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.

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.

Change of fucosylated IgG2 Fc-glycoforms in pancreatitis and pancreatic adenocarcinoma: a promising disease-classification model

The fixed constant (Fc) region of IgG is subject to changes in glycosylation state in response to diseases. On the basis of sera from 75 healthy controls, 75 pancreatitis (PT) patients, and 75 pancreatic adenocarcinoma (PAC) patients, we analyzed six fucosylated glycoforms of IgG₂ (G0F, G1F, G2F, G0FN, G1FN, and G2FN), by matrix-assisted laser desorption/ionization–Fourier-transform ion cyclotron resonance mass spectrometry (MALDI–FTICR MS), to evaluate their use as biomarkers for pancreatic diseases. Compared with healthy controls, significant increases in agalactosylated glycoforms and decreases in galactosylated glycoforms were observed for PT and PAC patients. Logistic regression analysis suggested that truncation of the sugar chain was prone to occur in PT and, especially, PAC patients. After participants were stratified by sex and age, receiver operating characteristic curve analysis revealed good overall sensitivity and specificity for discrimination of PAC and PT patients from healthy controls. A combination of G0F and galactosylation also had acceptable power for differentiating PAC patients from PT patients.

Analysis of native and APTS-labeled N-glycans by capillary electrophoresis/time-of-flight mass spectrometry

The glycosylation of proteins is of particular interest in biopharmaceutical applications. The detailed characterization of glycosylation based on the released carbohydrates is mandatory since the protein stability, folding, and efficacy are strongly dependent on the structural diversity inherent in the glycan moieties of a glycoprotein. For glycan pattern analysis, capillary electrophoresis with laser-induced fluorescence using 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-labeled glycans is used frequently. In this paper, a robust capillary electrophoresis–mass spectroscopy method both for the analysis of APTS-labeled glycans and unlabeled charged glycans is presented. The background electrolyte consists of 0.7 M ammonia and 0.1 M ε-aminocaproic acid in water/methanol 30:70 (v/v). High separation efficiency including separation of structural isomers was obtained. The method was validated in terms of reproducibility and linearity. Submicromolar sensitivity is achieved with linearity up to 24 μM. The ability to analyze APTS-labeled, as well as unlabeled, charged glycans enables the determination of labeling and ionization efficiency: APTS-labeled glycans show a factor of three better ionization efficiency compared to non-labeled native glycans. The presented method is applied to the analysis of pharmaceutical products. Furthermore, the system can be applied to the analysis of 2-ANSA-labeled glycans, though separation efficiency is limited.

Rapid Screening for Potential Epitopes Reactive with a Polycolonal Antibody by Solution-Phase H/D Exchange Monitored by FT-ICR Mass Spectrometry

The potential epitopes of a recombinant food allergen protein, cashew Ana o 2, reactive to polyclonal antibodies, were mapped by solution-phase amide backbone H/D exchange (HDX) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Ana o 2 polyclonal antibodies were purified in the serum from a goat immunized with cashew nut extract. Antibodies were incubated with recombinant Ana o 2 (rAna o 2) to form antigen:polyclonal antibody (Ag:pAb) complexes. Complexed and uncomplexed (free) rAna o 2 were then subjected to HDX-MS analysis. Four regions protected from H/D exchange upon pAb binding are identified as potential epitopes and mapped onto a homologous model.

Individuality Normalization when Labeling with Isotopic Glycan Hydrazide Tags (INLIGHT): A Novel Glycan-Relative Quantification Strategy

The Individuality Normalization when Labeling with Isotopic Glycan Hydrazide Tags (INLIGHT) strategy for the sample preparation, data analysis, and relative quantification of N-linked glycans is presented. Glycans are derivatized with either natural (L) or stable-isotope labeled (H) hydrazide reagents and analyzed using reversed phase liquid chromatography coupled online to a Q Exactive mass spectrometer. A simple glycan ladder, maltodextrin, is first used to demonstrate the relative quantification strategy in samples with negligible analytical and biological variability. It is shown that after a molecular weight correction attributable to isotopic overlap and a post-acquisition normalization of the data to account for any systematic bias, a plot of the experimental H:L ratio versus the calculated H:L ratio exhibits a correlation of unity for maltodextrin samples mixed in different ratios. We also demonstrate that the INLIGHT approach can quantify species over four orders of magnitude in ion abundance. The INLIGHT strategy is further demonstrated in pooled human plasma, where it is shown that the post-acquisition normalization is more effective than using a single spiked-in internal standard. Finally, changes in glycosylation are able to be detected in complex biological matrices, when spiked with a glycoprotein. The ability to spike in a glycoprotein and detect change at the glycan level validates both the sample preparation and data analysis strategy, making INLIGHT an invaluable relative quantification strategy for the field of glycomics.

