Journal of Chromatography B, 1047 (2017) 131–140

Contents lists available at ScienceDirect

Journal of Chromatography B

jou rnal homepage: www.elsevier.com/locate/chromb

MALDI imaging mass spectrometry analysis—A new approach for

protein mapping in brain lesions

a,b,1 a,1 c

Giuseppina Maccarrone , Sandra Nischwitz , Sören-Oliver Deininger ,

a d,e d

Joachim Hornung , Fatima Barbara König , Christine Stadelmann ,

b,1 a,f,∗,1

Christoph W. Turck , Frank Weber

a

Max Planck Institute of Psychiatry, Kraepelinstr. 2-10, 80804 Munich, Germany

b

Department of Translational Research in Psychiatry, Max Planck Institute of Psychiatry, Germany

c

Bruker Daltonik GmbH, Fahrenheitstr. 4, 28359 Bremen, Germany

d

Institute of Neuropathology, University Medical Center Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany

e

Institut für Pathologie, Klinikum Kassel, Mönchebergstr. 41-43, 34125 Kassel, Germany

f

Medical Park Bad Camberg, Obertorstr. 100-102, 65520 Bad Camberg, Germany

a r t i c l e i n f o a b s t r a c t

Article history: Multiple sclerosis is a disease of the characterized by recurrent inflammatory

Received 21 February 2016

demyelinating lesions in the early disease stage. Lesion formation and mechanisms leading to lesion

Accepted 1 July 2016

remyelination are not fully understood. Matrix Assisted Laser Desorption Ionisation Mass Spectrom-

Available online 1 July 2016

etry imaging (MALDI–IMS) is a technology which analyses proteins and in tissue, preserves

their spatial localization, and generates molecular maps within the tissue section. In a pilot study we

Keywords:

employed MALDI imaging mass spectrometry to profile and identify peptides and proteins expressed in

MALDI imaging mass spectrometry

normal-appearing white matter, grey matter and multiple sclerosis brain lesions with different extents

LC–ESI–MS/MS

of remyelination. The unsupervised clustering analysis of the mass spectra generated images which

Multiple sclerosis

Demyelination reflected the tissue section morphology in luxol fast blue stain and in basic protein immunohis-

Remyelination tochemistry. Lesions with low remyelination extent were defined by compounds with molecular weight

Thymosin beta-4 smaller than 5300 Da, while more completely remyelinated lesions showed compounds with molecular

weights greater than 15,200 Da. An in-depth analysis of the mass spectra enabled the detection of cortical

lesions which were not seen by routine luxol fast blue histology. An ion mass, mainly distributed at the

rim of multiple sclerosis lesions, was identified by liquid chromatography and tandem mass spectrom-

etry as beta-4, a protein known to be involved in cell migration and in restorative processes.

The ion mass of thymosin beta-4 was profiled by MALDI imaging mass spectrometry in brain slides of

12 multiple sclerosis patients and validated by immunohistochemical analysis. In summary, our results

demonstrate the ability of the MALDI–IMS technology to map proteins within the brain parenchyma and

multiple sclerosis lesions and to identify potential markers involved in multiple sclerosis pathogenesis

and/or remyelination.

© 2016 Elsevier B.V. All rights reserved.

1. Introduction

Multiple sclerosis is a disease of the central nervous system

that is characterized by recurrent inflammatory, demyelinated

lesions—at least in the early stages. The mechanisms of lesion for-

mation and lesion repair, especially remyelination are not fully

Abbreviation: MALDI–IMS, matrix assisted laser desorption ionisation–imaging

mass spectrometry; LC–ESI–MSMS, liquid chromatography–electro spray ionisation understood.

 

tandem mass spectrometry; LFB, luxol fast blue; CNP-ase, 2 ,3 -cyclic nucleotide

In the early disease stages multiple sclerosis is characterised



3 -phosphodiesterase; PLP, proteolipid protein.

∗ by focal lympho- and monocytic infiltrations, in the later stages

Corresponding author at: Max Planck Institute of Psychiatry, Kraepelinstr. 2,

microglial activation and ongoing neurodegeneration prevail [1].

D-80804 Munich, Germany.

Remyelination which occurs to some extent in most multiple scle-

E-mail address: [email protected] (F. Weber).

1

These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.jchromb.2016.07.001

1570-0232/© 2016 Elsevier B.V. All rights reserved.

132 G. Maccarrone et al. / J. Chromatogr. B 1047 (2017) 131–140

rosis lesions is an important mechanism that protects axons and peptides in human multiple sclerosis brain tissues including lesions

may prevent chronic disability [2,3]. The extent of remyelination with different extents of remyelination.

varies in different lesions of an individual multiple sclerosis patient.

