Molecula ly imp in ed polyme s coupled
o mass spec ome ic de ec ion
o me allo hionein sensing
VANĚČKOVÁ, T.; POMPEIANO VANÍČKOVÁ, L.; TVRDOŇOVÁ, M.; POMORSKI, A.;
KRĘŻEL, A.; VACULOVIČ, T.; KANICKÝ, V.; VACULOVIČOVÁ, M.; ADAM, V.
Talan a
2019, ol. 198, June 2019, pp. 224-229
ISSN: 0039-9140
DOI: h ps://doi.o g/10.1016/j. alan a.2019.01.089
Accep ed manusc ip
© 2018. This manusc ip e sion is made a ailable unde he CC-BY-NC-ND 4.0 license
(h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/),
doi: h ps://doi.o g/ 10.1016/j. alan a.2019.01.089
Final e sion a ailable om
h ps://www.sciencedi ec .com/science/a icle/pii/S0039914019301109
dspace. u b .cz
1
Molecula ly imp in ed polyme s coupled o mass spec ome ic de ec ion
o me allo hionein sensing
Te eza Vanecko a1,2, Lucie Pompeiano Vanicko a1,2, Michaela T dono a3, Adam
Pomo ski4, A u K ężel4, Tomas Vaculo ic3,5, Vik o Kanicky3,5, Ma ke a Vaculo ico a1,2,
Voj ech Adam1,2*
1Depa men o Chemis y and Biochemis y, Mendel Uni e si y in B no, Zemedelska 1, CZ-
613 00 B no, Czech Republic
2Cen al Eu opean Ins i u e o Technology, B no Uni e si y o Technology, Pu kyno a 123,
CZ-612 00 B no, Czech Republic
3 Depa men o Chemis y, Facul y o Science, Masa yk Uni e si y, Kamenice 753/5, CZ-625
00 B no, Czech Republic
4Depa men o Chemical Biology, Facul y o Bio echnology, Uni e si y o W oclaw, Jolio -
Cu ie 14a, 50-383 W ocław, Poland
5 Cen al Eu opean Ins i u e o Technology, Masa yk Uni e si y, Kamenice 753/5, CZ-625 00
B no, Czech Republic
* Co esponding au ho : Voj ech Adam, Depa men o Chemis y and Biochemis y, Mendel
Uni e si y in B no, Zemedelska 1, CZ-613 00 B no, Czech Republic; E-mail:
oj [email protected]; phone: +420-5-4513-3350; ax: +420-5-4521-2044
2
Abs ac
We epo a acile me hod o de ec ion o me allo hionein (MT), a p omising clinically
ele an bioma ke , in spiked plasma samples. This me hod, o he i s ime, in eg a es
molecula ly imp in ed polyme s as pu i ica ion/p e ea men s ep wi h ma ix assis ed plasma
deso p ion/ioniza ion ime-o - ligh mass spec ome ic de ec ion and wi h lase abla ion
induc i ely coupled plasma mass spec ome y o analysis o MTs. The p epa ed MT-
imp in ed polydopamine laye showed high binding capaci y and speci ic ecogni ion
p ope ies owa d he empla e. Op imal monome (dopamine) concen a ion was ound o be
16 mM o dopamine. This expe imen al se up allows o measu e µM concen a ions o MT
ha a e p esen in blood as his can be used o clinical s udies ecognizing MT as ma ke o
a ious diseases including umou one. P esen ed app oach no only p o ides as sample
h oughpu bu also a oids he limi a ions o me hods based on use o an ibodies (e.g. high
p ice, c oss- eac i i y, limi ed a ailabili y in some cases, e c.).
Keywo ds
Me allo hionein; molecula ly imp in ed polyme s; polydopamine; MALDI-TOF-MS; LA-
ICP-MS
3
In oduc ion
Me allo hioneins (MTs) a e low molecula mass (< 7kDa), cys eine- ich p o eins,
ubiqui ously p esen in p ac ically all euka yo es [1-3]. MTs pe o m a wide ange o
unc ions in an o ganism including essen ial me al homeos asis, i.e. Zn2+ and Cu+, hea y me al
de oxi ica ion, i.e. Cd2+, sca enging o eac i e oxygen species and egula ion o ansc ip ion
[4-8]. The ele a ed concen a ion o MTs has been obse ed in blood and/o issues coming
om pa ien s wi h a ious umou diseases (e.g., colon, b eas , li e , kidney, lung,
nasopha ynx, o a y, sali a y gland, p os a e, hy oid, and u ina y bladde cance ) as i was
e iewed [9, 10], demons a ing ha MTs a e p omising oncosupp esso s [11-13].
