Mass spec ome ic imaging o cys eine ich
p o eins in human skin
POMPEIANO VANÍČKOVÁ, L.; GURÁŇ, R.; KOLLAR, S.; EMRI, G.;
KŘÍŽKOVÁ, S.; DO, T.; HEGER, Z.; ZÍTKA, O.; ADAM, V.
In e na ional Jou nal o Biological Mac omolecules
2019, ol. 125, Ma ch 2019, pp. 270-277
ISSN: 0141-8130
DOI: h ps://doi.o g/10.1016/j.ijbiomac.2018.11.272
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.ijbiomac.2018.11.272
Final e sion a ailable om
h ps://www.sciencedi ec .com/science/a icle/pii/S0141813018356241
dspace. u b .cz
1
Mass Spec ome ic Imaging o Cys eine Rich P o eins in Human Skin
Lucie Vanicko aa,b, Roman Gu ana,b, Sándo Kollá c, Gab iella Em id, Sona K izko aa,b,
Tomas Doa, Zbynek Hege a,b, Ond ej Zi kaa,b, Voj ech Adama,b,*
a Depa men o Chemis y and Biochemis y, Mendel Uni e si y in B no, B no, Czech
Republic
b Cen al Eu opean Ins i u e o Technology, B no Uni e si y o Technology, B no, Czech
Republic
c Kenézy Gyula Coun y Hospi al, Deb ecen, Hunga y
d Depa men o De ma ology, Uni e si y o Deb ecen, Deb ecen, Hunga y
*Co esponding au ho : [email p o ec ed]
2
Abs ac
Looking insigh pa hological p ocesses, me allo hioneins (MTs) a e conside ed o be po en ial
bioma ke s o moni o ing o a de elopmen o a ious ypes o malignan disease, such as
cance . The ea ly iden i ica ion o he MT bioma ke s in biological issues could be impo an
ool o he es ima ion o app op ia e clinical he apy. The e o e he e we in es iga ed he
applica ion o ma ix assis ed lase deso p ion/ioniza ion mass spec ome y imaging (MALDI
MSI) oge he wi h immunohis ochemical analyses (IHC) using MT-1/2 an ibody o MT
de ec ion in o malin- ixed pa a in-embedded (FFPE) biopsy specimens o human skin.
P incipal componen analyses e ealed di e ences in he pep ide/p o ein p o iles sepa a ing
heal hy skin om he ca cinoma specimens. S a is ically signi ican ion peaks a m/z 6038,
6300, 6676, and 7026 we e mo e equen ly de ec ed in squamous cell ca cinoma (SCC),
basal cell ca cinoma (BCC) and melanoma. Using IHC, we ound ha MT-1/2 was
signi ican ly highe in SCC and melanoma compa ed o heal hy skin. Su p isingly,
signi ican ly low le els o MT-1/2 we e ound in BCC. On one side, he esul s indica e
impo an ole o MTs in melanoma occu ence and p og ession, as on he second side, he e
a e hidden p ocesses associa ed wi h MTs based on di e ences o he occu ence o he MS
peaks, which could be associa ed wi h cycling o MTs iso o ms.
