Glycoprotein Characterization Combining Intact Protein and Glycan Analysis by Capillary Electrophoresis-Electrospray Ionization-Mass Spectrometry

Balaguer, Elvira; Neusüß, Christian

doi:10.1021/ac060376g

Cited by 120 publications

(109 citation statements)

References 31 publications

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“…4,8,10,43,44 These modifications were mainly observed in highly sialylated glycans and include acetylation of sialic acids, N-acetylneuraminic (Neu5Ac) / N-glycolylneuraminic acid (Neu5Gc) variation, and elongation of the glycan chains because of the presence of LacNAc repeats. Up to three modifications occurred per glycan.…”

Section: Detection Of Glycan Modifications In Rhuepomentioning

confidence: 99%

Simple Capillary Electrophoresis–Mass Spectrometry Method for Complex Glycan Analysis Using a Flow-Through Microvial Interface

et al. 2014

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A flow-through microvial is used to interface capillary electrophoresis and mass spectrometry (CE-MS) to develop a method for simultaneous profiling both neutral and sialylated glycans without derivatization or labeling. The CE separation was performed at near-zero electroosmotic flow in a capillary with neutral, hydrophilic coating, using 50 mM ammonium acetate in 20% methanol (pH 3.1) as the background electrolyte. The method was optimized with reversed CE polarity and negative ion ESI-MS. Enzymatically released N-glycans from human immunoglobulin G (IgG) were used as the test sample. The approach was also used to study the more complex N-glycans from recombinant human erythropoietin (rHuEPO) expressed in Chinese hamster ovary (CHO) cells. Glycoscreening of rHuEPO was performed using a triple quadrupole MS and an ultrahigh resolution TOF-MS. The high sensitivity and high mass accuracy of the TOF-MS revealed the presence of more than 70 glycans. Three mono-and di-sialylated tetra-antennary N-glycans and one mono-sialylated tri-antennary N-glycan of rHuEPO are reported for the first time. Further glycan heterogeneity was identified of the highly sialylated N-glycans of rHuEPO by extensive acetylation, Neu5Ac/Neu5Gc variation and the presence of N-acetyl-lactosamine repeats. For comparative purposes, porous graphitic carbon-based LC-MS/MS was also used to glycoprofile rHuEPO. This work demonstrates the potential of CE-MS to provide a comprehensive glycosylation profile with detailed features of the secondary glycan modifications. The CE-MS based method eliminates the need to label the N-glycans, as well as the requirement to desialylate before analysis, and could complement other established techniques for glycan characterization of therapeutic glycoproteins.

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Section: Detection Of Glycan Modifications In Rhuepomentioning

confidence: 99%

Simple Capillary Electrophoresis–Mass Spectrometry Method for Complex Glycan Analysis Using a Flow-Through Microvial Interface

et al. 2014

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“…After the CE separation of glycosylated and nonglycosylated peptides, the eluting peptides were automatically spotted onto a MALDI target and their sequences analyzed by MS and MS/MS. A second study reported the analysis of intact glycoproteins and underivatized glycans by CE-MS (121). The CE separation of intact proteins was performed in capillaries coated with either Polybrene or UltraTrol.…”

