Application of MALDI-TOF mass spectrometry for study on fibrillar and oligomeric aggregates of alpha-synuclein
Aim. To study the -synuclein (ASN) aggregates of different structural origin, namely amyloid fibrils and spherical oligomers, in comparison with a native protein. Methods. MALDI TOF mass spectrometry and atomic for- ce microscopy (AFM). Results. The mass spectra of native and fibrillar ASN have simi...
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| Date: | 2014 |
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Інститут молекулярної біології і генетики НАН України
2014
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| Cite this: | Application of MALDI-TOF mass spectrometry for study on fibrillar and oligomeric aggregates of alpha-synuclein / O.V. Severinovskaya, V.B. Kovalska, M.Yu. Losytskyy, V.V. Cherepanov, V. Subramaniam, S.M. Yarmoluk // Вiopolymers and Cell. — 2014. — Т. 30, № 3. — С. 190-196. — Бібліогр.: 26 назв. — англ. |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine| _version_ | 1859542811975614464 |
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| author | Severinovskaya, O.V. Kovalska, V.B. Losytskyy, M.Yu. Cherepanov, V.V. Subramaniam, V. Yarmoluk, S.M. |
| author_facet | Severinovskaya, O.V. Kovalska, V.B. Losytskyy, M.Yu. Cherepanov, V.V. Subramaniam, V. Yarmoluk, S.M. |
| citation_txt | Application of MALDI-TOF mass spectrometry for study on fibrillar and oligomeric aggregates of alpha-synuclein / O.V. Severinovskaya, V.B. Kovalska, M.Yu. Losytskyy, V.V. Cherepanov, V. Subramaniam, S.M. Yarmoluk // Вiopolymers and Cell. — 2014. — Т. 30, № 3. — С. 190-196. — Бібліогр.: 26 назв. — англ. |
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| container_title | Вiopolymers and Cell |
| description | Aim. To study the -synuclein (ASN) aggregates of different structural origin, namely amyloid fibrils and spherical oligomers, in comparison with a native protein. Methods. MALDI TOF mass spectrometry and atomic for- ce microscopy (AFM). Results. The mass spectra of native and fibrillar ASN have similar character, i. e. they are characterized by the well pronounced peak of protein molecular ion, the low molecular weight associates, and rather low contain of fragmentation products. The spectrum of oligomeric aggregate is characterized by the high contain of fragmentation products, low intensity of protein molecular ion and the absence of peaks of associates. Conclusions. The difference between the spectra of fibrillar and oligomeric ASN could be explained, first, by the different content of the «residual» monomeric ASN and the protein degradation products in the studied samples, and, second, by the different structure-depended mechanisms of the protein degradation induced by the laser ionization. We suggested that the MALDI-TOF mass spectroscopy is a method useful for the investigation of ASN aggregation and characterization of its high order self-associates; besides, there is an interest in estimating the potency of the MALDI-TOF for the analysis of aggregation of various amyloidogenic proteins.
Мета. Вивчення агрегатів альфа-синуклеїну (ASN) різного структурового походження, а саме – амілоїдних фібрил і сферичних олігомерів порівняно з нативним білком. Методи. MALDI-TOF мас-спектрометрія та атомно-силова мікроскопія (АFМ). Результати. Мас-спектри нативного і фібрилярного ASN мають подібний характер – для них характерні інтенсивний пік молекулярного іона білка, піки низькомолекулярних асоціатов та досить незначний вміст продуктів фрагментації білка. У той же час у спектрі олігомерних агрегатів спостерігаються висока концентрація продуктів фрагментації білка, низька інтенсивність молекулярного іона та відсутність піків самоасоціатів. Висновки. Різницю між спектрами фибрилярного та олігомерного ASN можна пояснити як наявністю у зразках «залишкового» ASN і продуктів деградації білка, так і різними структурово залежними механізмами руйнування цих двох видів агрегатів при лазерній десорбції/іонізації. MALDI-TOF мас-спектрометрію можна запропонувати як метод вивчення агрегації та аналізу високомолекулярних агрегатів ASN. Також представляє інтерес визначення ефективності цього методу для дослідження агрегатів різних амілоїдогенних білків.
Цель. Изучение агрегатов альфа-синуклеина (ASN) различной структуры, а именно – амилоидных фибрилл и сферических олигомеров в сравнении с нативным белком. Методы. MALDI-TOF масс-спектрометрия и атомно-силовая микроскопия (АFМ). Результаты. Масс-спектры нативного и фибриллярного ASN имеют подобный характер – для них характерны интенсивный пик молекулярного иона белка, пики низкомолекулярных ассоциатов, а также достаточно незначительное содержание продуктов фрагментации белка. В то же время в спектре олигомерных агрегатов наблюдаются высокая концентрация продуктов фрагментации белка, молекулярный ион низкой интенсивности и отсутствие пиков ассоциатов белка. Выводы. Различие между спектрами фибриллярного и олигомерного ASN можно объяснить как содержанием «избытка» ASN и продуктов деградации белка, так и различными структурно-зависимыми механизмами разрушениями этих двух видов агрегатов при лазерной десорбции/ионизации. MALDI-TOF масс-спектрометрию можно предложить в качестве метода изучения агрегации и анализа высокомолекулярных агрегатов ASN. Также представляет интерес определение эффективности MALDI-TOF для исследования агрегатов различных амилоидогенных белков.