Nume ous analy ical app oaches ha e been sugges ed o de ec ion and de e mina ion o MTs
as e iewed elsewhe e [14, 15]. Mo e ecen ly, a me hod based on he enzyme-linked
immunoso ben assay and he eal ime polyme ase chain eac ion was used [16]. Ano he
s udy epo ed a mic o luidic MT elec ochemical immunosenso u ilizing supe pa amagne ic
aga ose beads [17], whe e a cu e i ing app oach was be used o ol ammog ams o a ious
iso o ms o MTs [18]. Mass spec ome y coupled wi h ma ix assis ed lase
deso p ion/ioniza ion (MALDI) o induc i ely coupled plasma (ICP) ioniza ion was also used
o de e mina ion o MTs [19]. Recen ly, bo om-up mass spec ome y-based app oach o
human MT iso o ms quan i ica ion has been de eloped [20], ne e heless combina ions o
hese ypes o de ec ion wi h biological molecule ecogni ion elemen s a e needed.
Molecula ly imp in ed polyme s (MIPs) a e bio ecogni ion su aces wi h high a ini y
owa ds desi ed empla e. Named as na u al ecep o mimics, MIPs a e being conside ed as an
al e na i e o biological ecep o s, such as enzymes, an ibodies o ap ame s [21, 22]. Thei
majo ad an ages co e p edic able speci ic ecogni ion, low cos , ease o p epa a ion, good
mechanical/chemical s abili y, and eusabili y [23]. Al hough MIPs ha e been success ully
applied using wide ange o small molecules [24], imp in ing o biomac omolecules, such as
4
p o eins, aces challenges. Mac omolecula empla es ha e a endency o adso b o polyme s,
whe e i is no i ial o emo e hem om he polyme ma ix, as hey p oduce he e ogeneous
si es, and may be sensi i e o dena u a ion o p esence o o ganic sol en s, which a e usually
essen ial o o ma ion o polyme s [22, 25]. To da e, only ew s udies epo ed combina ion
o molecula imp in ing echnology oge he wi h MALDI ime-o - ligh mass spec ome y
(MALDI-TOF-MS) [26-33]. Howe e , de e mina ion o MT using molecula ly imp in ing
echnology combined wi h mass spec ome ic de ec ion (nei he MALDI no ICP) has no
been epo ed ye .
In he p esen s udy, we de eloped an easy me hod o MT pu i ica ion om a complex
ma ix using MT-selec i e PDA laye combined ollowed wi h MALDI-TOF-MS and LA-
ICP-MS de ec ion echniques (Fig. 1). The analy ical pe o mance o he senso was
e alua ed. The ob ained esul s e eal a new pe spec i e in ecogni ion and sepa a ion o his
empla e, which is a po en ial ma ke o diseases.
Ma e ial and me hods
Ma e ials
Dopamine hyd ochlo ide, albumin om human se um (≥ 97%), T izma® (TRIS base), and
lysozyme om chicken egg whi e we e pu chased om Sigma-Ald ich company (S . Louis,
MO, USA). Ace ic acid (99.8%) and hyd ochlo ic acid ( eagen g ade, 35%) was ob ained
om PENTA (Ch udim, Czech Republic). Amicon® Ul a 0.5 mL 50K Cen i ugal Fil e s
we e pu chased om Me ck Millipo e (Bille ica, MA, USA). T is-HCl bu e (20 mM, pH
8.5) was p epa ed om T izma® and hyd ochlo ic acid was used o pH adjus men . Aqueous
solu ion o ace ic acid (3%, / ) was used as a washing bu e . Deionized wa e used du ing
he expe imen s was p epa ed wi h a Milli-Q wa e pu i ica ion sys em (Millipo e, Mil o d,
MA, USA).

5
P epa a ion o MT-imp in ed polydopamine laye
Polydopamine (PDA) MIP was p epa ed by sel -polyme iza ion inspi ed by wo ks o [34, 35].