Keywo ds
me allo hionein; squamous cell ca cinoma; melanoma; MALDI MSI; immunohis ochemis y
3
1. In oduc ion
Ou expe ise on p o ein spa ial de ec ion has e ol ed massi ely in las wo decades
especially due o echniques o mass spec ome y (MS), liquid ch oma og aphy (LC),
immunohis ochemis y, ac iona ion and mul iplex assays [1-5]. Recen ly he p o eomics
echnologies ha e been applied o he analysis o o malin- ixed, pa a in-embedded (FFPE)
issues [6]. Gi en he s abili y o FFPE issues ollowing ixa ion, la ge numbe o hospi als
keep a chi es o hese issues oge he wi h pa ien diagnosis and ou come [1]. Me hods o
LC-MS/MS and 2D gel elec opho esis ha e been p e iously used o he iden i ica ion o
p o eins om FFPE issues [7, 8]. Fu he mo e, lase mic odissec ion me hod (LM) and/o
hyd ogel-based me hod ep esen o he p o eomic app oaches allowing o in es iga ion o
egions o in e es wi hin a la ge issue sec ions [9-11]. Fo he o dina y p o eomic
p ocedu e, i is necessa y o ex ac su icien amoun o issue p o eins o enable he analyses
and p o ein iden i ica ion. Rega ding he abo e men ioned echniques o LC-MS/MS and 2D
gel elec opho esis, none o hem p o ides spa ially egis e ed da a o he iden i ied
pep ides/p o eins [11]. Ma ix assis ed lase deso p ion/ioniza ion mass spec ome y imaging
(MALDI MSI) is able o analyse he spa ial dis ibu ion o molecules a o nea he su ace o
complex subs a es and ha e been widely used o bioma ke disco e y, umou classi ica ion
and o de e mina ion o he o igin o he p ima y umou [12, 13]. In addi ion, MSI is
compa ible wi h his opa hological assessmen o de ine diagnosis and suppo he ollowing
he apeu ic managemen [14, 15]. Immunohis ochemis y (IHC) is a classical echnique o
s udy p o ein localiza ion in issues, as well as o con i m a p o ein's iden i y, and i is
he e o e complimen a y o MALDI MSI s udies [16].
Due o i s e icien abili y o g ow, in ade and me as asise, malignan melanomas a e
conside ed as one o he mos agg essi e malignancies esul ing in 80% o mo ali ies om
skin cance . The ea men o me as a ic melanoma is s ill limi ed, ega dless ecen successes
4
o a ge ed he apies. Fo his eason unde s anding o disease p og ession, aiming a
p edic ion o eac ion o ce ain he apeu ic ea men s and iden i ica ion o new he apeu ic
objec i es is highly impo an [1].
Me allop o eins ep esen di e se class o p o eins wi h me al-binding si es p o iding a
s uc u al, egula o y and ca aly ic unc ion(s). An in e es ing g oup o me allop o eins,
known as ma ke s o me al poisoning and po en ial ma ke s o umou diseases, comp ises a
amily o me allo hioneins (MTs) [17, 18]. In mammals, hese low molecula mass cys eine
ich p o eins ha e ou iden i ied iso o ms (MT-1 o MT-4) a ying in exp ession and
localiza ion wi hin indi idual issues. Zn2+ na u ally binds o he mammalian MT a i s α- and
β-domains, ne e heless i may be subs i u ed o ano he me al ion, which has a highe
a ini y o hiola e. I is no su p ising ha mammalian MTs a e in ol ed in de oxi ying,
main aining homeos asis and s o age o me al ions. Based on ecen indings one may
summa ize ha MTs ha e c i ical egula o y impac on apop osis, immunomodula ion,
ansc ip ion egula ion, p oli e a ion and enzyme ac i a ion ia he adminis a ion o Zn ions
o p o eins and egula ion o he concen a ion o ee Zn2+. Based on hese ac s, MTs a e
conside ed as impo an bioma ke s o zinc s a us and me al exposu e and impo an ly hey
migh se e o he p ognosis o ce ain umou diseases. In addi ion, an inc eased con en o
hea y me als and MTs in umou issues can be connec ed o he inc eased in asi eness and
me as asizing o a umou cells [19-21]. All hese indings indica e ha MTs could ha e a
g ea po en ial as diagnos ic, p ognos ic and p edic i e cance bioma ke s, howe e , i
emains hidden and undisco e ed. To con i m such hypo heses, obus analy ical echniques
a e needed. De ec ion and de e mina ion o me allo hioneins a e challenging asks o
analy ical chemis y, when elec ochemis y, immunochemis y, mass spec ome ic and
o he s a e o en used [22, 23], howe e , o he bes o ou knowledge, an app oach o
de ec ion o hese p o eins spa ially in a issue is missing.

5
The e o e, a goal o he p esen s udy was o in es iga e he abili y/sui abili y o MALDI MSI
combined wi h IHC o de ec ion o biological molecules in human skin issues.