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STRUCTURE AND FUNCTION OF BIOPOLYMERS
UDC 577.336 + 667.287.4 + 543.51
Application of MALDI-TOF mass spectrometry
for study on fibrillar and oligomeric aggregates of
alpha-synuclein
O. V. Severinovskaya1, V. B. Kovalska2, M. Yu. Losytskyy2, V. V. Cherepanov3,
V. Subramaniam4, S. M. Yarmoluk2
1O. O. Chuiko Institute of Surface Chemistry, NAS of Ukraine
17, Generala Naumova Str., Kyiv, Ukraine, 03164
2Institute of Molecular Biology and Genetics, NAS of Ukraine
150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03143
3Institute of Physics, NAS of Ukraine
46, Nauky Ave., Kyiv, Ukraine, 03028
4Nanobiophysics Group, MESA + Institute for Nanotechnology and MIRA Institute for Biomedical Technology, University of Twente
PO Box 217, 7500 AE Enschede, The Netherlands
severinovskaya.o@gmail.com
Aim. To study the �-synuclein (ASN) aggregates of different structural origin, namely amyloid fibrils and sphe-
rical oligomers, in comparison with a native protein. Methods. MALDI TOF mass spectrometry and atomic for-
ce microscopy (AFM). Results. The mass spectra of native and fibrillar ASN have similar character, i. e. they are
characterized by the well pronounced peak of protein molecular ion, the low molecular weight associates, and
rather low contain of fragmentation products. The spectrum of oligomeric aggregate is characterized by the high
contain of fragmentation products, low intensity of protein molecular ion and the absence of peaks of associates.
Conclusions. The difference between the spectra of fibrillar and oligomeric ASN could be explained, first, by the
different content of the «residual» monomeric ASN and the protein degradation products in the studied samples,
and, second, by the different structure-depended mechanisms of the protein degradation induced by the laser ioni-
zation. We suggested that the MALDI-TOF mass spectroscopy is a method useful for the investigation of ASN ag-
gregation and characterization of its high order self-associates; besides, there is an interest in estimating the po-
tency of the MALDI-TOF for the analysis of aggregation of various amyloidogenic proteins.
Keywords: alpha-synuclein, MALDI-TOF, amyloid fibril, oligomeric aggregate, AFM.
Introduction. Pathogenesis of some harmful disorders
among them neurodegenerative disorders (Parkinson,
Alzheimer’s diseases), prion diseases, type II diabetes
is associated with the spontaneous uncontrolled protein
aggregation, particularly with the formation of amyloid
fibrils. Besides, a wide range of proteins, not involved in
a certain disease, are able to form amyloid aggregates
[1]. Thefore, the study on the protein aggregation is an
actual biomedical task.
Among the methods of protein analysis the MALDI-
TOF mass spectrometry is known as a popular and ver-
satile tool [2]. In the proteomics, particularly in the stu-
dies on protein non-controlled aggregation, MALDI-
TOF is mostly applied for the identification of protein
origin by a proteolysis-based mass mapping method
[3, 4].
The method of direct (excluding proteolysis diges-
tion) MALDI-TOF has a low descriptiveness for the
characterization of the protein high-molecular aggre-
gates and was applied to analyze the fibrillization in-
190
ISSN 0233–7657. Biopolymers and Cell. 2014. Vol. 30. N 3. P. 190–196 doi: http://dx.doi.org/10.7124/bc.000895
� Institute of Molecular Biology and Genetics, NAS of Ukraine, 2014
termediates and side products, e. g. the misfolded be-
ta-lactoglobuline dimers and protein degradation pro-
ducts [5].
Alpha-synuclein (ASN) is a small natively unfolded
protein that plays a central role in the etiology of Parkin-
son’s disease. It forms amyloid fibrils that are found in
Lewy bodies, i. e. cell depositions in the brain that are
characteristic of this disease [6, 7]. Besides, during the
aggregation process ASN forms small oligomeric inter-
mediates, which are considered as presumably toxic
species [8–10].
Here we report the examination of the ASN aggre-
gates populations of different structural origin, namely
amyloid fibrils and spherical oligomers, by the method
of MALDI-TOF mass spectrometry. We suggest that the
filamentous amyloid fibrils and spherical oligomers due
to the distinct folding of the protein molecules and asso-
ciation/degradation on the aggregation pathway could
possess the mass spectra of different character. Besides,
we intend to evaluate the applicability of MALDI-TOF
as a method for the analysis of protein aggregates and
study on the aggregation process.
Materials and methods. Reagents. Recombinant
human wild-type ASN was expressed and purified in
10 mM Tris-HCl, 50 mM NaCl (pH 7.4) as described ear-
lier [11]. ASN amyloid fibrils and oligomers were obtai-
ned according to [11] and [12] correspondingly.
MALDI-TOF studies. The samples of native, fibril-
lar and oligomeric ASN were prepared for the MALDI-
TOF analysis as follows: matrix material (12 mg of 3,
5-dimethoxy-4-hydroxycinnamic (sinapinic) acid) was
dissolved in 1 ml of water/acetonytyl solution 1:1 (v/v)
with addition of 0.1 % (v/v) of trifluoroacetic acid. The
obtained solution was incubated during 10 min at 30 °C
in ultrasonic bath up to the full dissolving of the acid.
[13]. The 50 µmol protein samples in 0,05 M Tris-HCl
buffer (pH 7.9) with concentration of 1mg/ml were
then added to the matrix solution in 1:1 ratio. Small ali-
quots of the mixture were applied to the steel probe tips
and dried. Mass analysis was performed on the «Auto-
flex II» («Bruker Daltonics», Germany) MALDI-TOF
mass spectrometer with nitrogen laser (� = 337 nm).