Identification of 34 N-glycan isomers in human serum by capillary electrophoresis coupled with laser-induced fluorescence allows improving glycan biomarker discovery

Alterations in glycosylation have been observed in many human diseases and specific changes in glycosylation have been proposed as relevant diagnostic information. Capillary electrophoresis coupled with laser-induced fluorescence (CE-LIF) is a robust method to quantify desialylated N-glycans that are labeled with 8-aminopyrene-1,3,6-trisulfonic acid prior to analysis. To date, only a maximum of 12 glycan structures, the most abundant ones, have been identified by CE-LIF to characterize glycome modulations of total serum in the course of the diseases. In most forms of cancer, findings using CE-LIF were limited to the increase of triantennary structures carrying a Lewis^x epitope. In this work, we identified 32 linkage and positional glycan isomers in healthy human serum using exoglycosidase digestions as well as standard glycoproteins, for which we report the assignment of novel structures. It was possible to identify and quantify 34 glycan isomers in the serum of primary epithelial ovarian cancer patients (EOC). Reduced levels of diantennary structures and of high-mannose 5 were statistically significant in the EOC samples, and also, elevated branching as well as increased antennary fucosylation were observed. For the first time, we could demonstrate that not only antennary fucosylation was of relevance in tetraantennary structures but also core-fucosylated tetraantennary N-glycans were statistically increased in EOC patients. The results of the current study provide an improved dataset to be used in glycan biomarker discovery. Graphical abstract

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Reliable Determination of Site-Specific In Vivo Protein N-Glycosylation Based on Collision-Induced MS/MS and Chromatographic Retention Time

Site-specific glycopeptide mapping for simultaneous glycan and peptide characterization by MS is difficult because of the heterogeneity and diversity of glycosylation in proteins and the lack of complete fragmentation information for either peptides or glycans with current fragmentation technologies. Indeed, multiple peptide and glycan combinations can readily match the same mass of glycopeptides even with mass errors less than 5 ppm providing considerably ambiguity and analysis of complex mixtures of glycopeptides becomes quite challenging in the case of large proteins. Here we report a novel strategy to reliably determine site-specific N-glycosylation mapping by combining collision-induced dissociation (CID)-only fragmentation with chromatographic retention times of glycopeptides. This approach leverages an experimental pipeline with parallel analysis of glyco- and deglycopeptides. As the test case we chose ABCA4, a large integral membrane protein with 16 predicted sites for N-glycosylation. Taking advantage of CID features such as high scan speed and high intensity of fragment ions together combined with the retention times of glycopeptides to conclusively identify the non-glycolytic peptide from which the glycopeptide was derived, we obtained virtually complete information about glycan compositions and peptide sequences, as well as the N-glycosylation site occupancy and relative abundances of each glycoform at specific sites for ABCA4. The challenges provided by this example provide guidance in analyzing complex relatively pure glycoproteins and potentially even more complex glycoprotein mixtures.

Determination of the molecular weight of poly(ethylene glycol) in biological samples by reversed-phase LC–MS with in-source fragmentation

PEGylation has been widely used to improve the biopharmaceutical properties of therapeutic proteins and peptides. Previous studies have used multiple analytical techniques to determine the fate of both the therapeutic molecule and unconjugated poly(ethylene glycol) (PEG) after drug administration. A straightforward strategy utilizing liquid chromatography–mass spectrometry (LC–MS) to characterize high-molecular weight PEG in biologic matrices without a need for complex sample preparation is presented. The method is capable of determining whether high-MW PEG is cleaved in vivo to lower-molecular weight PEG species. Reversed-phase chromatographic separation is used to take advantage of the retention principles of polymeric materials whereby elution order correlates with PEG molecular weight. In-source collision-induced dissociation (CID) combined with selected reaction monitoring (SRM) or selected ion monitoring (SIM) mass spectrometry (MS) is then used to monitor characteristic PEG fragment ions in biological samples. MS provides high sensitivity and specificity for PEG and the observed retention times in reversed-phase LC enable estimation of molecular weight. This method was successfully used to characterize PEG molecular weight in mouse serum samples. No change in molecular weight was observed for 48 h after dosing.