The underlying mechanisms leading to this discrepancy and the 2. Materials and methods

lack of remyelination in many lesions are not fully understood

[4]. Several studies, however, suggest that successful regeneration 2.1. Brain tissue samples

depends on a signaling environment conducive to remyelina-

tion, which is provided in the context of acute inflammation [5]. The multiple sclerosis brain tissues were provided by the UK

Macrophages are mainly found in acute and chronic active multi- Multiple Sclerosis Tissue Bank, Division of Neuroscience and Men-

ple sclerosis lesions with ongoing inflammation and are involved tal Health Imperial College, London with fully informed consent

in demyelination but also in remyelination processes of the central from the donor and the next of kin. The brain tissue includes

nervous system [6,7]. Nerve growth factors, proteases, and other normal-appearing white matter, grey matter and lesions. The brain

proteins that affect myelin stability or have anti-oxidative activi- tissue was collected post-mortem with the post mortem time less

ties seem to play an important role in either promoting or inhibiting than 24 h. Cerebral hemispheres were coronally sliced, blocked and

remyelination [8–11]. However, the exact mechanisms of remyeli- frozen by immersion in cold liquid iso-pentane and then stored at

◦

nation and the proteins involved remain elusive. −75 C.

An ongoing disease process is reflected by qualitative and quan-

titative molecular alterations. In case of multiple sclerosis the

2.2. Discovery samples

changes of protein localisation and composition in the brain lesions

reflects the alteration of myelination and the effect of inflammation.

For the pilot study brain tissue slides from 2 brain blocks labelled

Thus molecular maps and relative abundance profiling of proteins

A and B of a 71-year-old female patient with secondary progressive

and protein fragments expressed in lesions with different extents

multiple sclerosis were used. The patient had initially a relapsing

of remyelination are of great value for an improved understanding

and remitting disease course for 20 years, then secondary progres-

of multiple sclerosis aetiology.

sive for about 14 years. A magnetic resonance imaging (MRI) scan of

Typically, in neuropathology protein analysis is performed by

the brain showed lesions suggestive of multi-focal demyelination.

immunohistochemistry and two-dimensional polyacrylamide gel

No other neurological disorders were noted. Diagnosis of multiple

electrophoresis [12,13]. Recently, mass spectrometry-based meth-

sclerosis was confirmed post-mortem by histopathology.

ods have been applied for the identification of lesion-specific

proteins of distinct histopathological types of multiple sclerosis

2.3. Validation samples

brain lesions [14]. Although this type of analysis results in com-

prehensive proteomic profiles, information about protein location,

For the MALDI Imaging and immunohistochemical validation

the main focus in neuropathological analyses, is not captured. An

studies brain samples from 12 multiple sclerosis patients were

important technological advance in protein analyses that addresses

used. Clinical characteristics and the death tissue-preservation

this shortcoming is Matrix Assisted Laser Desorption Ionisation

interval are summarized in Table 1.

Imaging Mass Spectrometry (MALDI–IMS). This technique yields

a tri-dimensional image of proteins and peptides capturing molec-

2.4. Histological analysis

ular masses, relative abundances and spatial coordinates [15–17].

MALDI-IMS is an unbiased approach to look for potential disease ◦

The brain tissue was sliced at −18 C into 10 ␮m thick sec-

related proteins and peptides (complementing immunohistochem- ◦

tions using a microtome (Leica, CM30050), and stored at −20 C.

istry). In the present study our main focus was to investigate the

To identify lesions, standard hematoxylin and eosin and Luxol Fast

capability of MALDI–IMS to map proteins and peptides associated

Blue (LFB) (Sigma, Solvent Blue 38) were used to visualise

either with remyelination or resulting from demyelination pro-

myelin, and counterstaining with cresyl violet (Sigma, Cresyl-Violet

cesses which occur during the development of multiple sclerosis

Acetate) or hematoxylin and periodic acid-Schiff were performed.

lesions. We applied this new technology to profile proteins and

To determine the extent of remyelination in multiple sclerosis

lesions, LFB-stained brain sections were scanned with a Color View

Table 1

Clinical characteristics and death tissue-preservation interval of multiple sclerosis patients used in the validation studies.