Section: Protein and Peptide Analysismentioning

confidence: 99%

Capillary Electrophoresis in Bioanalysis

2008

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Science reports ∼4800 hits on capillary electrophoresis (CE) including 410 reviews. Because of the prominence of CE techniques in bioanalysis, we decided make them the focus of this review and selected 200 hundred papers representing advances in this field. The selected papers cover advances in CE theory, instrumentation, and methodologies that are specific to various analytes of biological origin or relevance. The group of analytes includes nucleic acids, proteins and peptides, carbohydrates, lipids, single cells, and bioparticles. In addition, we have included advances in the use of CE to define functional assays or to investigate biomolecular interactions. The use of microfabricated devices for CE analysis was not included because this is already covered in other review. Technique Developments Separation SchemesDetermining the velocity of the electroosmotic flow (EOF) and how it changes during an electrophoretic separation is still an important research topic. A simple method for EOF measurements using so-called thermal marks was reported (1). Here, a tungsten filament caused punctual heating at the capillary wall and caused a perturbation in the electrolyte concentration. A sequence of these "thermal marks" then migrated with the EOF until each mark reached and was detected by a conductivity detector. The feasibility of using thermal marks as internal EOF standards in different separation systems was thereby demonstrated.Isoelectric focusing separates amphoteric analytes such as proteins or peptides by the differences in their isoelectric points. Most of the reports on capillary isoelectric focusing (cIEF) describe an initial focusing phase after which the focused zones are mobilized and detected. A dynamic cIEF method for protein analysis was reported (2). This technique made it possible to control each protein's position and focused width by moving the pH gradient within the capillary through manipulation of the electric fields. An important advantage of this approach is the capability of collecting focused analytes from the central section, suggesting that there may be great potential for introducing selectively focused proteins to a second separation dimension such as LC or CE.Micellar electrokinetic capillary chromatography (MEKC) is typically incompatible with electrospray mass spectrometry (ESI-MS) because the nonvolatile surfactants in the micellar phase result in complicated adduct formation and loss of sensitivity during the electrospray process and because the presence of the organic solvent needed for electrospray may cause instability in the micellar phase. These drawbacks were overcome by using synthetic polymeric surfactants that can work as a pseudostationary phase and provide stable electrospray (3). The polymeric surfactant was made by polymerizing three amino acidderived (L-leucinol, L-isoleucinol, L-valinol) sulfated chiral surfactants. These polymeric

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“…After the CZE-UV and CZE-MS analysis of the intact protein glycans from human, AGP were released by enzymatic deglycosylation which was performed with peptide N-glycosidase F, as described elsewhere [23]. The sample containing the glycans, the deglycosylated protein, and the partially deglycosylated protein was analyzed by CZE-MS.…”

Section: Sample Treatmentmentioning

confidence: 99%

Statistical evaluation of CZE‐UV and CZE‐ESI‐MS data of intact α‐1‐acid glycoprotein isoforms for their use as potential biomarkers in bladder cancer

et al. 2010

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α-1-acid glycoprotein (AGP) is a highly heterogeneous protein that presents a vast number of isoforms (molecules of the protein differing in its peptidic and/or glycosidic moieties). In the last years, several authors have studied the potential use of AGP as a cancer biomarker. These studies focus on the correlation of different features of AGP structure (i.e. fucosylation, antennarity) with cancer or on the total protein blood concentration. In this study, the potential of CZE-UV and CZE-ESI-MS analysis of intact AGP isoforms to study the correlation of this protein with bladder cancer is shown. Samples from 16 individuals (eight healthy, eight bladder cancer) were analyzed and characterized in great detail including data on intact protein isoforms and on released glycans. The analytical data were evaluated employing different statistical techniques (ANOVA; principal component analysis, PCA; linear discriminant analysis; and partial least squares-discriminant analysis). Statistical differences between the two groups of study were observed. The best results were obtained by linear discriminant analysis of the CZE-ESI-MS data for intact AGP isoforms (93.75% of correct classification). Due to MS characterization, it can be observed that differences between the samples are mainly due to higher abundance of AGP isoforms containing tri- and tetra-antennary fucosylated oligosaccharides in cancer patients. The results show the great potential of CE-MS in combination with advanced data processing for the use of intact protein isoforms as disease biomarkers.

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Glycoprotein Characterization Combining Intact Protein and Glycan Analysis by Capillary Electrophoresis-Electrospray Ionization-Mass Spectrometry

Cited by 120 publications

References 31 publications

Simple Capillary Electrophoresis–Mass Spectrometry Method for Complex Glycan Analysis Using a Flow-Through Microvial Interface

Simple Capillary Electrophoresis–Mass Spectrometry Method for Complex Glycan Analysis Using a Flow-Through Microvial Interface

Capillary Electrophoresis in Bioanalysis

Statistical evaluation of CZE‐UV and CZE‐ESI‐MS data of intact α‐1‐acid glycoprotein isoforms for their use as potential biomarkers in bladder cancer

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