2. Ma e ials and Me hods
2.1. Cha ac e iza ion o he samples
This s udy was app o ed by he Kenézy Gyula Coun y Hospi al E hics Commi ee (Deb ecen,
Hunga y), and in o med consen was ob ained om all subjec s. No mal skin was de i ed
om an 83-yea -old emale pa ien , who had a ke a oacan homa on he o ea m. The esec ion
ma gin ee o any umou cells was used o he s udy. Basal cell cance (basalioma solidum
e cys icum) was localized on he shoulde o a 61-yea -old male pa ien . Supe icially
sp eading malignan melanoma wi h B eslow umou hickness 2.76 mm, Cla k le el III, 0-
2/hp mi o ic ac i i y (pT3a) was emo ed om he high o a 47-yea -old male pa ien . G ade
2 cu aneous squamous cell cance o an 86-yea -old emale pa ien was localized on he nose.
Melanocy ic ne us was emo ed om he ches o a 45-yea -old emale pa ien .
2.2. His ology
Fo malin- ixed pa a in-embedded issue blocks o a ious ypes o skin cance s we e
selec ed o MALDI MSI. Blocks o ne us and no mal skin adjacen o a benign skin umou
we e also included. All cases chosen o he s udy we e diagnosed by a boa d-ce i ied
de ma opa hologis . 4-µm sec ions we e p epa ed om he issue blocks and s ained wi h
haema oxylin-eosin o alida ion. The ea e , 5-µm sec ions we e cu and moun ed on o ITO-
coa ed glass slides. A e d ying, slides ha e been s o ed a oom empe a u e un il analysed.
2.3. Immunohis ochemis y
6
4-µm sec ions moun ed on coa ed (o cha ged) glass slides we e depa a inized and
ehyd a ed. Endogenous pe oxidase was blocked in 1% ( / ) H2O2-me hanol o 20 min a
oom empe a u e. An igen e ie al was achie ed by p essu e-cooking he issue samples in
10 mM ci a e bu e (pH = 6). A e he slides we e cooled (> 10 min), hey we e incuba ed
wi h p ima y an ibody o 1 hou a oom empe a u e (MT-1/2 – clone E9, 1:50). Fo
isualisa ion o he eac ion, he ho se adish pe oxidase de ec ion sys em and pe oxide/DAB
as subs a es we e used. The slides we e coun e s ained wi h haema oxylin.
2.4. MALDI MSI expe imen
The mass spec ome y imaging was pe o med on a MALDI-TOF/TOF mass spec ome e
B uke ul a leX eme (B uke Dal onik GmbH, B emen, DE). B oadband mass esol ing
powe (RP) is up o 40,000 and he mass accu acy is 1ppm. In o al 10 ITO glass slides
con aining 20 issue sec ions we e analysed. MALDI ma ix was sp ayed on o ITO glass
slides con aining depa a inized skin samples applying ImageP epTM s anda d p og ams
(B uke Dal onik GmbH, B emen, DE). Sinapinic acid (SA) and 2,5-dihyd oxybenzoic acid
(DHB) we e used as MALDI ma ices as ollows: 10 mg mL-1 SA in ace oni ile/wa e (60:40,
/ ) wi h 0.2% i luo oace ic acid (TFA) and 30 mg mL-1 DHB in me hanol/wa e (50:50,
/ ) wi h 0.2% TFA. SA ma ix was applied o he analyses o in ac p o eins, while DHB
oge he wi h on- issue ypsiniza ion we e used o pep ide mapping. P io analysis he slides
we e d ied in acuum desicca o o 15 min. Scanned images o issue slices we e loaded in o
FlexImaging 3.0 so wa e (B uke Dal onik GmbH, B emen, DE) and MALDI adap e wi h
wo ITO glass slides was loaded in o mass spec ome e . The posi ion o MALDI adap e was
augh acco ding o whi e eaching ma ks on ITO glass slides. Regions o acquisi ion we e
highligh ed by mouse poin e in FlexImaging and 50 μm as e wid h was chosen. The
calib a ion was done ex e nally using a pep ide and p o ein s anda d mix u e in he m/z ange
7
0.5–20 kDa (B uke Dal onik GmbH, B emen, DE). The in ensi y o each scan, o e he en i e
mass ange acqui ed, was mapped on he issue sec ion image, allowing he isualiza ion o
he loca ion o each m/z alue de ec ed. A lase powe was se 5–10% abo e he h eshold.