Patient Age [years] Gender Disease duration Disease course MS specific treatment Cause of death Death-Tissue

[years] (except steroids) preservation

interval [h]

MS049 75 m 38 SPMS none aspiration pneumonia 8

MS055 47 f 32 SPMS none pneumonia 15

MS066 86 f more than 50 N/A none pneumonia 21

MS080 71 f 35 SPMS none bowel 24 blockage/post-operative

comlpications

MS094 42 f 6 PP none pneumonia 11

MS098 57 m 33 SPMS none sepsis secondary to urinary 21

tract infection

MS105 73 m N/A N/A N/A pneumonia 8

MS107 38 m 16 SPMS vitamin B12 injections, tricyclic pneumonia 19

anti-depressants and secret remedies

MS114 52 f 15 SPMS none Pneumonia, sepsis, 12

pulmonary embolism

MS136 40 m 9 SPMS interferone beta 1a Respiratory failure, sepsis 10

MS154 34 f 11 SPMS none pneumonia 12

MS249 59 f 42 SPMS none Chest infection 8

G. Maccarrone et al. / J. Chromatogr. B 1047 (2017) 131–140 133

digital camera (Soft Imaging System, Münster, Germany) mounted 2.7. Statistical analysis

on an Olympus BX51 microscope (Olympus, Tokyo, Japan). Total

white matter, total lesion area (including remyelinated parts of The statistical analyses and data processing of the MALDI spec-

the lesion) as well as remyelinated lesion areas were measured tra were performed using the ClinProTools 2.2 software package

®

using Analysis software (Soft Imaging System GmbH, Münster, (Bruker Daltonik, Germany). First the mass spectra were internally

Germany). The percentage of remyelinated lesion area was calcu- aligned on common peaks and normalised with respect to the total

lated by dividing the total lesion area by the remyelinated lesion ion counts. Then an average spectrum was created and used for the

area and then multiplying by 100. peak picking process. An unsupervised hierarchical statistical anal-

To determine the distribution of Proteolipid Protein (PLP) and ysis was performed as previously described [19]. Briefly, the data

  

2 ,3 -Cyclic Nucleotide 3 -Phosphodiesterase (CNPase), cryostat were first pre-processed by Principal Component Analysis and then

sections were air-dried, incubated for 1 h in chloroform, and sub- the spectra were clustered applying unit variance scaling, Euclidean

sequently fixed for 30 min in 4% paraformaldehyde and for epitope distance metric and Ward linkage method. The clustered mass

retrieval in 10 mM citric acid buffer (pH 6.0) in a steamer. Tis- spectra were illustrated by dendrogram with color-code branches

sue sections were incubated in phosphate buffered saline solution whose visualization generated a tissue MALDI image. The color-

◦

containing 0.3% H2O2, for 10 min at 4 C. After incubation with code image was obtained with the support of the flexImaging 2.1

10% fetal calf serum in phosphate buffered saline solution, pri- software (Bruker Daltonik, Germany).

mary antibodies, i.e. mouse anti-CNPase (CNPase, clone SMI91, A supervised analysis was performed on selected spectra from

Sternberger Monoclonals Inc., Lutherville, MD, USA) or mouse anti- regions of interest, predefined in the histopathological images and

PLP (PLP; 1:500, clone: plpc1, Serotec, Oxford, UK), were applied designated as lesion 1 (L1), lesion 2 (L2), white matter (WM) and

overnight. Macrophages/activated microglia were detected by grey matter (GM). The statistical analysis was performed on the ion

KiM1P immunohistochemistry following the protocol described in signal intensities of the average spectrum calculated for each region

Radzun [18]. Antibody binding was detected with biotinylated don- of interest by applying algorithms embedded in the ClinProTools 2.2

key anti-mouse antibodies (Calbiochem), streptavidin-peroxidase software. The distributions of the ion mass signals resulting from

(Sigma) and visualised with diaminobenzidine (Sigma). The sec- the statistical analysis were manually ascertained by displaying the

tions were counterstained with , then dehydrated ion mass distribution within the tissue slides using the flexImag-

and mounted. ing 2.1 software. The ion mass distribution was displayed applying

Thymosin beta-4 was detected with a primary antibody (Rabbit a colour-coded gradient system to highlight the ion mass and its

polyclonal anti-thymosin beta-4-antibody; Peninsula Laborato- abundance.

ries, San Carlos, CA, USA), a biotinylated secondary antibody and

a horseradish-peroxidase/DAB detection system (Super Sensitive 2.8. Protein marker identification

