Development of Capillary Electrophoretic Method with
Fluorescence Detection for Beta-Secretase Activity Assays with
FRET-Labeled Substrate

a 2018

Development of Capillary Electrophoretic Method with Fluorescence Detection for Beta-Secretase Activity Assays with FRET-Labeled Substrate

ŘEMÍNEK, Roman, Jan SCHEJBAL a Zdeněk GLATZ

Základní údaje

Originální název

Development of Capillary Electrophoretic Method with Fluorescence Detection for Beta-Secretase Activity Assays with FRET-Labeled Substrate

Název česky

Vývoj kapilárně elektroforetické metody s fluorescenční detekcí pro studium aktivity beta-sekretázy s FRET značeným substrátem

Autoři

ŘEMÍNEK, Roman, Jan SCHEJBAL a Zdeněk GLATZ

Vydání

The 11th International Symposium on Drug Analysis and the 29th International Symposium on Pharmaceutical and Biomedical Analysis (DA-PBA), 2018, 2018

Další údaje

Jazyk

angličtina

Typ výsledku

Konferenční abstrakt

Obor

10609 Biochemical research methods

Stát vydavatele

Česká republika

Utajení

není předmětem státního či obchodního tajemství

Odkazy

URL

Organizační jednotka

Přírodovědecká fakulta

Klíčová slova česky

kapilární elektroforéza, Alzheimerova choroba, beta-sekretáza, inhibice, fluorescenční detekce, FRET

Klíčová slova anglicky

capillary electrophoresis, Alzheimer's disease, beta-secretase, inhibition, fluorescent detection, FRET

Příznaky

Mezinárodní význam, Recenzováno

Změněno: 3. 1. 2019 09:42, Ing. Mgr. Roman Řemínek, Ph.D.

Anotace

ORIG CZ

V originále

Alzheimer's disease (AD) represents a degenerative brain disorder that is characterized by a progressive loss of memory and other cognitive abilities seriously affecting a person's ability to carry out daily activities. Currently available medications temporarily improve accompanying symptoms; however, a cure to treat the disease itself has not been introduced yet. Recent studies suggest beta-secretase, an aspartic-acid protease playing a key role in the development of neurotoxic amyloid plagues in the patients' brain tissue, as a promising druggable target to slow down or even stop the progression of AD. Screenings of potential beta-secretase inhibitors are commonly performed by using systems based on Förster resonance energy transfer (FRET). The information gathered from these assays can be negatively affected by low solubility of fluorescently labeled substrates and non-specific interferences with measured signal leading to a risk of false positive/negative results and need of data confirmation by further tests though. For these reasons the main aim of the presented study was to develop a method for activity studies of beta-secretase based on the capillary electrophoresis with fluorescence detection. Separation of a reaction product increases the assay sensitivity and diminishes the risk of interactions with other sample components. Separation conditions were optimized using standards of the QXL® 520/ HiLyteTM Fluor 488 FRET substrate and HiLyteTM 488 reaction product obtained from commercially available SensoLyte® 520 beta-secretase assay kit. An Agilent 7100 CE system equipped with Picometrics Zetalif LEDIF detector was used to perform all analyses. In the final method, the reaction product labeled with HiLyteTM 488 dye was separated by application of 30 kV (positive polarity) inside the uncoated fused-silica capillary (45 cm effective length, 50 m id) thermostated at 25 °C. 45 mM SDS prepared in 65 mM tetraborate buffer (pH 9.2) was used as BGE. The analyte was monitored at excitation/emission = 490 nm/520 nm. The capillary was rinsed with 0.1 M NaOH for 1 min, deionized water for 1 min, and BGE for 2 min before every analysis. This procedure was sufficient to ensure good method repeatability despite sample containing the enzyme and substrate and products peptides was directly injected into the capillary. The optimized method was validated and applied for activity assays of beta-secretase. The results obtained were in a good agreement with literature and confirmed the practical applicability of the developed method.

Česky

Byla zavedena nová metoda pro studium aktivity beta-sekretázy pomocí kapilární elektroforézy s fluorescenční detekcí.