Spectra were obtained for the mass range 7 000 to 100
000 m/z. in positive ion reflectron registration mode.
The spectra were obtained by summing the data of 100
laser shots.
Atomic force microscopy studies. The structure of
ASN aggregates was studied by means of AFM («Sol-
ver Pro M» system, NT-MDT, RF). The scanning rate
was 30 �m/s. For the formation of subnanolayer consis-
ting of separate nanoobjects, the reaction solutions we-
re diluted 30 times with bidistilled water. Then a drop
of the solution was applied on the freshly cleaved surfa-
ce of mica. The AFM measurements were carried out in
a tapping mode at ambient conditions after the full eva-
poration of the solvent. The AFM probes of type NSG01
(NT-MDT) were used. The average diameter values of
ASN aggregates were determined based on their heights
in the AFM images.
Results and discussion. Structural peculiarities of
native, fibrillar and oligomeric ASN. ASN is a small
(140 amino acids) protein that is known to be natively
unfolded in solutions. The protein's amino acid sequen-
ce consists of a basic N-terminal region (residues 1–95)
containing repeats of highly conserved KTKEGV hexa-
meric motif, and an acidic C-terminal region (residues
96–140) [14], the first 100 residues are predicted to ha-
ve �-helical propensity [15].
Amyloid fibrils are stable protein aggregates, with
cross-� filament structure where the protein molecules,
making up the �-sheet, are arranged perpendicular to
the fibril axis [16]. The core of the ASN fibril contains
mainly the protein residues of the basic region (appro-
ximately residues 38–95 [17]). We have estimated the
characteristics of the ASN fibrils population by AFM.
These fibrils are both linear and branched long filaments
with the diameter of single filament about 3–8 nm (Fig.
1). The bundles of fibrils formed by overlapping of sing-
le filaments were observed in the studied sample as well.
The strong fluorescent sensitivity of the amyloid-
specific cyanine dye 7519 to the ASN fibrils was pre-
viously reported [18].
The ASN oligomers are known as spherical protein
aggregates of the size in the range 2–20 nm [19–21], be-
sides, the structures with annular and tubular morpho-
logy have also been reported [21–23].
Our AFM study demonstrated the ASN oligomer
population to be mostly the species of the height from 3
till 6 nm (Fig. 2). Besides, the structures formed due to
the coalescence of these aggregates are observed; the
diameter of such «super-oligomeric» structures being
up to 10 nm.
191
MALDI-TOF MASS SPECTROMETRY FOR STUDY ON AGGREGATES OF �-SYNUCLEIN
192
SEVERINOVSKAYA O. V. ET AL.
Despite the oligomer aggregates are mainly consi-
dered to possess beta-sheet structure [24, 25], the pre-
sence of �-helical content has been shown as well [19].
The difference between the secondary structure motifs
of amyloid fibrils and oligomeric aggregates is indi-
cated by the different sensitivity of amyloid-specific
cyanine dyes to these ASN formations [26]. The noti-
ceably lower fluorescent response of the dyes on the
presence of oligomers comparing to fibrils is explained
by the lower content of beta-pleated regions accessible
for the dye molecules.
MALDI-TOF studies. The mass spectra of native,
fibrillar and oligomeric ASN are presented in Fig. 3.
All spectra contain the peaks corresponding to the pro-
tein molecular ion about 14460 m/z and a wide range of
protein fragmentation peaks, but only two of these peaks
H
ei
g
h
t,
n
m
2
4
0
6
2 1040 6 8 µm
a b
µm0 62 4 8 10
0
6
4
2
8
10
8
Fig. 1. AFM image of amyloid fibril of ASN (a) and Z-profile along the line marked on the image (b)
µm0 62 4 8
0
6
4
2
8
a b
2 840 6 µm
2
4
0
6
8
H
ei
g
h
t,
n
m
10
Fig. 2. AFM image of oligomer aggregates of ASN (a) and Z-profile along the line marked on the image (b)
(about 13720 and 10630 m/z) are common for all protein
forms.
The peaks corresponding to low-order associates
were detected only for native and fibrillar ASN. Thus, we
suggest that the protein mass-spectra depend on its
association degree and folding type (native ASN, oligo-
mer or fibril).
M o l e c u l a r i o n a n d p r o t e i n a s s o c i a t e s.
In the mass spectrum of native ASN the molecular ion
peak with molecular mass about14460 m/z is the most
intensive and highly resolved, it contains satellite shoul-
der corresponding to an associate of ASN molecule with
the matrix one (sinapic acid). The mass spectrum is cha-
racterized by insignificant content of the protein frag-
193
MALDI-TOF MASS SPECTROMETRY FOR STUDY ON AGGREGATES OF �-SYNUCLEIN
Fragment Mass, m/z
Native Oligomer Fibril
I, a. u. I, a. u. I, a. u.
[5M]
+
72170 280 – –
[4M]
+
57720 734 – –
[3M]
+
43260 2234 – 739
[2M]
+
28860 9808 – 2681
[M]
+
14460 66459 1672 40648
F1 13720 6441 1167 9148
F2 10630 3379 3427 8407
N o t e. Mass – observed mass of the peaks; I – intensity of peaks; a. u. – arbitrary units; [M]
+
– molecular ion; [nM]
+
(where n = 2–5) – molecular
associates of ASN containing n protein molecules; F1 and F2 – fragments that are common for all protein forms.
