Migration time correction for the analysis of derivatized amino acids and oligosaccharides by micellar capillary electrochromatography

Li, Xing‐Fang; Ren, Hongji; Le, X. Chris; Qi, Ming; Ireland, Ian D.; Dovic̀hi, Norman J.

doi:10.1016/s0021-9673(99)00893-6

Cited by 38 publications

(48 citation statements)

References 17 publications

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“…The generation of a fluorescence signal by commercial ampholytes was reported by Righetti in 1975, and appears to persist in the current reagents [36]. The features in the background electropherogram focus quite tightly with a width of less than 1 s. Their emission spectrum shifts with excitation wavelength.…”

Section: Calibration Curve and Detection Limits For Labeled Proteinmentioning

confidence: 74%

Attomole protein analysis by CIEF with LIF detection

2009

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We have coupled CIEF with an LIF detector that is based on a post-column sheath flow cuvette. We employed Chromeo P503 as a fluorogenic reagent to label proteins before analysis. This reagent reacts with the epsilon-amine of lysine residues, preserving the cationic nature of the residue; labeled proteins generate extremely sharp peaks in CIEF. A set of four standard proteins generated a linear relationship between migration time and pI. A protein homogenate prepared from a Barrett's esophagus cell line resolved over 100 components in a 40 min separation. Detection limits for Chromeo P503-labeled beta-lactoglobulin were 5 amol injected into the capillary. Fluorescent impurities present in the ampholytes generated a large background signal that degraded the detection limit by four orders of magnitude compared with other forms of capillary electrophoresis with this detector.

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Section: Calibration Curve and Detection Limits For Labeled Proteinmentioning

confidence: 74%

Attomole protein analysis by CIEF with LIF detection

2009

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“…2, this IS migrated earlier than the EOF marker (that corresponds to the negative peak in the figure), indicating that the sign of its electro- phoretical mobility is opposite to that of EPO bands. It is known that among the characteristics of a substance to be considered as a good IS, its electrophoretical properties should be very similar to that of the studied analyte to compensate the effects of nonideality on the electrophoretic mobility, and that the more different is the mobility of a compound respect to that of the marker, the worse is the precision [12,16]. In our case, finding an adequate IS was not an easy task.…”

Section: Performance Of the Different Migration Parametersmentioning

confidence: 83%

“…Some of the studies include the comparison of different parameters such as migration time, relative migration, electrophoretic mobili-ty, actual mobility, and relative mobility [11][12][13][14][15][16]. A different approach makes use of the effective electrophoretic mobility of known markers to determine the electrophoretic mobility of the compound of interest [17,18].…”

Section: Introductionmentioning

confidence: 99%

Selection of migration parameters for a highly reliable assignment of bands of isoforms of erythropoietin separated by capillary electrophoresis

et al. 2004

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Capillary zone electrophoresis of samples of recombinant human erythropoietin is performed. An in-house computer program is developed to compare the reliability of different migration parameters to assign the close migration bands of isoforms of erythropoietin. The migration time relative to the electroosmotic flow marker and the effective electrophoretic mobility are selected as the most accurate parameters. Percentages of correct assignment of bands higher than 99% are obtained with these parameters even when changes in operational factors are introduced. The chosen parameters have been applied to assign bands of isoforms in commercial samples of alpha- and beta-epoetin. The same capillary electrophoresis method has been applied to separate bands of isoforms of an erythropoietin analogue, darbepoetin alpha, the novel erythropoiesis-stimulating protein.

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“…The migration times presented in Table 3 are raw data. Reproducibility could be improved slightly by normalizing the migration times using two-peak method [36], although the effect is less dramatic at low pH were the electroosmotic flow is essentially nil, so this was not done. Mean RSD: 1.5% (n = 3)…”

Section: Reproducibility Of Cze Analysismentioning

confidence: 99%

Comparison of two glutaraldehyde immobilization techniques for solid‐phase tryptic peptide mapping of human hemoglobin by capillary zone electrophoresis and mass spectrometry

et al. 2004

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Stabilization of proteolytic enzymes, especially by immobilization, is of considerable interest because of their potential applications in medicine and the chemical and pharmaceutical industries. We report here a detailed comparison of two procedures for trypsin immobilization using the same homobifunctional agent, glutaraldehyde, for the purpose of peptide mapping. These methods include covalent coupling either to controlled pore glass (solid support) or via a cross-linking reaction (without any solid support). The immobilized trypsin preparations were characterized by the determination of immobilization efficiency, which ranged from 68 to > 95%, and measurement of apparent kinetic parameters toward a synthetic peptide-like substrate. Batch digestions of whole denaturated human normal adult hemoglobin (HbA) were performed to obtain peptide maps by capillary zone electrophoresis (CZE). Migration time reproducibility of the CZE maps was excellent, with a mean relative standard deviation of 1.5%. Moreover, the two immobilized enzyme preparations showed excellent reproducibility for repeated digestions. Matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry was also used for peptide mass mapping of denaturated HbA digested using the two immobilized trypsin preparations. Even though the two immobilized trypsin preparations do not behave identically, similar sequence coverages of 57% and 61% (for the two HbA chains merged) were achieved for the support-based and cross-linked trypsin preparations, respectively.

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Migration time correction for the analysis of derivatized amino acids and oligosaccharides by micellar capillary electrochromatography

Cited by 38 publications

References 17 publications

Attomole protein analysis by CIEF with LIF detection

Attomole protein analysis by CIEF with LIF detection

Selection of migration parameters for a highly reliable assignment of bands of isoforms of erythropoietin separated by capillary electrophoresis

Comparison of two glutaraldehyde immobilization techniques for solid‐phase tryptic peptide mapping of human hemoglobin by capillary zone electrophoresis and mass spectrometry

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