Difficulties associated with the structural analysis of proteins susceptible to form aggregates: the case of tau protein as a biomarker of Alzheimer's disease

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00023752%3A_____%2F16%3A43914828" target="_blank" >RIV/00023752:_____/16:43914828 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/00216275:25310/16:39902239

Výsledek na webu

<a href="http://onlinelibrary.wiley.com/doi/10.1002/jssc.201501045/epdf" target="_blank" >http://onlinelibrary.wiley.com/doi/10.1002/jssc.201501045/epdf</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1002/jssc.201501045" target="_blank" >10.1002/jssc.201501045</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Difficulties associated with the structural analysis of proteins susceptible to form aggregates: the case of tau protein as a biomarker of Alzheimer's disease

Popis výsledku v původním jazyce

Mass spectrometry coupled with bioaffinity separation techniques is considered a powerful tool for studying protein interactions. This work is focused on epitope analysis of tau protein, which contains two VQIXXK aggregation motifs regarded as crucial elements in the formation of paired helical filaments, the main pathological characteristics of Alzheimer's disease. To identify major immunogenic structures, the epitope extraction technique utilizing protein fragmentation and magnetic microparticles functionalized with specific antibodies was applied. However, the natural adhesiveness of some newly generated peptide fragments devalued the experimental results. Beside presumed peptide fragment specific to applied monoclonal anti-tau antibodies, the epitope extraction repeatedly revealed inter alia tryptic fragment 299-HVPGGGSVQIVYKPVDLSK-317 containing the fibril-forming motif 306-VQIVYK-311. The tryptic fragment pro-aggregation and hydrophobic properties that might contribute to adsorption phenomenon were examined by Thioflavin S and reversed-phase chromatography. Several conventional approaches to reduce the non-specific fragment sorption onto the magnetic particle surface were performed, however with no effect. To avoid methodological complications, we introduced an innovative approach based on altered proteolytic digestion. Simultaneous fragmentation of tau protein by two immobilized proteases differing in the cleavage specificity (TPCK-trypsin and α-chymotrypsin) led to the disruption of motif responsible for undesirable adhesiveness and enabled us to obtain undistorted structural data.

Název v anglickém jazyce

Difficulties associated with the structural analysis of proteins susceptible to form aggregates: the case of tau protein as a biomarker of Alzheimer's disease

Popis výsledku anglicky

Mass spectrometry coupled with bioaffinity separation techniques is considered a powerful tool for studying protein interactions. This work is focused on epitope analysis of tau protein, which contains two VQIXXK aggregation motifs regarded as crucial elements in the formation of paired helical filaments, the main pathological characteristics of Alzheimer's disease. To identify major immunogenic structures, the epitope extraction technique utilizing protein fragmentation and magnetic microparticles functionalized with specific antibodies was applied. However, the natural adhesiveness of some newly generated peptide fragments devalued the experimental results. Beside presumed peptide fragment specific to applied monoclonal anti-tau antibodies, the epitope extraction repeatedly revealed inter alia tryptic fragment 299-HVPGGGSVQIVYKPVDLSK-317 containing the fibril-forming motif 306-VQIVYK-311. The tryptic fragment pro-aggregation and hydrophobic properties that might contribute to adsorption phenomenon were examined by Thioflavin S and reversed-phase chromatography. Several conventional approaches to reduce the non-specific fragment sorption onto the magnetic particle surface were performed, however with no effect. To avoid methodological complications, we introduced an innovative approach based on altered proteolytic digestion. Simultaneous fragmentation of tau protein by two immobilized proteases differing in the cleavage specificity (TPCK-trypsin and α-chymotrypsin) led to the disruption of motif responsible for undesirable adhesiveness and enabled us to obtain undistorted structural data.

Druh

J_x - Nezařazeno - Článek v odborném periodiku (Jimp, Jsc a Jost)

CEP obor

CB - Analytická chemie, separace

OECD FORD obor

—

Projekt

Výsledek vznikl pri realizaci vícero projektů. Více informací v záložce Projekty.

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Rok uplatnění

2016

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Journal of Separation Science

ISSN

1615-9306

e-ISSN

—

Svazek periodika

39

Číslo periodika v rámci svazku

4

Stát vydavatele periodika

DE - Spolková republika Německo

Počet stran výsledku

9

Strana od-do

799-807

Kód UT WoS článku

000371163300023

EID výsledku v databázi Scopus

2-s2.0-84954290607