Table 1
Protein fragments and associates observed in mass spectra of native and aggregated ASN
Fragment (Forme) I [M]+/I[M]+ – [1–40] I [M]+/I[M]+ – [1–7] [M]+ [2M]+ [3M]+ [4M]+ [5M]+
Native 0.05 0.09 1 0.14 0.03 0.01 0.004
Oligomer 2.04 2.04 1
Fibril 0.20 0.22 1 0.06 0.02
Table 2
Ratio between intensities of ASN fragments and molecular ion peaks
m/z
a b c
20000 40000 60000 80000
0
20000
40000
60000
[4M]
+[3M]
+
[2M]
+
[M]
+
I, a. u
20000 40000 60000 80000
0
1000
2000
3000
[M
+
]
I, a.u.
20000 40000 60000 80000
0
10000
20000
30000
40000
[3M]
+
[2M]
+
[M]
+
I, a.u.
Fig. 3. General mass spectrum of native (a), oligomeric (b) and fibrillar (c) ASN
mentation products. The series of peaks with the inten-
sities decreasing upon the mass increase are detected in
the high mass region; they correspond to the protein mo-
lecular associates from dimer to pentamer with molecu-
lar masses about 28860, 43260, 57720 and 72170 m/z,
correspondingly (Table 1, 2). The association is caused
by intermolecular interactions and is typically obser-
ved in the MALDI-TOF mass spectra of different pro-
teins (albumin, insulin, lysozime etc.) [4, 13], the forma-
tion of ASN dimer, trimer and tetramer was also descri-
bed [15].
In the fibrillar protein spectrum, similarly to that of
the native ASN, the peak of molecular ion is the most in-
tensive. The low order associates are also presented in
the spectrum, but the number of aggregated molecules
(only dimers and trimers were observed) and intensity of
peaks are lower than for the native protein. On the other
hand, the number and intensity of the peaks of fragmen-
tation products for the fibrillar ASN is enhanced as com-
pared to the native protein.
The mass spectrum of oligomer significantly differs
from that of native and fibrillar protein. It is characteri-
zed by a very high fragmentation degree (the peak of
molecular ion is less intensive than the main peaks of
fragmentation products) and by the absence of associa-
tes peaks.
T h e f r a g m e n t a t i o n p r o d u c t s. In the mass
spectrum of the native, fibrillar and oligomeric ASN the
wide range of protein fragmentation product is detected
(Fig. 4, Table 1, 2). However, only two of them with mo-
lecular masses about 13720 and 10630 m/z (F1 and F2
correspondingly), are common for all protein forms.
The content of fragmentation products in the native
ASN mass spectrum is quite low, the peaks with m/z of
about 13980, 13720, 10830 and 10630 correspond to
the major fragmentation products of ASN molecule.
In the case of fibrillar ASN the content of protein
fragmentation products is much higher than in the mass
spectrum of native protein, the major peaks with m/z of
about 13720, 12950, 11870, 10830, 10630, 9680 and
9300 were detected.
In the oligomeric ASN spectrum the wide poorly re-
solved bands of protein fragmentation products domi-
nate. The two peaks of about 11130 m/z and 13980 m/z
194
SEVERINOVSKAYA O. V. ET AL.
a b c
10000 12000 14000
0
20000
40000
60000
[F2]
[F1]
[M]
+
I, a.u
10000 12000 14000
0
1000
2000
3000
[F2]
[F1]
[M
+
]
I, a.u.
m/z
10000 12000 14000
0
10000
20000
30000
40000
[F2] [F1]
[M]
+
I, a.u.
Fig. 4. Representative mass spectra of native (a), oligomeric (b) and fibrillar ASN (c) in molecular mass region 9000–15000 m/z
10 20 30 40 50 60
MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH VATVAEKTK
70 80 90 100 110 120
EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP
130 140
DNEAYEMPSE EGYQDYEPEA
Fig. 5. Sequence of human ASN (from uniprot.db, entry P37840)
are twice more intensive than this of the molecular ion
of ASN. Another intensive peak of about 10630 m/z is
also present in the spectra of native and fibrillar ASN.
The fragment with m/z about 13720 that is common for
all protein forms has low intensity in spectra of oligome-
ric aggregate.
Explanations of a distinct character of the fibrillar
and oligomeric ASN mass-spectra. We could propose
two explanations of the distinctions in the mass-spectra
of fibrillar and oligomeric ASN.
T h e f i r s t e x p l a n a t i o n is that the similarity
of the native and fibrillar ASN mass specta is caused by
the presence of an excess of non-aggregated protein in
the fibrillar ASN sample. Since the ASN monomeric
molecules are easily ionized they give a high intensive
peak of the molecular ion and associates.
The distinction between the fragmentation of fibrils
and oligomers could be caused by the presence of spe-
cific degradation products formed during the aggrega-
tion reaction or later storage of the samples.
A n o t h e r p o s s i b l e e x p l a n a t i o n is dis-
similarity in the folding of protein molecules in the amy-
loid fibrils and the oligomeric species and different struc-
ture of these aggregates.
It may be supposed that due to their regular ladder-
like structure the degradation of the amyloid fibrils upon
ionization in a large extent occurs through the tearing
off the intact protein molecules from the end (one by one)
of the filament. These protein molecules are responsib-
le for an intensive peak of the molecular ion and peaks of
the low-ordered associates.
Besides, the laser-induced degradation could occur
through a break in the protein chain, that leads to the ap-
pearance of new intensive bands of the protein frag-
mentation products. This mechanism is proved by high-
er number, content and lower resolution of the protein
fragment peaks in the spectrum of fibrils comparing
with the native ASN.