MALDI MSI o p o eins was pe o med in linea posi i e mode in he m/z ange 1–20 kDa.
MALDI MSI o yp ic pep ides was pe o med in e lec o posi i e mode in he m/z ange
0.5–5 kDa. 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. The m/z images we e gene a ed and isualized using SCiLS Lab
2014b so wa e (SCiLS – B uke Dal onik GmbH, B emen, DE). Final co ec ions and
p epa a ions o he MALDI MSI images we e ca ied ou in GIMP 2.8 so wa e.
Fo on- issue ypsiniza ion, one ITO glass slide wi h each ype o issue was used – in o al, 5
ITO glass slides we e used o ypsiniza ion. App oxima ely, 300 μL o ypsin solu ion (20
μg mL-1 in 40 mM ammonium bica bona e, 0.1 mM HCl and 9% ace oni ile ( / )) pe one
slide was applied by ImageP ep. Then, he slide was kep inside small box wi h humid
a mosphe e o 15 hou s a 37 °C. Fu he , DBH ma ix solu ion was applied. P io MSI
analysis he slide was d ied in acuum desicca o o 15 min. The LIFT cell was used o
MS/MS analysis o de ec ed pep ides [24].
2.5. S a is ical analysis o MSI da a
MSI da a om FlexImaging we e con e ed and uploaded in o SCiLS Lab so wa e ha was
used o pipeline p e-p ocessing, segmen a ion and s a is ical analysis, mainly p incipal
componen analysis (PCA) wi h uni a iance scaling, Ande son-Da ling no mali y es and
K uskal-Wallis es . The da a ea men was pe o med as desc ibed in de ail in ou p e ious
s udy [25].
3. Resul s
8
The ep esen a i e p o ein (Fig. 1A) and pep ide (Fig. 1B) p o iles o he i e ypes o human
skin issues we e cha ac e ised using his ology-di ec ed MALDI MSI app oach o speci ied
egions o in e es (ROIs). The mean p o ein peak in ensi ies we e conside ably lowe han he
mean peak in ensi ies o pep ides as exp essed in pseudo-gel iews. To in es iga e he
di e ences among he p o ein/pep ide p o iles, PCA o ROIs was applied (Fig. 2). The i s
wo axes coun ed o 63.82% and 50.75% o he o al a iabili y o p o eins and pep ides,
espec i ely. P o ein p o iles o heal hy skin (HS) issue and nae us pigmen osus (NP) we e
simila , while he ca cinoma issues o basal cell ca cinoma (BCC), squamous cell ca cinoma
(SCC) and malignan melanoma (MM) we e sepa a ed om he heal hy specimens among he
i s axis (Fig. 2A). The o e all p o ein p o ile simila i ies among he ROIs o he h ee ypes
o skin cance issues esul ed in o e lapping o he BCC, SCC, and MM. The ela i e
in ensi ies o ion peaks a m/z 4376, 4879, 5154, 6038, 6300, 6676, 7026 and 14065 a ied
signi ican ly and we e esponsible o he sepa a ion o heal hy skin issues om cance
issues (Fig. 2B). Pep ide p o iles we e able o di e en ia e SCC om he emaining issue
ypes along he PC1 axis, while HS, BCC and MM we e pa ially sepa a e om each o he
along he PC2 axis (Fig. 2C). Pep ide m/z speci ic o MM we e 1106 and 1461, o SCC
issue was cha ac e is ic he p esence o m/z a 1199, 2182, and 2421. BCC and HS issues
we e sepa a ed om each o he by speci ic pep ides a m/z 1586 and 616, espec i ely (Fig.
2D).
