• Je něco špatně v tomto záznamu ?

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

L. Hromadkova, R. Kupcik, B. Jankovicova, T. Rousar, D. Ripova, Z. Bilkova,

. 2016 ; 39 (4) : 799-807. [pub] 20160112

Jazyk angličtina Země Německo

Typ dokumentu časopisecké články, Research Support, N.I.H., Extramural, práce podpořená grantem

Perzistentní odkaz   https://www.medvik.cz/link/bmc17014201

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.

Citace poskytuje Crossref.org

000      
00000naa a2200000 a 4500
001      
bmc17014201
003      
CZ-PrNML
005      
20170427105505.0
007      
ta
008      
170413s2016 gw f 000 0|eng||
009      
AR
024    7_
$a 10.1002/jssc.201501045 $2 doi
035    __
$a (PubMed)26644024
040    __
$a ABA008 $b cze $d ABA008 $e AACR2
041    0_
$a eng
044    __
$a gw
100    1_
$a Hromadkova, Lenka $u Department of Neurobiology and AD Center, National Institute of Mental Health, Klecany, Czech Republic. Faculty of Science, Charles University in Prague, Prague, Czech Republic. Department of Biological and Biochemical Sciences, University of Pardubice, Pardubice, Czech Republic.
245    10
$a Difficulties associated with the structural analysis of proteins susceptible to form aggregates: The case of Tau protein as a biomarker of Alzheimer's disease / $c L. Hromadkova, R. Kupcik, B. Jankovicova, T. Rousar, D. Ripova, Z. Bilkova,
520    9_
$a 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.
650    _2
$a adhezivita $7 D000268
650    _2
$a adsorpce $7 D000327
650    _2
$a Alzheimerova nemoc $x diagnóza $7 D000544
650    _2
$a aminokyselinové motivy $7 D020816
650    _2
$a monoklonální protilátky $x chemie $7 D000911
650    _2
$a biologické markery $x chemie $7 D015415
650    _2
$a chymotrypsin $x chemie $7 D002918
650    _2
$a epitopy $x chemie $7 D000939
650    _2
$a lidé $7 D006801
650    _2
$a magnetismus $7 D008280
650    _2
$a hmotnostní spektrometrie $x metody $7 D013058
650    _2
$a proteolýza $7 D059748
650    _2
$a spektrometrie hmotnostní - ionizace laserem za účasti matrice $7 D019032
650    _2
$a thiazoly $x chemie $7 D013844
650    _2
$a trypsin $x chemie $7 D014357
650    _2
$a proteiny tau $x chemie $7 D016875
655    _2
$a časopisecké články $7 D016428
655    _2
$a Research Support, N.I.H., Extramural $7 D052061
655    _2
$a práce podpořená grantem $7 D013485
700    1_
$a Kupcik, Rudolf $u Department of Biological and Biochemical Sciences, University of Pardubice, Pardubice, Czech Republic.
700    1_
$a Jankovicova, Barbora $u Department of Biological and Biochemical Sciences, University of Pardubice, Pardubice, Czech Republic.
700    1_
$a Rousar, Tomas $u Department of Biological and Biochemical Sciences, University of Pardubice, Pardubice, Czech Republic.
700    1_
$a Ripova, Daniela $u Department of Neurobiology and AD Center, National Institute of Mental Health, Klecany, Czech Republic.
700    1_
$a Bilkova, Zuzana $u Department of Biological and Biochemical Sciences, University of Pardubice, Pardubice, Czech Republic.
773    0_
$w MED00006463 $t Journal of separation science $x 1615-9314 $g Roč. 39, č. 4 (2016), s. 799-807
856    41
$u https://pubmed.ncbi.nlm.nih.gov/26644024 $y Pubmed
910    __
$a ABA008 $b sig $c sign $y a $z 0
990    __
$a 20170413 $b ABA008
991    __
$a 20170427105825 $b ABA008
999    __
$a ok $b bmc $g 1200666 $s 974979
BAS    __
$a 3
BAS    __
$a PreBMC
BMC    __
$a 2016 $b 39 $c 4 $d 799-807 $e 20160112 $i 1615-9314 $m Journal of separation science $n J Sep Sci $x MED00006463
LZP    __
$a Pubmed-20170413

Najít záznam

Citační ukazatele

Možnosti archivace