In b ie , dopamine was dissol ed in 1 mL T is-HCl bu e (20 mM, pH 8.5). Templa e
molecules o MT-1, MT-3 o lysozyme, o ini ial op imiza ion, we e hen mixed wi h he
s ock solu ion o DA a 1:1 ( / ) a io. The a ge concen a ion was a ied depending on he
expe imen . Nex , 1 µL o he polyme iza ion mix u e was applied on he MALDI a ge pla e
(B uke MTP Ancho Chip 384BCTM) o , in case o ini ial expe imen s, bo om o he 96 well-
pla e and le o polyme ize and d y a he oom empe a u e o 24 h. Subsequen ly, he
p epa ed polyme ic laye was o e laid wi h a sample solu ion and he incuba ion was ca ied
o 1 hou a oom empe a u e. The esul ing sel -assembled polyme was hen washed wi h
3% ace ic acid o emo e he bound MT o lysozyme. Final wash was pe o med wi h Milli-Q
wa e . Con ol, non-imp in ed polyme (NIP), was p epa ed unde he same condi ions
wi hou adding he empla e (MT/lysozyme). Each o he analysed polyme s was p epa ed in
iplica e.
Sample p epa a ion
S anda d solu ions
The coding sequences o human me allo hionein-1 (MT-1) (UniP o :P13640-2) and
me allo hionein-3 (MT-3) (UniP o :P25713-1) we e pu chased om Gensc ip (Pisca away,
NJ, USA) and inse ed in o he pTYB21 ec o (New England Biolabs, UK). P epa ed
plasmid ans o med in o BL21(DE3)pLysS E. coli cells. P o ein p oduc ion and pu i ica ion
was conduc ed as p e iously s a ed [36]. The ob ained ecombinan p o ein MT-1 used in he
MALDI-MS expe imen s had se en bound Zn(II) ions. Fo LA-ICP-MS expe imen s we e
p epa ed MT-1 wi h se en bound Cd(II) ions and MT-3 wi h se en bound Zn(II) ions.
6
Plasma sample p epa a ion
Whole blood was collec ed om a heal hy olun ee and hen he whole blood was
cen i uged o 10 min a 2000 c and he plasma was u he cen i uged o 30 min a 22000
c . The plasma was dilu ed 50× wi h T is-HCl bu e (20 mM, pH 8.5) and spiked wi h MT-1
o i s inal concen a ion o 5 μM. All subjec s ga e hei in o med consen o inclusion,
be o e pa icipa ing in he s udy. The s udy was conduc ed in acco dance wi h he Decla a ion
o Helsinki and he p o ocol was app o ed by he E hics Commi ee o Masa yk Uni e si y.
MALDI-TOF-MS
MIP/NIP we e analysed using MALDI-TOF-MS (ul a leX eme ins umen , B uke Dal onik
GmbH, B emen Ge many) equipped wi h a lase (ope a ing a wa eleng h o 355 nm wi h an
accele a ing ol age o 25 kV, a maximum ene gy o 43.2 μJ, and a epe i ion a e o
2000 Hz) in linea posi i e ion mode o da a acquisi ion. Th ee di e en o ganic ma ix
solu ions we e es ed, namely α-cyano-4-hyd oxycinnamic acid, sinapinic acid and 2,5-
dihyd oxybenzoic acid (DHB) (B uke Dal onik, B emen, Ge many), wi h DHB dilu ed in
0,1% i luo oace ic acid (Sigma-Ald ich) being conside ed he op imal solu ion, since less
backg ound was p oduced in he inal spec um [18]. Ma ix (0.5 µL) was applied on he
p epa ed MIP and/o NIP polyme ized laye (as p e iously desc ibed in sec ion 2.2) and d ied
unde a mosphe ic p essu e and ambien empe a u e (25 °C). The lase equency was se o
1000 Hz and lase ene gy was op imized p io o each measu emen . Calib a ion was done
ex e nally using a p o ein s anda d mix u e I and II (B uke Dal onics, B emen, Ge many) in
he ange o m/z 1–90 kDa. A o al o 500 spec a we e summed o each spo using he
Random Walk as e pa e n, wi h no e alua ion c i e ia and we e analysed wi h he Flex
Analysis so wa e (Ve sion 3.4).
7
Fluo escence spec ome y
Fluo escence spec ome ic measu emen s we e pe o med using In ini e M200 luo escence
mic opla e eade (Tecan, Männedo , CH). Polyme iza ion mix u e (50 µL) was deposi ed on
he bo om o he well o Co ning® 96 Well Clea Fla Bo om UV-T anspa en Mic opla e
(Co ning, NY, USA). Fluo escence emission o lysozyme was eco ded a λex = 280 nm and
λem = 330 nm wi h gain o he de ec o se o 100.
LA-ICP-MS
The analysis o MIP was pe o med by LA-ICP-MS se up ha consis s o LA sys em UP213
(NewWa e Resea ch, USA) emi ing lase adia ion wi h a wa eleng h o 213 nm wi h a
pulse wid h o 4.2 ns. The abla ed ma e ial was ca ied ou om an abla ion cell by a low o a
He (1.0 l/min) in o ICP-MS Agilen 7500CE (Agilen Technologies, Japan) wi h quad upole
analyze .