A rapid assay for on-site monitoring of infliximab trough levels: a feasibility study

Monitoring levels of biologicals against tumor necrosis factor (TNF) has been suggested to improve therapeutic outcomes in inflammatory bowel diseases (IBDs). This pilot study describes a rapid lateral flow (LF)-based assay for on-site monitoring of serum trough levels of humanized monoclonal antibody infliximab (IFX). The applied chromatographic method utilizes sequential flows of diluted serum, wash buffer, and an immunoglobulin generic label on LF strips with a Test line comprised of TNF-α. The successive flows permitted enrichment of IFX at the Test line before the label was applied. The label, luminescent upconverting phosphor (UCP) particles coated with protein-A, emits a 550-nm visible light upon excitation with 980-nm infrared light. IFX concentrations were determined through measurement of UCP fluorescence at the Test line. The assay was optimized to detect IFX levels as low as 0.17 μg/mL in serum. For patients with IBD, this limit is appropriate to detect levels associated with loss of response (0.5 μg IFX/mL). The assay was evaluated with clinical samples from patients with Crohn’s disease and correlated well within the physiologically relevant range from 0.17 to 10 μg/mL with an IFX-specific ELISA. Performance of the assay was further successfully validated with samples from blood donors, IFX negative IBD patients, and rheumatoid arthritis patients that had developed anti-IFX antibodies. Because of its generic nature, the assay is suited for detecting most therapeutic anti-TNF-α monoclonal antibodies.

Arsenic speciation in saliva of acute promyelocytic leukemia patients undergoing arsenic trioxide treatment

Arsenic trioxide has been successfully used as a therapeutic in the treatment of acute promyelocytic leukemia (APL). Detailed monitoring of the therapeutic arsenic and its metabolites in various accessible specimens of APL patients can contribute to improving treatment efficacy and minimizing arsenic-induced side effects. This article focuses on the determination of arsenic species in saliva samples from APL patients undergoing arsenic treatment. Saliva samples were collected from nine APL patients over three consecutive days. The patients received 10 mg arsenic trioxide each day via intravenous infusion. The saliva samples were analyzed using high-performance liquid chromatography coupled with inductively coupled plasma mass spectrometry. Monomethylarsonous acid and monomethylmonothioarsonic acid were identified along with arsenite, dimethylarsinic acid, monomethylarsonic acid, and arsenate. Arsenite was the predominant arsenic species, accounting for 71.8 % of total arsenic in the saliva. Following the arsenic infusion each day, the percentage of methylated arsenicals significantly decreased, possibly suggesting that the arsenic methylation process was saturated by the high doses immediately after the arsenic infusion. The temporal profiles of arsenic species in saliva following each arsenic infusion over 3 days have provided information on arsenic exposure, metabolism, and excretion. These results suggest that saliva can be used as an appropriate clinical biomarker for monitoring arsenic species in APL patients.

Classification of olive leaves and pulps according to their cultivar by using protein profiles established by capillary gel electrophoresis

A method to classify olive leaves and pulps according to their cultivar using protein profiles obtained by capillary gel electrophoresis (CGE) has been developed. For this purpose, proteins were extracted using an enzyme-assisted method, which provided higher protein recoveries than other previously described methods. Ten and nine common peaks, for leaf and pulp samples, respectively, were identified in the 12 cultivars studied in this work. In addition, and using linear discriminant analysis of the CGE data, olive leaf and pulp samples belonging to 12 cultivars from different Spanish regions were correctly classified with an excellent resolution among all the categories, which demonstrated that protein profiles were characteristic of each cultivar.