Návaznosti

GA16-06106S, projekt VaV

Název: Vysoce efektivní systém založený na kapilární elektroforéze pro screening inhibitorů beta-sekretázy jako terapeutického cíle pro Alzheimerovu chorobu (Akronym: Alzheimer)

Investor: Grantová agentura ČR, Vysoce efektivní systém založený na kapilární elektroforéze pro screening inhibitorů beta-sekretázy jako terapeutického cíle pro Alzheimerovu chorobu

Citovat

ŘEMÍNEK, Roman, Jan SCHEJBAL a Zdeněk GLATZ. Development of Capillary Electrophoretic Method with Fluorescence Detection for Beta-Secretase Activity Assays with FRET-Labeled Substrate. In The 11th International Symposium on Drug Analysis and the 29th International Symposium on Pharmaceutical and Biomedical Analysis (DA-PBA), 2018. 2018.

@proceedings{1482458,
   author = {Řemínek, Roman and Schejbal, Jan and Glatz, Zdeněk},
   booktitle = {The 11th International Symposium on Drug Analysis and the 29th International Symposium on Pharmaceutical and Biomedical Analysis (DA-PBA), 2018},
   keywords = {capillary electrophoresis, Alzheimer's disease, beta-secretase, inhibition, fluorescent detection, FRET},
   language = {eng},
   title = {Development of Capillary Electrophoretic Method with Fluorescence Detection for Beta-Secretase Activity Assays with FRET-Labeled Substrate},
   url = {https://kuleuvencongres.be/DA-PBA-2018/articles},
   year = {2018}
}

TY  - CONF
ID  - 1482458
AU  - Řemínek, Roman - Schejbal, Jan - Glatz, Zdeněk
PY  - 2018
TI  - Development of Capillary Electrophoretic Method with Fluorescence Detection for Beta-Secretase Activity Assays with FRET-Labeled Substrate
KW  - capillary electrophoresis, Alzheimer's disease, beta-secretase, inhibition, fluorescent detection, FRET
UR  - https://kuleuvencongres.be/DA-PBA-2018/articles
N2  - Alzheimer's disease (AD) represents a degenerative brain disorder that is characterized by a progressive loss of memory and other cognitive abilities seriously affecting a person's ability to carry out daily activities. Currently available medications temporarily improve accompanying symptoms; however, a cure to treat the disease itself has not been introduced yet. Recent studies suggest beta-secretase, an aspartic-acid protease playing a key role in the development of neurotoxic amyloid plagues in the patients' brain tissue, as a promising druggable target to slow down or even stop the progression of AD. Screenings of potential beta-secretase inhibitors are commonly performed by using systems based on Förster resonance energy transfer (FRET). The information gathered from these assays can be negatively affected by low solubility of fluorescently labeled substrates and non-specific interferences with measured signal leading to a risk of false positive/negative results and need of data confirmation by further tests though. For these reasons the main aim of the presented study was to develop a method for activity studies of beta-secretase based on the capillary electrophoresis with fluorescence detection. Separation of a reaction product increases the assay sensitivity and diminishes the risk of interactions with other sample components. Separation conditions were optimized using standards of the QXL® 520/ HiLyteTM Fluor 488 FRET substrate and HiLyteTM 488 reaction product obtained from commercially available SensoLyte® 520 beta-secretase assay kit. An Agilent 7100 CE system equipped with Picometrics Zetalif LEDIF detector was used to perform all analyses. In the final method, the reaction product labeled with HiLyteTM 488 dye was separated by application of 30 kV (positive polarity) inside the uncoated fused-silica capillary (45 cm effective length, 50 m id) thermostated at 25 °C. 45 mM SDS prepared in 65 mM tetraborate buffer (pH 9.2) was used as BGE. The analyte was monitored at excitation/emission = 490 nm/520 nm. The capillary was rinsed with 0.1 M NaOH for 1 min, deionized water for 1 min, and BGE for 2 min before every analysis. This procedure was sufficient to ensure good method repeatability despite sample containing the enzyme and substrate and products peptides was directly injected into the capillary. The optimized method was validated and applied for activity assays of beta-secretase. The results obtained were in a good agreement with literature and confirmed the practical applicability of the developed method.
ER  -

ŘEMÍNEK, Roman, Jan SCHEJBAL a Zdeněk GLATZ. Development of Capillary Electrophoretic Method with Fluorescence Detection for Beta-Secretase Activity Assays with FRET-Labeled Substrate. In \textit{The 11th International Symposium on Drug Analysis and the 29th International Symposium on Pharmaceutical and Biomedical Analysis (DA-PBA), 2018}. 2018.

Podrobný výpis o publikaci