Capillary electrophoresis-based analysis of phospholipid and glycosaminoglycan binding by human β2-glycoprotein I

Bohlin, Maria; Kogutowska, Ewa; Blomberg, Lars G.; Heegaard, Niels H. H.

doi:10.1016/j.chroma.2004.09.087

Cited by 16 publications

(16 citation statements)

References 39 publications

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“…It was previously found that by rinsing the separation capillary with HCl prior to each injection instead of NaOH the basic protein b 2 gpI could be reproducibly analyzed [9]. We here characterize this observation further by performing m EOF measurements after different pretreatment conditions.…”

Section: Dependency Of the Eof On Pretreatment Methodsmentioning

confidence: 53%

“…However, adsorption problems may be encountered even with coated capillaries [6,7]. This may be due to incomplete coverage of the surface and to the presence of precipitated proteins that originate from previous runs [7].Recently we showed that uncoated fused-silica capillaries, after rinsing with HCl, could be successfully used for electrophoresis of the basic protein b 2 -glycoprotein I (b 2 gpI) [9]. It has previously been shown that treatment of fused silica at low pH leads to decreased EOF, not only when subsequently separating at a low pH, but also, for some time, when separating at higher pH values -this is the hysteresis effect [10,11].…”

mentioning

confidence: 98%

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Utilizing the pH hysteresis effect for versatile and simple electrophoretic analysis of proteins in bare fused-silica capillaries

Bohlin¹,

Blomberg²,

Heegaard³

2005

Electrophoresis

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The analysis of peptides and proteins by CE is often desirable due to low sample consumption and possibilities for nondenaturing yet highly effective separations. However, adsorption to the inner surfaces of fused-silica capillaries often is detrimental to such analyses. This phenomenon is especially pronounced in the analysis of basic proteins and proteins containing exposed positively charged patches. To avoid wall interactions numerous buffer additives and static and dynamic wall coating principles have been devised. We previously showed (J. Chromatogr. A 2004, 1059, 215-222) that CE of the basic protein beta2-glycoprotein was rendered possible by an acidic pretreatment step, and we attributed this observation to the so-called pH hysteresis effect that influences the time for pH equilibration of the capillary wall and thus the effective wall charge and the electroosmotic mobility. We here investigate the effects of different pretreatment techniques on EOF values and on the rate of the deprotonation of silanol groups when performing the electrophoresis at neutral pH. We show the utility of this simple approach for the CE analysis of a number of basic proteins in plain silica capillaries at physiological pH.

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Section: Dependency Of the Eof On Pretreatment Methodsmentioning

confidence: 53%

mentioning

confidence: 98%

Utilizing the pH hysteresis effect for versatile and simple electrophoretic analysis of proteins in bare fused-silica capillaries

Bohlin¹,

Blomberg²,

Heegaard³

2005

Electrophoresis

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show abstract

“…The approach, however, is also a simple way of ensuring a more protonated capillary wall without resorting to pH extremes and may facilitate the analysis of proteins with a tendency to stick to the wall through ionic interactions involving positively charged side chains as was recently demonstrated by us in the analysis of the moderately basic serum protein b 2 -glycoprotein I. This protein could not be recovered for analysis and on-line binding studies at pH 8.2 in uncoated fused-silica capillaries unless analyzed in a capillary that was preconditioned at low pH [97].…”

Section: Peptides and Proteinsmentioning

confidence: 93%

Applications of affinity interactions in capillary electrophoresis

Heegaard

2003

Electrophoresis

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Capillary electrophoresis (CE) has proven useful for the study of reversible molecular interactions. This is because highly efficient and reproducible separations take place in an environment where molecular interactions may contribute to selectivity without being inhibited by adverse buffer conditions. Affinity CE may be used to estimate quantitative binding data (binding constants and in some cases binding stoichiometries and rate constants) for various molecular interactions. Specific binding interactions (e.g., based on antibodies or aptamers) may also be utilized to quantitatively measure specific analytes using CE. Applications within these areas are here reviewed with focus on the last three years and with emphasis on novel concepts as well as innovative methodology and technology. It is concluded that the affinity CE approach is of growing versatility and will continue to play an integral role in discovering, characterizing, and exploiting biomolecular interactions.

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“…In the case of a specific protein analyte, the glycosaminoglycan-binding plasma protein b 2 -glycoprotein I, the socalled pH-hysteresis effect of uncoated fused-silica capillaries [23,24] was exploited. In this way reproducible analyte recovery was achieved at neutral pH and binding studies could be carried out [25]. The pH-hysteresis effect refers to the slow deprotonization of silanol groups when brought to neutral pH from the acidic side compared with the faster equilibration when coming from the alkaline side.…”

Section: Proteins and Peptidesmentioning

confidence: 99%

Recent applications of affinity interactions in capillary electrophoresis

2006

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Systems biology depends on a comprehensive assignment and characterization of the interactions of proteins and polypeptides (functional proteomics) and of other classes of biomolecules in a given organism. High-capacity screening methods are in place for ligand capture and interaction screening, but a detailed dynamic characterization of molecular interactions under physiological conditions in efficiently separated mixtures with minimal sample consumption is presently provided only by electrophoretic interaction analysis in capillaries, affinity CE (ACE). This has been realized in different fields of biology and analytical chemistry, and the resulting advances and uses of ACE during the last 2.5 years are covered in this review. Dealing with anything from small divalent metal ions to large supramolecular assemblies, the applications of ACE span from low-affinity binding of broad specificity being exploited in optimizing selectivity, e.g., in enantiomer analysis to miniaturized affinity technologies, e.g., for fast processing immunoassay. Also, approaches that provide detailed quantitative characterization of analyte-ligand interaction for drug, immunoassay, and aptamer development are increasingly important, but various approaches to ACE are more and more generally applied in biological research. In addition, the present overview emphasizes that distinct challenges regarding sensitivity, parallel processing, information-rich detection, interfacing with MS, analyte recovery, and preparative capabilities remain. This will be addressed by future technological improvements that will ensure continuing new applications of ACE in the years to come.

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Capillary electrophoresis-based analysis of phospholipid and glycosaminoglycan binding by human β2-glycoprotein I

Cited by 16 publications

References 39 publications

Utilizing the pH hysteresis effect for versatile and simple electrophoretic analysis of proteins in bare fused-silica capillaries

Utilizing the pH hysteresis effect for versatile and simple electrophoretic analysis of proteins in bare fused-silica capillaries

Applications of affinity interactions in capillary electrophoresis

Recent applications of affinity interactions in capillary electrophoresis

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