Comparison and optimization of strategies for a more profound profiling of the sialylated N-glycoproteomics in human plasma using metal oxide enrichment

Glycosylation is an important posttranslational modification of proteins and plays a crucial role in both cellular functions and secretory pathways. Sialic acids (SAs), a family of nine-carbon-containing acidic monosaccharides, often terminate the glycan structures of cell surface molecules and secreted glycoproteins and perform an important role in many biological processes. Hence, a more profound profiling of the sialylated glycoproteomics may improve our knowledge of this modification and its effects on protein functions. Here, we systematically investigated different strategies to enrich the SA proteins in human plasma using a newly developed technology that utilizes titanium dioxide for sialylated N-glycoproteomics profiling by mass spectrometry. Our results showed that using a combination of a filter-aided sample preparation method, TiO₂ chromatography, multiple enzyme digestion, and two-dimensional reversed-phase peptide fractionation led to a more profound profiling of the SA proteome. In total, 982 glycosylation sites in 413 proteins were identified, among which 37.8 % were newly identified, to establish the largest database of sialic acid containing proteins from human plasma.

Structural Feature Ions for Distinguishing N- and O-Linked Glycan Isomers by LC-ESI-IT MS/MS

Glycomics is the comprehensive study of glycan expression in an organism, cell, or tissue that relies on effective analytical technologies to understand glycan structure–function relationships. Owing to the macro- and micro-heterogeneity of oligosaccharides, detailed structure characterization has required an orthogonal approach, such as a combination of specific exoglycosidase digestions, LC-MS/MS, and the development of bioinformatic resources to comprehensively profile a complex biological sample. Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS) has emerged as a key tool in the structural analysis of oligosaccharides because of its high sensitivity, resolution, and robustness. Here, we present a strategy that uses LC-ESI-MS/MS to characterize over 200 N- and O-glycans from human saliva glycoproteins, complemented by sequential exoglycosidase treatment, to further verify the annotated glycan structures. Fragment-specific substructure diagnostic ions were collated from an extensive screen of the literature available on the detailed structural characterization of oligosaccharides and, together with other specific glycan structure feature ions derived from cross-ring and glycosidic-linkage fragmentation, were used to characterize the glycans and differentiate isomers. The availability of such annotated mass spectrometric fragmentation spectral libraries of glycan structures, together with such substructure diagnostic ions, will be key inputs for the future development of the automated elucidation of oligosaccharide structures from MS/MS data.

Separation of dansylated 17β-estradiol, 17α-estradiol, and estrone on a single HPLC column for simultaneous quantitation by LC–MS/MS

We show here that baseline separation of dansylated estrone, 17β-estradiol, and 17α-estradiol can be done, contrary to previous reports, within a short run time on a single RP-LC analytical column packed with particles bonded with phenyl-hexyl stationary phase. The chromatographic method coupled with isotope dilution tandem MS offers a simple assay enabling the simultaneous analysis of these analytes. The method employs ¹³C-labeled estrogens as internal standards to eliminate potential matrix effects arising from the use of deuterated estrogens. The assay also offers adequate accuracy and sensitivity to be useful for biological samples. The practical applicability of the validated method is demonstrated by the quantitative analyses of in vivo samples obtained from rats treated with Premarin®.

Metabolomic profiling of mice urine and serum associated with trans-trans 2, 4-decadienal induced lung lesions by liquid chromatography-mass spectrometry

Metabolomics has become an important tool in clinical research and the diagnosis of human disease. Intratracheal instillation of trans-trans 2,4-decadienal (tt-DDE), a major component in cooking oil fumes, has been demonstrated to cause lung lesions in mice at 8 weeks after treatment. The objective of this study was to identify any changes in metabolite profiles associated with the development of tt-DDE-induced lung lesions. Using a metabolomics strategy involving a liquid chromatography–mass spectrometry-based approach in conjunction with principal component analysis and confirmation by liquid chromatography triple quadrupole tandem mass spectrometry, we have demonstrated that the amino acid profiles of the urine and serum of tt-DDE-treated mice are changed. Ten amino acids were significantly reduced in serum of tt-DDE-treated mice at 8 weeks after treatment. Our results suggest that amino acid profiles may be useful as an early indicator of the presence of tt-DDE-induced lung lesions.

