Characterization of Glycopeptides from Recombinant Coagulation Factor VIIa by High-Performance Liquid Chromatography and Capillary Zone Electrophoresis Using Ultraviolet and Pulsed Electrochemical Detection

Weber, Paul L.; Kornfelt, Troels; Klausen, Niels Kristian; Lunte, Susan M.

doi:10.1006/abio.1995.1119

Cited by 44 publications

(19 citation statements)

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“…CE-PAD has been also used to analyze mixtures of oligosaccharides obtained from bovine fetuin, to monitor the desialylation process used in the characterization of the oligosaccharides, to resolve a series of glycopeptides obtained from recombinant coagulation factor VIIa and to analyze a tryptic digest of recombinant tissue plasminogen activator. Information about peptide glycosylation along with a brief review of prior applications of CE-PAD has been published [27,28]. Calystegines are polyhydroxyalkaloids with a nortropane skeleton.…”

Section: Capillary Electrophoresis With Pulsed Electrochemical Detectionmentioning

confidence: 99%

Coupling Capillary Electrophoresis and Pulsed Electrochemical Detection

Garcı́a

Henry

2005

Electroanalysis

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Pulsed electrochemical detection (PED) is an excellent method for detection of analytes that normally foul electrodes. In PED, the detection electrode is first cleaned at a high positive potential, then reactivated at a negative potential dissolving the surface oxide, and finally used to oxidize the analyte at a moderate positive potential. Due to the advantages and versatility of PED, many different variations of the detection waveform can be found in literature. This review focuses on application of PED to CE and in particular, the most commonly used modes: pulsed amperometric detection (PAD) and integrated pulsed amperometric detection (iPAD).

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Section: Capillary Electrophoresis With Pulsed Electrochemical Detectionmentioning

confidence: 99%

Coupling Capillary Electrophoresis and Pulsed Electrochemical Detection

Garcı́a

Henry

2005

Electroanalysis

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“…For complex-type N-glycosylation only partial resolution of isoforms was reported by several authors and sometimes only broad signals were obtained [55,59,61,62,65]. Higher resolution of isoforms has been presented, though not all effects to obtain a successful separation are yet understood: Separation by charge or sialylation only can be achieved by CE for both N-and O-glycopeptides [18,51]. For example, Birdwell et al [58] observed the collapsing of five signals representing five sialoforms of a purified glycopeptide into one signal upon treatment with sialidase.…”

Section: The Separation Of Different Peptides Depends On Theirmentioning

confidence: 94%

“…A hint on glycopeptides is often seen in relatively broad signals in a specific migration time region, indicating microheterogeneity [52]. Various methods have been applied to allow their identification: use of exo-and endoglycosidases [50,57,58,64] (see Table 2), sample fractionation from CE to subsequent MS analysis or Edman sequencing [50,53,57], prefractionation by, e.g., LC [50,51,58,61,64,78], prefractionation by lectin affinity columns [49,64], and filtration with a low molecular weight cut-off membrane of large glycopeptides involving a disulfide bridge prior to reduction [65].…”

Section: Detection and Identification Of Glycopeptides In Cementioning

confidence: 99%

Protein glycosylation analysis with capillary-based electromigrative separation techniques

Pattky

Hühn

2010

Bioanal Rev

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An impressive complexity is associated with glycoproteins due to the microheterogeneity of glycosylation as posttranslational modification giving rise to a vast number of isoforms. The full characterization of glycoproteins is difficult to achieve, and a number of analytical methods have to be combined for a detailed understanding of glycosylation. In this review, we focus on capillary electromigrative separation techniques in the formats capillary electrophoresis, micellar electrokinetic chromatography, and capillary sieving electrophoresis. These separation techniques can be applied to all levels of glycosylation analysis including intact glycoproteins, glycopeptides, and released glycans. We here discuss the separation characteristics for each method and the information that they can provide for each level. Detection issues, especially laser-induced fluorescence detection and mass spectrometry are taken into account. In addition, tables provide an overview on the achievements made from the very beginning of glycosylation research by electromigrative separation techniques. From the literature presented here it is clear, that glycosylation analysis by electromigrative separation techniques is on the edge of transition of basic research and method development towards applications. First proof-ofprinciple studies for in-depth glycoprotein characterization and clinical diagnosis are described. However, this overview also shows that many basic aspects of separation have not yet been fully understood and more research is necessary to be able to fully use the capabilities of electromigrative separation techniques.

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“…It was not long after that the first reports of the detection of carbohydrates by CE with PAD utilizing a Au microelectrode were published [48][49][50][51]. Wen and Cassidy [52] reported detection by PAD for a series of metal ions separated by CE, describing sub-micromolar limits of detection for the majority of metals analyzed.…”

Section: Microelectrodes and Microchip Pedmentioning

confidence: 97%