In p o ein p o iles, he PCA and K uskal-Wallis es indica ed wel e s a is ically signi ican
(p < 0.001) ion peaks (Supplemen a y Table 1) ha we e used o he o ma ion o he MSI
maps (Figs. 3, S1 and S2) and in ensi y box plo s (Fig. 4s, S3 and S4). P ima ily, we
his ologically examined he analysed samples (Fig. 3A), depa a inised hem (Fig. 3B) and
de ec ed he spa ial dis ibu ion o ou p o ein ion peaks a m/z 6038, 6300, 6676 and 7026
(Fig. 3C). The MSI images oge he wi h he in ensi y box plo s demons a e ha all ou
15
Cap ions o Figu es
Figu e 1
Pseudo-gel iew o mean mass spec a om (A) p o ein ROIs, and (B) pep ides ROIs in i e
di e en skin issues. The iangles indica e m/z depic ed in he PCA loading plo s in Fig. 2.
Figu e 2
(A) PCA sco es plo o p o ein ROIs. (B) PCA loading plo o p o ein ROIs. (C) PCA sco es
plo o pep ide ROIs. (D) PCA loadings plo o pep ide ROIs. G een – no mal skin, pu ple –
nae us pigmen osus ROI, blue – squamous cell ca cinoma ROI, ed – basal cell ca cinoma
ROI, o ange – malignan melanoma. Numbe s in he loading plo s indica e s a is ically
signi ican m/z (p < 0.001).
Figu e 3
His ological and MSI analysis o i e di e en skin issues, namely no mal skin, nae us
pigmen osus, squamous cell ca cinoma (SCC), basal cell ca cinoma (BCC), and malignan
melanoma (MM). (A) Rep esen a i e his ological pho omic og aph showing he pa icula
issue ype. (B) Scanned pic u es o he pa icula issue ype a e he depa a iniza ion
oge he wi h selec ed egions o in e es (ROIs) used o MSI analysis. (C) P o ein maps o
ion peaks a m/z 6038, 6300, 6676 and 7026 gene a ed as “[m/z] ± 10 Da”, scale ba = 1 cm.
Figu e 4
In ensi y box plo s o selec ed p o ein m/z alues (A) 6038, (B) 6300, (C) 6676 and (D) 7026
in i e di e en skin issues.
Figu e 5

16
Immunohis ochemical (IHC), his ological and MSI analysis o squamous cell ca cinoma
(SCC), basal cell ca cinoma (BCC) and malignan melanoma (MM). (A) Rep esen a i e
pho omic og aph showing inc eased (b own colou ) o dec eased MT-1/2 immuno eac i i y
in speci ic skin issues. (B) Magni ied his ological pho omic og aph showing he selec ed a ea
( ed ec angle) o he pa icula issue ype. (C) Magni ied pho omic og aph showing
inc eased (b own colou ) o dec eased MT-1/2 immuno eac i i y in speci ic skin issues in he
selec ed a ea ( ed ec angle). (D) P o ein maps o ion peaks a m/z 6038, 6300, 6676 and
7026 o e exp essed in SSC, BCC and MM in he selec ed a ea ( ed ec angle), gene a ed as
“[m/z] ± 10 Da”. Scale ba = 2 mm.
17
Supplemen a y Table
Table S1
S a is ically signi ican ion peaks iden i ied in p o ein p o iles ha accoun o he a ia ion
be ween i e egions o in e es (ROIs) in he s udied skin specimens.
Supplemen a y Cap ions o Figu es
Figu e S1
P o ein maps o m/z 3376, 3447, 4376 and 4879 Da. All m/z images we e gene a ed as “[m/z]
± 10 Da”.
Figu e S2
P o ein maps o m/z 5154, 9323, 11422 and 14065 Da. All m/z images we e gene a ed as
“[m/z] ± 10 Da”.
Figu e S3
In ensi y box plo s in all ROIs o m/z (A) 3376, (B) 3447, (C) 4376 and (D) 4879 Da.
Figu e S4
In ensi y box plo s in all ROIs o m/z (A) 5154, (B) 9323, (C) 11422 and (D) 14065 Da.
18
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2197-2203.
No mal skin
Nae us
pigmen osus
Squamous cell
ca cinoma
Basal cell
ca cinoma
Malignan
melanoma
No mal skin
Nae us
pigmen osus
Squamous cell
ca cinoma
Basal cell
ca cinoma
Malignan
melanoma
A
B

A B C IHC His ology