The large content of fragmentation products in the
spectrum of the oligomeric aggregates and their low
resolution may be explained by the poor ionization and
poor stability of these structures. We assume that due to
their spherical shape the oligomeric aggregates degra-
ded through the lost of protein fragments from the ope-
ned surface regions. The suggestion about the hindered
ability of degradation of the oligomeric aggregate through
the tearing off a whole protein molecule is supported by
the very low intensity of molecular ion.
Î. Â. Ñåâåðèíîâñüêà, Â. Á. Êîâàëüñüêà, Ì. Þ. Ëîñèöüêèé,
Â. Â. ×åðåïàíîâ, Â. Ñóáðàìàí³àì, Ñ. Ì. ßðìîëþê
Âèêîðèñòàííÿ ìåòîäó MALDI-TOF ìàññ-ñïåêòðîìåò𳿠äëÿ
âèâ÷åííÿ ô³áðèëÿðíèõ òà îë³ãîìåðíèõ àãðåãàò³â àëüôà-ñèíóêëå¿íó
Ðåçþìå
Ìåòà. Âèâ÷åííÿ àãðåãàò³â àëüôà-ñèíóêëå¿íó (ASN) ð³çíîãî ñòðóê-
òóðîâîãî ïîõîäæåííÿ, à ñàìå – àì³ëî¿äíèõ ô³áðèë ³ ñôåðè÷íèõ
îë³ãîìåð³â ïîð³âíÿíî ç íàòèâíèì á³ëêîì. Ìåòîäè. MALDI-TOF
ìàñ-ñïåêòðîìåòð³ÿ òà àòîìíî-ñèëîâà ì³êðîñêîï³ÿ (ÀFÌ). Ðå-
çóëüòàòè. Ìàñ-ñïåêòðè íàòèâíîãî ³ ô³áðèëÿðíîãî ASN ìàþòü
ïîä³áíèé õàðàêòåð – äëÿ íèõ õàðàêòåðí³ ³íòåíñèâíèé ï³ê ìîëåêó-
ëÿðíîãî ³îíà á³ëêà, ï³êè íèçüêîìîëåêóëÿðíèõ àñîö³àòîâ òà äîñèòü
íåçíà÷íèé âì³ñò ïðîäóêò³â ôðàãìåíòàö³¿ á³ëêà. Ó òîé æå ÷àñ ó
ñïåêòð³ îë³ãîìåðíèõ àãðåãàò³â ñïîñòåð³ãàþòüñÿ âèñîêà êîíöåíò-
ðàö³ÿ ïðîäóêò³â ôðàãìåíòàö³¿ á³ëêà, íèçüêà ³íòåíñèâí³ñòü ìîëåêó-
ëÿðíîãî ³îíà òà â³äñóòí³ñòü ï³ê³â ñàìîàñîö³àò³â. Âèñíîâêè. гç-
íèöþ ì³æ ñïåêòðàìè ôèáðèëÿðíîãî òà îë³ãîìåðíîãî ASN ìîæíà
ïîÿñíèòè ÿê íàÿâí³ñòþ ó çðàçêàõ «çàëèøêîâîãî» ASN ³ ïðîäóêò³â
äåãðàäàö³¿ á³ëêà, òàê ³ ð³çíèìè ñòðóêòóðîâî çàëåæíèìè ìåõàí³ç-
ìàìè ðóéíóâàííÿ öèõ äâîõ âèä³â àãðåãàò³â ïðè ëàçåðí³é äåñîðá-
ö³¿/³îí³çàö³¿. MALDI-TOF ìàñ-ñïåêòðîìåòð³þ ìîæíà çàïðîïîíó-
âàòè ÿê ìåòîä âèâ÷åííÿ àãðåãàö³¿ òà àíàë³çó âèñîêîìîëåêóëÿðíèõ
àãðåãàò³â ASN. Òàêîæ ïðåäñòàâëÿº ³íòåðåñ âèçíà÷åííÿ åôåêòèâ-
íîñò³ öüîãî ìåòîäó äëÿ äîñë³äæåííÿ àãðåãàò³â ð³çíèõ àì³ëî¿äî-
ãåííèõ á³ëê³â.
Êëþ÷îâ³ ñëîâà: àëüôà-ñèíóêëå¿í, MALDI-TOF, àì³ëî¿äíà ô³áðè-
ëà, îë³ãîìåðí³ àãðåãàòè, ÀFÌ.
