowing the measurement of sulfated glycans by substituting the sulfate group with a deuteromethyl group. Sulfated glycan samples are initially permethylated before the methanolytic cleavage of their sulfate groups. Desulfated and permethylated glycans are then subjected to another permethylation step using deuteromethyl iodide to label the hydroxyl groups resulting from methanolysis. The number of attached sulfate groups is subsequently calculated from the mass-shift resulting from the chemical cleavage of these sulfate groups. The position of the sulfate substitution is then determined by collision-induced dissociation (CID) tandem mass spectrometry of permethylated and permethylated plus deuteromethylated samples. The described approach was initially optimized and validated using linear standard glycans, while its effectiveness has also been demonstrated here for the N-glycans derived from bovine thyroid-stimulating hormone (bTSH).     

Correlating Glycoforms of DC‐SIGN with Stability Using a Combination of Enzymatic Digestion and Ion Mobility Mass Spectrometry

The immune scavenger protein DC‐SIGN interacts with glycosylated proteins and has a putative role in facilitating viral infection. How these recognition events take place with different viruses is not clear and the effects of glycosylation on the folding and stability of DC‐SIGN have not been reported. Herein, we report the development and application of a mass‐spectrometry‐based approach to both uncover and characterise the effects of O‐glycans on the stability of DC‐SIGN. We first quantify the Core 1 and 2 O‐glycan structures on the carbohydrate recognition and extracellular domains of the protein using sequential exoglycosidase sequencing. Using ion mobility mass spectrometry, we show how specific O‐glycans, and/or single monosaccharide substitutions, alter both the overall collision cross section and the gas‐phase stability of the DC‐SIGN isoforms. We find that rather than the mass or length of glycoprotein modifications, the stability of DC‐SIGN is better correlated with the number of glycosylation sites.