Mechanistic Study on Electron Capture Dissociation of the Oligosaccharide-Mg2+ Complex

Electron capture dissociation (ECD) has shown great potential in structural characterization of glycans. However, our current understanding of the glycan ECD process is inadequate for accurate interpretation of the complex glycan ECD spectra. Here, we present the first comprehensive theoretical investigation on the ECD fragmentation behavior of metal-adducted glycans, using the cellobiose-Mg₂₊ complex as the model system. Molecular dynamics simulation was carried out to determine the typical glycan-Mg₂₊ binding patterns and the lowest-energy conformer identified was used as the initial geometry for density functional theory-based theoretical modeling. It was found that the electron is preferentially captured by Mg₂₊ and the resultant Mg_+? can abstract a hydroxyl group from the glycan moiety to form a carbon radical. Subsequent radical migration and α-cleavage(s) result in the formation of a variety of product ions. The proposed hydroxyl abstraction mechanism correlates well with the major features in the ECD spectrum of the Mg₂₊-adducted cellohexaose. The mechanism presented here also predicts the presence of secondary, radical-induced fragmentation pathways. These secondary fragment ions could be misinterpreted, leading to erroneous structural determination. The present study highlights an urgent need for continuing investigation of the glycan ECD mechanism, which is imperative for successful development of bioinformatics tools that can take advantage of the rich structural information provided by ECD of metal-adducted glycans.

Non-destructive depth profile reconstruction of bio-engineered surfaces by parallel-angle-resolved X-ray photoelectron spectroscopy

In the present, contribution angle-resolved X-ray photoelectron spectroscopy (AR-XPS) was proposed as a useful tool to address the challenge of probing the near-surface region of bio-active sensor surfaces. A model bio-functionalised surface was characterised by parallel AR-XPS and commercially available Thermo Avantage-ARProcess software was used to generate non-destructive concentration depth profiles of protein-functionalised silicon oxide substrates. At each step of the functionalisation procedure, the surface composition, the overlayer thickness, the in-depth organisation and the in-plane homogeneity were evaluated. The critical discussion of the generated profiles highlighted the relevance of the information provided by PAR-XPS technique.

Structural Documentation of Glycan Epitopes: Sequential Mass Spectrometry and Spectral Matching

Documenting mass spectral data is a fundamental aspect of accepted protocols. In this report, we contrast MSⁿ sequential disassembly spectra obtained from natural and synthetic glycan epitopes. The epitopes considered are clusters found on conjugate termini of lipids and N- and O-glycans of proteins. The latter are most frequently pendant through a CID-labile HexNAc glycosidic linkage. The synthetic samples were supplied by collaborating colleagues and commercial sources and usually possessed a readily released reducing-end linker, a by-product of synthesis. All samples were comparably methylated, extracted, and MSⁿ disassembled to compare their linkage and branching spectral details. Both sample types provide B-ion type fragments early in a disassembly pathway and their compositions are a suggestion of structure. Further steps of disassembly are necessary to confirm the details of linkage and branching. Included in this study were various Lewis and H antigens, 3- and 6-linked sialyl-lactosamine, NeuAc-2,8-NeuAc dimer, and Galα1,3Gal. Sample infusion provided high quality spectral data whereas disassembly to small fragments generates reproducible high signal/noise spectra for spectral matching. All samples were analyzed as sodium adducted positive ions. This study includes comparability statistics and evaluations on several mass spectrometers.

N-Glycosylamine-mediated isotope labeling for mass spectrometry-based quantitative analysis of N-linked glycans

N-Linked glycosylation is a major protein modification involved in many essential cellular functions. Methods capable of quantitative glycan analysis are highly valuable and have been actively pursued. Here we describe a novel N-glycosylamine-based strategy for isotopic labeling of N-linked glycans for quantitative analysis by use of mass spectrometry (MS). This strategy relies on the primary amine group on the reducing end of freshly released N-linked glycans for labeling, and eliminates the need for the harsh labeling reaction conditions and/or tedious cleanup procedures required by existing methods. By using NHS-ester amine chemistry we used this strategy to label N-linked glycans from a monoclonal antibody with commercially available tandem mass tags (TMT). Only duplex experiments can be performed with currently available TMT reagents, because quantification is based on the intensity of intact labeled glycans. Under mild reaction conditions, greater than 95 % derivatization was achieved in 30 min and the labeled glycans, when kept at ?20 °C, were stable for more than 10 days. By performing glycan release, TMT labeling, and LC–MS analysis continuously in a single volatile aqueous buffer without cleanup steps, we were able to complete the entire analysis in less than 2 h. Quantification was highly accurate and the dynamic range was large. Compared with previously established methods, N-glycosylamine-mediated labeling has the advantages of experimental simplicity, efficient labeling, and preserving glycan integrity.