Î. Â. Ñåâåðèíîâñêàÿ, Â. Á. Êîâàëüñêàÿ, Ì. Þ. Ëîñèöêèé,
Â. Â. ×åðåïàíîâ, Â. Ñóáðàìàíèàì, Ñ. Í. ßðìîëþê
Èñïîëüçîâàíèå ìåòîäà MALDI-TOF ìàññ-ñïåêòðîìåòðèè
äëÿ èçó÷åíèÿ ôèáðèëëÿðíûõ è îëèãîìåðíûõ àãðåãàòîâ
àëüôà-ñèíóêëåèíà
Ðåçþìå
Öåëü. Èçó÷åíèå àãðåãàòîâ àëüôà-ñèíóêëåèíà (ASN) ðàçëè÷íîé
ñòðóêòóðû, à èìåííî – àìèëîèäíûõ ôèáðèëë è ñôåðè÷åñêèõ îëè-
ãîìåðîâ â ñðàâíåíèè ñ íàòèâíûì áåëêîì. Ìåòîäû. MALDI-TOF
ìàññ-ñïåêòðîìåòðèÿ è àòîìíî-ñèëîâàÿ ìèêðîñêîïèÿ (ÀFÌ). Ðå-
çóëüòàòû. Ìàññ-ñïåêòðû íàòèâíîãî è ôèáðèëëÿðíîãî ASN èìå-
þò ïîäîáíûé õàðàêòåð – äëÿ íèõ õàðàêòåðíû èíòåíñèâíûé ïèê
ìîëåêóëÿðíîãî èîíà áåëêà, ïèêè íèçêîìîëåêóëÿðíûõ àññîöèàòîâ,
à òàêæå äîñòàòî÷íî íåçíà÷èòåëüíîå ñîäåðæàíèå ïðîäóêòîâ
ôðàãìåíòàöèè áåëêà. Â òî æå âðåìÿ â ñïåêòðå îëèãîìåðíûõ àãðå-
ãàòîâ íàáëþäàþòñÿ âûñîêàÿ êîíöåíòðàöèÿ ïðîäóêòîâ ôðàãìåí-
òàöèè áåëêà, ìîëåêóëÿðíûé èîí íèçêîé èíòåíñèâíîñòè è îòñóò-
ñòâèå ïèêîâ àññîöèàòîâ áåëêà. Âûâîäû. Ðàçëè÷èå ìåæäó ñïåêò-
ðàìè ôèáðèëëÿðíîãî è îëèãîìåðíîãî ASN ìîæíî îáúÿñíèòü êàê
ñîäåðæàíèåì «èçáûòêà» ASN è ïðîäóêòîâ äåãðàäàöèè áåëêà,
òàê è ðàçëè÷íûìè ñòðóêòóðíî-çàâèñèìûìè ìåõàíèçìàìè ðàçðó-
øåíèÿìè ýòèõ äâóõ âèäîâ àãðåãàòîâ ïðè ëàçåðíîé äåñîðáöèè/èî-
íèçàöèè. MALDI-TOF ìàññ-ñïåêòðîìåòðèþ ìîæíî ïðåäëîæèòü
â êà÷åñòâå ìåòîäà èçó÷åíèÿ àãðåãàöèè è àíàëèçà âûñîêîìîëåêó-
ëÿðíûõ àãðåãàòîâ ASN. Òàêæå ïðåäñòàâëÿåò èíòåðåñ îïðåäåëå-
195
MALDI-TOF MASS SPECTROMETRY FOR STUDY ON AGGREGATES OF �-SYNUCLEIN
196
SEVERINOVSKAYA O. V. ET AL.
íèå ýôôåêòèâíîñòè MALDI-TOF äëÿ èññëåäîâàíèÿ àãðåãàòîâ ðàç-
ëè÷íûõ àìèëîèäîãåííûõ áåëêîâ.
Êëþ÷åâûå ñëîâà: àëüôà-ñèíóêëåèí, MALDI-TOF, àìèëîèäíàÿ
ôèáðèëëà, îëèãîìåðíûå àãðåãàòû, ÀFÌ.
REFERENCES
1. Green J, Goldsbury C, Mini T, Sunderji S, Frey P, Kistler J, Co-
oper G, Aebi U. Full-length rat amylin forms fibrils following
substitution of single residues from human amylin. J Mol Biol.
2003;326(4):1147–56.
2. Marvin LF, Roberts MA, Fay LB. Matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry in
clinical chemistry. Clin Chim Acta. 2003;337(1–2):11–21.
3. Nazabal A, Weber J. Characterization and quantitation of
antibody aggregates by high mass MALDI mass spectrometry. J
Biomol Tech. 2010; 21(3 Suppl): S36.
4. Metods in enzymology. Amyloid, prions and other protein aggre-
gates, part C. Eds I. Kheterpal, R. Wetzel. Amsterdam, Elsevier
Inc., 2006; Vol. 413. 375 p.
5. Hamada D, Dobson CM. A kinetic study of beta-lactoglobulin
amyloid fibril formation promoted by urea. Protein Sci. 2002;11
(10):2417–26.
6. Uversky VN. Neuropathology, biochemistry, and biophysics of
alpha-synuclein aggregation. J Neurochem. 2007;103(1):17–37.
7. Bartels AL, Leenders KL. Parkinson's disease: the syndrome, the
pathogenesis and pathophysiology. Cortex. 2009;45(8):915–21.
8. Lashuel HA, Petre BM, Wall J, Simon M, Nowak RJ, Walz T,
Lansbury PT Jr. Alpha-synuclein, especially the Parkinson's di-
sease-associated mutants, forms pore-like annular and tubular
protofibrils. J Mol Biol. 2002;322(5):1089–102.
9. Volles MJ, Lansbury PT Jr. Zeroing in on the pathogenic form of
alpha-synuclein and its mechanism of neurotoxicity in Parkin-
son's disease. Biochemistry. 2003;42(26):7871–8.
10. Volles MJ, Lee SJ, Rochet JC, Shtilerman MD, Ding TT, Kessler
JC, Lansbury PT Jr. Vesicle permeabilization by protofibrillar
alpha-synuclein: implications for the pathogenesis and
treatment of Parkinson's disease. Biochemistry. 2001;40(26):
7812–9.
11. van Raaij ME, Segers-Nolten IM, Subramaniam V. Quantitative
morphological analysis reveals ultrastructural diversity of amy-
loid fibrils from alpha-synuclein mutants. Biophys J. 2006;91
(11):L96–8.
