Noncovalently bilayer-coated capillaries for efficient and reproducible analysis of proteins by capillary electrophoresis

Catai, Jonatan R.; Tervahauta, Heli A.; Jong, Gerhardus J. de; Somsen, Govert W.

doi:10.1016/j.chroma.2005.06.001

Cited by 66 publications

(55 citation statements)

References 37 publications

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“…The LN coating is strongly adsorbing and can be applied in a precoating mode. Therefore, it was expected to be MS-compatible as other similar coatings [24,25]. The LN coating decreases the EOF to a value close to zero at a pH ,4.…”

Section: Separationmentioning

confidence: 97%

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Glycoform characterization of erythropoietin combining glycan and intact protein analysis by capillary electrophoresis – electrospray – time‐of‐flight mass spectrometry

et al. 2006

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Glycosylation of recombinant human erythropoietin (rHuEPO) is a post-translational process that alters biological activity, solubility and lifetime of the glycoprotein in blood, and strongly depends on the type of cell and the cell culture conditions. A fast and simple method providing extensive carbohydrate information about the glycans present in rHuEPO and other glycoproteins is needed in order to improve current methods in drug development or product quality control. Here, an improved method for intact rHuEPO glycoform characterization by CZE-ESI-TOF MS has been developed using a novel capillary coating and compared to a previous study. Both methods allow a fast separation in combination with accurate mass characterization of the single protein isoforms. The novel dynamic coating provides a separation at an EOF close to zero, enabling better separation. This results in an improved mass spectrometric resolution and the detection of minor isoforms. In order to assign an unequivocal carbohydrate composition to every intact glycoform, a CZE-ESI-MS separation method for enzymatically released underivatized N-glycans has been developed. The TOF MS allows the correct identification of the glycans due to its high mass accuracy and resolution. Therefore, glycan modifications such as acetylation, oxidation, sulfation and even the exchange of OH by NH(2) are successfully characterized. Information of the protein-backbone molecular mass has been combined with results from peptide analysis (revealing information about O-glycosylation) and from the glycan analysis, including the detection of as yet undescribed glycans containing four antennae and five sialic acids. This allows an unequivocal assignment of an overall glycosylation composition to the molecular masses obtained for the intact rHuEPO glycoforms.

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Section: Separationmentioning

confidence: 97%

“…CZE can be easily coupled to ESI-MS, by restricting the applicable electrolytes to volatile constituents. Moreover, in protein analysis, capillary coatings are mandatory in order to avoid protein adsorption to the capillary wall [24,25]. Few articles have been published on intact glycoprotein analysis [26][27][28].…”

Section: Introductionmentioning

confidence: 99%

Glycoform characterization of erythropoietin combining glycan and intact protein analysis by capillary electrophoresis – electrospray – time‐of‐flight mass spectrometry

et al. 2006

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“…So far, this type of coating, which generates a constant and significant cathodic EOF, has predominantly been applied for the analysis of low-molecular-weight compounds by CE-UV and, to a lesser extent, CE-MS [20][21][22][23]. Recently, we have demonstrated that capillaries coated with a bilayer of Polybrene (PB) and poly(vinyl sulfonate) (PVS) can also be successfully used for the fast and reproducible analysis of peptides and proteins by CE-UV [24,25]. High plate numbers were obtained when the PB-PVS coating was used in combination with BGEs of relatively high ionic strength.…”

Section: Introductionmentioning

confidence: 98%

Efficient and highly reproducible capillary electrophoresis–mass spectrometry of peptides using Polybrene‐poly(vinyl sulfonate)‐coated capillaries

et al. 2006

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The potential of capillaries noncovalently coated with a bilayer of oppositely charged polymers for the analysis of peptides by CE-MS was investigated. Bilayer coatings were produced by subsequently rinsing fused-silica capillaries with a solution of Polybrene (PB) and poly(vinyl sulfonate) (PVS). The PB-PVS coating showed to be fully compatible with MS detection causing no ionization suppression or background signals. The bilayer coating provided a considerable EOF at low pH, thereby facilitating the fast separation of peptides using a BGE of formic acid (pH 2.5). Under optimized CE-MS conditions, for enkephalin peptides high separation efficiencies were obtained with plate numbers in the range of 300,000-500,000. It is demonstrated that both the cancellation of the hydrodynamic capillary flow induced by the nebulizer gas and a sufficiently high-data acquisition rate are crucial for achieving these efficiencies. The overall performance of the CE-MS system using PB-PVS-coated capillaries was evaluated by the analysis of a tryptic digest of cytochrome c. The system provided an efficient separation of the peptide mixture, which could be effectively monitored by MS/MS detection allowing identification of at least 13 peptides within a time interval of 1.5 min. In addition, the PB-PVS coating proved to be very consistent yielding stable CE-MS patterns with highly favorable migration time reproducibilities (RSDs < 1% over a 3-day period).

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“…Capillary electrophoresis (CE) can be used to carry out more specific protein analysis [17][18][19][20][21] and in the current work was able to resolve the albumin peak as a major protein from the other proteins in the deproteinated plasma samples. However, the semipolar solvents precipitated certain amounts of salts from the urine samples; the remaining salts in the sample supernatant would be expected to govern the electrical current properties of the final preparation after subtracting the blank contribution.…”

Section: Introductionmentioning

confidence: 87%

Comparison of different water-miscible solvents for the preparation of plasma and urine samples in metabolic profiling studies

Alzweiri

Watson

Robertson

et al. 2008

Talanta

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This version is available at https://strathprints.strath.ac.uk/7602/ Strathprints is designed to allow users to access the research output of the University of Strathclyde. Unless otherwise explicitly stated on the manuscript, Copyright © and Moral Rights for the papers on this site are retained by the individual authors and/or other copyright owners. Please check the manuscript for details of any other licences that may have been applied. You may not engage in further distribution of the material for any profitmaking activities or any commercial gain. You may freely distribute both the url (https://strathprints.strath.ac.uk/) and the content of this paper for research or private study, educational, or not-for-profit purposes without prior permission or charge.Any correspondence concerning this service should be sent to the AbstractThe Lowry method and a capillary electrophoresis method were used to analyse protein residues in the supernatant after solvent deproteination of plasma. Acetonitrile and acetone were much more effective than methanol and ethanol at reducing the levels of proteins in plasma. The ability of different solvents to decrease levels of phospholipids in plasma samples was assessed using electrospray ionisation mass spectrometry (MS). Phospholipid signals can obscure differences between samples in general metabolite profiling (i.e. non-target compound) studies. Acetonitrile was much more effective than methanol in reducing the MS signal due to phospholipids in plasma which is a consequence of the poor solubility of phospholipids in acetonitrile. The capability of the solvents at reducing salts in urine samples was also studied by using an amperometric method. Using this approach little difference was detected between methanol, ethanol, acetonitrile and acetone in their ability to desalt urine samples.

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Noncovalently bilayer-coated capillaries for efficient and reproducible analysis of proteins by capillary electrophoresis

Cited by 66 publications

References 37 publications

Glycoform characterization of erythropoietin combining glycan and intact protein analysis by capillary electrophoresis – electrospray – time‐of‐flight mass spectrometry

Glycoform characterization of erythropoietin combining glycan and intact protein analysis by capillary electrophoresis – electrospray – time‐of‐flight mass spectrometry

Efficient and highly reproducible capillary electrophoresis–mass spectrometry of peptides using Polybrene‐poly(vinyl sulfonate)‐coated capillaries

Comparison of different water-miscible solvents for the preparation of plasma and urine samples in metabolic profiling studies

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