“Getting the best sensitivity from on-capillary fluorescence detection in capillary electrophoresis” – A tutorial

Galievsky, Victor A.; Stasheuski, Alexander S.; Krylov, Sergey N.

doi:10.1016/j.aca.2016.06.015

“…Such sensitivity can be routinely achieved with fluorescence detection, [24] but remains ac hallenge with MS detection. In contrast, ACTIS is perfectly applicable to stable complexes.T he upper level of the K d value is limited by protein solubility and can be roughly defined as one tenth of the highest achievable protein concentration in as olution.…”

Section: Angewandte Chemiementioning

confidence: 99%

“…Fore xample,a ccurate determination of K d % 1nm (characteristic for high-affinity drugs) will require detecting 0.1 nm ligand with as ignal to noise ratio of about 100. Such sensitivity can be routinely achieved with fluorescence detection, [24] but remains ac hallenge with MS detection. [25] As in other separation-based methods, [8,9] surface adsorption phenomena will distort separagrams and affect the accuracy of K d values determined with ACTIS.However,the time required for TIS is equal to the characteristic time of transverse diffusion of the ligand and much shorter than that of the protein and protein-ligand complex.…”

Section: Angewandte Chemiementioning

confidence: 99%

Transient Incomplete Separation Facilitates Finding Accurate Equilibrium Dissociation Constant of Protein–Small Molecule Complex

Sisavath

¹

,

Rukundo

²

,

Leblanc³

et al. 2019

Self Cite

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Current practical methods for finding the equilibrium dissociation constant, K d ,o fp rotein-small molecule complexes have inherent sources of inaccuracy.I ntroduced here is "accurate constant via transient incomplete separation" (ACTIS), which appears to be free of inherent sources of inaccuracy.C onceptually,as hort plug of the pre-equilibrated protein-small molecule mixture is pressure-propagated in ac apillary,c ausing fast transient incomplete separation of the complex from the unbound small molecule.Asuperposition of signals from these two components is measured near the capillary exit and used to calculate af raction of unbound small molecule,which,inturn, is used to calculate K d . Herein the validity of ACTIS is proven theoretically,i ts accuracy is verified by computer simulation, and its practical use is demonstrated. ACTIS has the potential to become ar eference-standardm ethod for determining K d values of protein-small molecule complexes.Reversible binding of proteins (P) to small-molecule ligands (L) plays an important role in the regulation of cellular processes. [1] In addition, most therapeutic targets are proteins, [2] and drugs are developed to form stable PL complexes with them:Complex stability is characterized by the equilibrium dissociation constant K d ,which is defined as:

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“…Different aspects of LIF detection in CE (light sources, detection elements, optimization of excitation conditions, photostability of the fluorophores, spectral filtering, and fluorescent labels) including the possibilities to further improve the LODs of on‐capillary LIF detectors were in detail analyzed in the tutorial article . Comparison of the pulsed lasers and continuous light sources have shown that continuous lasers and LEDs are more suitable for LIF detection in CE since they provide linear calibration curves, whereas pulsed lasers give polynomial ones .…”

Section: Detectionmentioning

confidence: 99%

Recent developments in capillary and microchip electroseparations of peptides (2015–mid 2017)

Kašička

¹

2017

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The review brings a comprehensive overview of recent developments and applications of high performance capillary and microchip electroseparation methods (zone electrophoresis, isotachophoresis, isoelectric focusing, affinity electrophoresis, electrokinetic chromatography, and electrochromatography) to analysis, microscale isolation, purification, and physicochemical and biochemical characterization of peptides in the years 2015, 2016, and ca. up to the middle of 2017. Advances in the investigation of electromigration properties of peptides and in the methodology of their analysis (sample preseparation, preconcentration and derivatization, adsorption suppression and EOF control, and detection) are described. New developments in particular CE and CEC methods are presented and several types of their applications to peptide analysis are reported: qualitative and quantitative analysis, determination in complex (bio)matrices, monitoring of chemical and enzymatical reactions and physical changes, amino acid, sequence and chiral analysis, and peptide mapping of proteins. Some micropreparative peptide separations are shown and capabilities of CE and CEC methods to provide important physicochemical characteristics of peptides are demonstrated.

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“…For example, accurate determination of K d ≈1 n m (characteristic for high‐affinity drugs) will require detecting 0.1 n m ligand with a signal to noise ratio of about 100. Such sensitivity can be routinely achieved with fluorescence detection, but remains a challenge with MS detection …”

Section: Figurementioning

confidence: 99%

Transient Incomplete Separation Facilitates Finding Accurate Equilibrium Dissociation Constant of Protein–Small Molecule Complex

Sisavath

¹

,

Rukundo

²

,

Leblanc³

et al. 2019

Self Cite

View full text Add to dashboard Cite

Current practical methods for finding the equilibrium dissociation constant, K d ,o fp rotein-small molecule complexes have inherent sources of inaccuracy.I ntroduced here is "accurate constant via transient incomplete separation" (ACTIS), which appears to be free of inherent sources of inaccuracy.C onceptually,as hort plug of the pre-equilibrated protein-small molecule mixture is pressure-propagated in ac apillary,c ausing fast transient incomplete separation of the complex from the unbound small molecule.Asuperposition of signals from these two components is measured near the capillary exit and used to calculate af raction of unbound small molecule,which,inturn, is used to calculate K d . Herein the validity of ACTIS is proven theoretically,i ts accuracy is verified by computer simulation, and its practical use is demonstrated. ACTIS has the potential to become ar eference-standardm ethod for determining K d values of protein-small molecule complexes.Reversible binding of proteins (P) to small-molecule ligands (L) plays an important role in the regulation of cellular processes. [1] In addition, most therapeutic targets are proteins, [2] and drugs are developed to form stable PL complexes with them:Complex stability is characterized by the equilibrium dissociation constant K d ,which is defined as:

show abstract

“Getting the best sensitivity from on-capillary fluorescence detection in capillary electrophoresis” – A tutorial

Cited by 51 publications

References 114 publications

Transient Incomplete Separation Facilitates Finding Accurate Equilibrium Dissociation Constant of Protein–Small Molecule Complex

Transient Incomplete Separation Facilitates Finding Accurate Equilibrium Dissociation Constant of Protein–Small Molecule Complex

Recent developments in capillary and microchip electroseparations of peptides (2015–mid 2017)

Transient Incomplete Separation Facilitates Finding Accurate Equilibrium Dissociation Constant of Protein–Small Molecule Complex

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