12. van Rooijen BD, Claessens MM, Subramaniam V. Lipid bilayer
disruption by oligomeric alpha-synuclein depends on bilayer
charge and accessibility of the hydrophobic core. Biochim Bio-
phys Acta. 2009;1788(6):1271–8.
13. Dekina SS, Romanovska II, Gromovoy TYu. Influence of poly-
mers on lysozyme molecules association. Biopolym Cell. 2011;
27(6):442–445.
14. Sode K, Ochiai S, Kobayashi N, Usuzaka E. Effect of reparation
of repeat sequences in the human alpha-synuclein on fibrillation
ability. Int J Biol Sci. 2006;3(1):1–7.
15. Wang W, Perovic I, Chittuluru J, Kaganovich A, Nguyen LT, Li-
ao J, Auclair JR, Johnson D, Landeru A, Simorellis AK, Ju S, Co-
okson MR, Asturias FJ, Agar JN, Webb BN, Kang C, Ringe D,
Petsko GA, Pochapsky TC, Hoang QQ. A soluble �-synuclein
construct forms a dynamic tetramer. Proc Natl Acad Sci USA.
2011;108(43):17797–802.
16. Rambaran RN, Serpell LC. Amyloid fibrils: abnormal protein as-
sembly. Prion. 2008;2(3):112-7.
17. Cho MK, Kim HY, Fernandez CO, Becker S, Zweckstetter M.
Conserved core of amyloid fibrils of wild type and A30P mutant
�-synuclein. Protein Sci. 2011;20(2):387–95.
18. Volkova KD, Kovalska VB, Yu Losytskyy M, Veldhuis G, Se-
gers-Nolten GM, Tolmachev OI, Subramaniam V, Yarmoluk SM.
Studies of interaction between cyanine dye T-284 and fibrillar al-
pha-synuclein. J Fluoresc. 2010;20(6):1267–74.
19. Apetri MM, Maiti NC, Zagorski MG, Carey PR, Anderson VE.
Secondary structure of alpha-synuclein oligomers: characteri-
zation by raman and atomic force microscopy. J Mol Biol. 2006;
355(1):63–71.
20. Conway KA, Lee SJ, Rochet JC, Ding TT, Williamson RE, Lans-
bury PT Jr. Acceleration of oligomerization, not fibrillization, is
a shared property of both alpha-synuclein mutations linked to
early-onset Parkinson's disease: implications for pathogenesis
and therapy. Proc Natl Acad Sci U S A. 2000;97(2):571–6.
21. Lashuel HA, Petre BM, Wall J, Simon M, Nowak RJ, Walz T,
Lansbury PT Jr. Alpha-synuclein, especially the Parkinson's di-
sease-associated mutants, forms pore-like annular and tubular
protofibrils. J Mol Biol. 2002;322(5):1089–102.
22. Ding TT, Lee SJ, Rochet JC, Lansbury PT Jr. Annular alpha-
synuclein protofibrils are produced when spherical protofibrils
are incubated in solution or bound to brain-derived membranes.
Biochemistry. 2002;41(32):10209–17.
23. Quist A, Doudevski I, Lin H, Azimova R, Ng D, Frangione B,
Kagan B, Ghiso J, Lal R. Amyloid ion channels: a common
structural link for protein-misfolding disease. Proc Natl Acad
Sci USA. 2005;102(30):10427–32.
24. Volles MJ, Lee SJ, Rochet JC, Shtilerman MD, Ding TT, Kessler
JC, Lansbury PT Jr. Vesicle permeabilization by protofibrillar
alpha-synuclein: implications for the pathogenesis and treatment
of Parkinson's disease. Biochemistry. 2001;40(26):7812–9.
25. Kaylor J, Bodner N, Edridge S, Yamin G, Hong DP, Fink AL.
Characterization of oligomeric intermediates in alpha-synuclein
fibrillation: FRET studies of Y125W/Y133F/Y136F alpha-sy-
nuclein. J Mol Biol. 2005;353(2):357–72.
26. Kovalska VB, Losytskyy MY, Tolmachev OI, Slominskii YL, Se-
gers-Nolten GM, Subramaniam V, Yarmoluk SM. Tri- and penta-
methine cyanine dyes for fluorescent detection of �-synuclein
oligomeric aggregates. J Fluoresc. 2012;22(6):1441–8.