The MIP and NIP we e abla ed wi h ollowing abla ion pa ame e s: lase beam diame e o
110 μm, he epe i ion a e o 10 Hz; lase beam luence o 6 J/cm2, he scan speed o
400 μm/s and dis ance be ween indi idual lines o 100 μm. The analy es MT-1 and MT-3
we e moni o ed by measu ing o iso ope 111Cd and 66Zn, espec i ely.
Resul s and discussion
Op imiza ion o MIPs p epa a ion
To add ess limi a ions o p o ein imp in ing, se e al s a egies ha e been in es iga ed
including me al ion-coo dina ion polyme iza ion, p o ein epi ope app oach, and su ace
imp in ing [22, 25, 37]. The la e app oach u ilizes polydopamine (PDA), one o he mos
a ou able polyme s conside ing i s g een chemis y s a us and acile p epa a ion. Dopamine
8
(DA), a unc ional monome , can o m a hin, sel -polyme izing ilm on a wide a ie y o
ma e ials in a weak alkaline en i onmen (pH > 8) [38]. I has been shown ha PDA o ms
hin ilms by he spon aneous polyme iza ion in he p esence o oxygen; howe e some o he
polyme iza ion me hods in ol ing adicals o ma ion ha e been de eloped [39]. DA,
commonly in ol ed in human body as a neu o ansmi e , is also a small-molecule mimicking
he adhesi e p o eins. I s mul i unc ional g oups and p ope ies o hyd ophilici y and
biocompa ibili y make i sui able o imp in ing o p o eins. I can be sel -polyme ized unde
mild condi ions ( oom empe a u e, pH 8.0) esul ing in o ma ion o an adhe en
polydopamine ilm. Fo example, app oach o imp in ing p o eins using PDA coa ing o
Fe3O4 nanopa icles has been epo ed [40]. Xia e al. sugges ed an app oach o p o ein
ecogni ion and sepa a ion using PDA-coa ed molecula ly imp in ed silica nanopa icles [41].
The e o e, i is belie ed ha polydopamine MIPs a e expedien and app op ia e ma e ials
applicable in he iden i ica ion o p o eins.
The simple oxida i e polyme iza ion was employed in his s udy. Fo non-co alen
imp in ing, he op imal a io o empla e o unc ional monome (T/M) has o be achie ed
empi ically [42]. The e o e, he concen a ion o monome (dopamine) was es ed in
concen a ions o 16, 32, 65, and 130 mM (da a no shown). The unc ionali y o he
imp in ed polyme was ini ially es ed u ilizing lysozyme as a empla e. The globula
glycop o ein lysozyme (Lys) plays and impo an ole in li ing o ganisms. Conside ing i s
excellen an ibac e ial p ope y, Lys is widely used in medical and ood indus y. Thus, he
de elopmen o an e ec i e pu i ica ion me hod o Lys is b oadly aluable [43]. Based on
he in insic luo escence o Lys, he e iciency o he polyme ic laye p epa a ion was
e alua ed. Acco ding o he luo escence spec ome y measu emen s, he dopamine
concen a ion o 16 mM was chosen as he mos app op ia e due o he highes binding yield.
15
Figu es
Figu e 1
O e all wo k low o molecula ly imp in ed polyme (MIP) o ma ion, sampling and MALDI-
MS de ec ion o MT-1.

16
Figu e 2
LA-ICP-MS quan i ica ion o 66Zn o di e en MT-1 concen a ions in he sample ex ac ed
by MIP ( ed) and NIP (g ey). 66Zn quan i ica ion in MIP a e empla e emo al is shown in
blue.
17
Figu e 3
LA-ICP-MS analyses o MIP and NIP using he MT-1 (Cd) and MT-3 (Zn) empla es. (A)
Sample con aining MT-3(Zn) was applied o MIP/NIP c ea ed wi h empla e MT-1(Cd). (B)
Sample con aining MT-1(Cd) was applied o MIP/NIP c ea ed wi h empla e MT-3(Zn), (C)
Sample con aining MT-3(Zn) and MT-1(Cd) was applied o MIP/NIP c ea ed wi h empla e
MT-1(Cd).
18
Figu e 4
S acked o e lay o MALDI-TOF-MS da a using MT-MIP pu i ica ion o he plasma spiked
wi h 5 μM MT-1 ( ed), in compa ison wi h NIP pu i ica ion (blue), MT-1 peak (m/z 6071.5)
is ma ked wi h (*).
19
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