Received 31.12.13
|
| id | nasplib_isofts_kiev_ua-123456789-154302 |
| institution | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| issn | 0233-7657 |
| language | English |
| last_indexed | 2025-11-26T00:21:59Z |
| publishDate | 2014 |
| publisher | Інститут молекулярної біології і генетики НАН України |
| record_format | dspace |
| spelling | Severinovskaya, O.V. Kovalska, V.B. Losytskyy, M.Yu. Cherepanov, V.V. Subramaniam, V. Yarmoluk, S.M. 2019-06-15T12:32:39Z 2019-06-15T12:32:39Z 2014 Application of MALDI-TOF mass spectrometry for study on fibrillar and oligomeric aggregates of alpha-synuclein / O.V. Severinovskaya, V.B. Kovalska, M.Yu. Losytskyy, V.V. Cherepanov, V. Subramaniam, S.M. Yarmoluk // Вiopolymers and Cell. — 2014. — Т. 30, № 3. — С. 190-196. — Бібліогр.: 26 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.000895 https://nasplib.isofts.kiev.ua/handle/123456789/154302 577.336 + 667.287.4 + 543.51 Aim. To study the -synuclein (ASN) aggregates of different structural origin, namely amyloid fibrils and spherical oligomers, in comparison with a native protein. Methods. MALDI TOF mass spectrometry and atomic for- ce microscopy (AFM). Results. The mass spectra of native and fibrillar ASN have similar character, i. e. they are characterized by the well pronounced peak of protein molecular ion, the low molecular weight associates, and rather low contain of fragmentation products. The spectrum of oligomeric aggregate is characterized by the high contain of fragmentation products, low intensity of protein molecular ion and the absence of peaks of associates. Conclusions. The difference between the spectra of fibrillar and oligomeric ASN could be explained, first, by the different content of the «residual» monomeric ASN and the protein degradation products in the studied samples, and, second, by the different structure-depended mechanisms of the protein degradation induced by the laser ionization. We suggested that the MALDI-TOF mass spectroscopy is a method useful for the investigation of ASN aggregation and characterization of its high order self-associates; besides, there is an interest in estimating the potency of the MALDI-TOF for the analysis of aggregation of various amyloidogenic proteins. Мета. Вивчення агрегатів альфа-синуклеїну (ASN) різного структурового походження, а саме – амілоїдних фібрил і сферичних олігомерів порівняно з нативним білком. Методи. MALDI-TOF мас-спектрометрія та атомно-силова мікроскопія (АFМ). Результати. Мас-спектри нативного і фібрилярного ASN мають подібний характер – для них характерні інтенсивний пік молекулярного іона білка, піки низькомолекулярних асоціатов та досить незначний вміст продуктів фрагментації білка. У той же час у спектрі олігомерних агрегатів спостерігаються висока концентрація продуктів фрагментації білка, низька інтенсивність молекулярного іона та відсутність піків самоасоціатів. Висновки. Різницю між спектрами фибрилярного та олігомерного ASN можна пояснити як наявністю у зразках «залишкового» ASN і продуктів деградації білка, так і різними структурово залежними механізмами руйнування цих двох видів агрегатів при лазерній десорбції/іонізації. MALDI-TOF мас-спектрометрію можна запропонувати як метод вивчення агрегації та аналізу високомолекулярних агрегатів ASN. Також представляє інтерес визначення ефективності цього методу для дослідження агрегатів різних амілоїдогенних білків. Цель. Изучение агрегатов альфа-синуклеина (ASN) различной структуры, а именно – амилоидных фибрилл и сферических олигомеров в сравнении с нативным белком. Методы. MALDI-TOF масс-спектрометрия и атомно-силовая микроскопия (АFМ). Результаты. Масс-спектры нативного и фибриллярного ASN имеют подобный характер – для них характерны интенсивный пик молекулярного иона белка, пики низкомолекулярных ассоциатов, а также достаточно незначительное содержание продуктов фрагментации белка. В то же время в спектре олигомерных агрегатов наблюдаются высокая концентрация продуктов фрагментации белка, молекулярный ион низкой интенсивности и отсутствие пиков ассоциатов белка. Выводы. Различие между спектрами фибриллярного и олигомерного ASN можно объяснить как содержанием «избытка» ASN и продуктов деградации белка, так и различными структурно-зависимыми механизмами разрушениями этих двух видов агрегатов при лазерной десорбции/ионизации. MALDI-TOF масс-спектрометрию можно предложить в качестве метода изучения агрегации и анализа высокомолекулярных агрегатов ASN. Также представляет интерес определение эффективности MALDI-TOF для исследования агрегатов различных амилоидогенных белков. en Інститут молекулярної біології і генетики НАН України Вiopolymers and Cell Structure and Function of Biopolymers Application of MALDI-TOF mass spectrometry for study on fibrillar and oligomeric aggregates of alpha-synuclein Використання методу MALDI-TOF масс-спектрометрії для вивчення фібрилярних та олігомерних агрегатів альфа-синуклеїну Использование метода MALDI-TOF масс-спектрометрии для изучения фибриллярных и олигомерных агрегатов альфа-синуклеина Article published earlier |
| spellingShingle | Application of MALDI-TOF mass spectrometry for study on fibrillar and oligomeric aggregates of alpha-synuclein Severinovskaya, O.V. Kovalska, V.B. Losytskyy, M.Yu. Cherepanov, V.V. Subramaniam, V. Yarmoluk, S.M. Structure and Function of Biopolymers |
| title | Application of MALDI-TOF mass spectrometry for study on fibrillar and oligomeric aggregates of alpha-synuclein |
| title_alt | Використання методу MALDI-TOF масс-спектрометрії для вивчення фібрилярних та олігомерних агрегатів альфа-синуклеїну Использование метода MALDI-TOF масс-спектрометрии для изучения фибриллярных и олигомерных агрегатов альфа-синуклеина |
| title_full | Application of MALDI-TOF mass spectrometry for study on fibrillar and oligomeric aggregates of alpha-synuclein |
| title_fullStr | Application of MALDI-TOF mass spectrometry for study on fibrillar and oligomeric aggregates of alpha-synuclein |
| title_full_unstemmed | Application of MALDI-TOF mass spectrometry for study on fibrillar and oligomeric aggregates of alpha-synuclein |
| title_short | Application of MALDI-TOF mass spectrometry for study on fibrillar and oligomeric aggregates of alpha-synuclein |
| title_sort | application of maldi-tof mass spectrometry for study on fibrillar and oligomeric aggregates of alpha-synuclein |
| topic | Structure and Function of Biopolymers |
| topic_facet | Structure and Function of Biopolymers |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/154302 |
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