Polymer-HRP IHC Detection System/DAB, BioGenex Fremont, CA,

USA), according to the protocol above. 2.8.1. Sample preparation

White matter and cortical lesion areas of tissue slides (10 ␮m

thickness) from brain block B, indicated in Fig. 3A as L2 and GM*

2.5. Sample preparation for MALDI–IMS

(cortical lesions), respectively, were manually cut. Three L2 and

GM* tissue areas each were pooled and subjected to tissue lysis

The brain tissue was sliced (10 ␮m) using a microtome instru-

◦ and protein extraction protocols. Briefly, the tissue samples were

ment (Leica, CM30050) at −18 C and transferred onto precooled

− ◦ immersed into chilled 0.1% trifluoroacetic acid aqueous solution

( 20 C) Indium-Tin-Oxide (ITO) coated slides (Bruker, Bremen,

(200 ␮l) and ultra-sonicated for 15 min in an ice bath. The homog-

Germany). Afterwards, the slides were warmed on the hand inside ◦

enized sample was centrifuged at 20,000g for 15 min at 4 C. The

the cryostat and then desiccated (45 min) in a vacuum desiccator. ◦

supernatant was collected and stored at −20 C, while the pellet

The dried slides were washed (15 s) in 70% ethanol (Sigma–Aldrich,

was immersed in 50% acetonitrile, 0.1% trifluoroacetic acid aqueous

Germany) and in 96% ethanol (15 s) and then desiccated again

solution (120 ␮l) and ultra-sonicated for 15 min in an ice bath. The

(45 min). The dried tissue slides were wrapped in aluminium foil

◦ extracted proteins were separated by centrifugation at 20,000g for

−

and stored at 80 C. ◦

15 min at 4 C. The supernatant in 50% acetonitrile was lyophilized

◦

and stored at −20 C.

2.6. MALDI–IMS analysis

The L2 and GM* (cortical lesions) extracts in 0.1% trifluoroacetic

acid aqueous solution were washed three times with 50 mM

The tissue slides were scanned with a Nikon Coolscan 5 ED

ammonium acetate using Vivaspin cartridge (CO 3 kDa, Sartorius,

imager (Nikon). The images were used to delimit the measure-

Germany). The samples were centrifuged at 5000 rpm for 30 min

ment area for the MALDI spectra acquisition. Afterwards the tissue

and concentrated down to 20 ␮l.

slides were coated with the matrix using the ImagePrep station

Subsequently, the protein extracts were chromatographed

(Bruker Daltonik, Bremen, Germany) following the manufacturer’s

using a micro column mRP-C18 (4.5 × 50 mm, Agilent Technologies,

◦

protocol. Sinapinic acid (Bruker Daltonik; 10 mg/ml) in 60:40 (v/v)

USA) conditioned at 80 C with an analytical HPLC HP1100 cou-

acetonitrile:water (Merck, Germany) and 0.2% trifluoroacetic acid

pled to an AS1200 fraction collector (Agilent Technologies, USA).

(Fluka, Germany) were used as matrix. Subsequently, a thin gold

For protein separation 0.1% trifluoroacetic acid aqueous solution

film was deposited onto the tissue slides using an Agar Auto Sputter

(solution A) and 0.1% trifluoroacetic acid in acetonitrile (solution

Coater instrument (Plano GmbH, Wetzlar, Germany). MALDI-IMS

B) were used as eluents at a flow rate of 750 ␮l/min using the fol-

TM

measurements were performed using a linear AutoFlex II mass

lowing gradient elution: eluent B, 0–3% in 7 min, 3–22% in 23 min,

spectrometer (Bruker Daltonik, Bremen, Germany) equipped with

22–38% in 5 min, 38–97% in 2 min. The eluate was monitored at

TM

the Smart-beam laser at 100 Hz and laser beam of 85 ␮m diame-

220 nm and 30 s fractions were collected into a 96 microtiter plate.

◦

ter. The spectra were acquired in the linear mode in the mass range

The collected fractions were lyophilized and stored at −20 C.

of 2–30 kDa, lateral resolution of 200–500 ␮m, under control of the

The L2 and GM* (cortical lesions) protein extracts in 50% ace-

flexImaging 2.1 software (Bruker Daltonik). The MALDI instrument

tonitrile were dissolved in Laemmli buffer and separated by sodium

was externally calibrated using the calibration standard mixture

dodecyl sulphate-polyacrylamide gel electrophoresis (15% resolv-

purchased from Bruker Daltonik, Germany.

ing gel and 5% stacking gel) at 120 V. The gel was stained overnight

134 G. Maccarrone et al. / J. Chromatogr. B 1047 (2017) 131–140

Fig. 1. MALDI imaging mass spectrometry (MALDI-IMS) of human tissue samples workflow. The frozen tissue is sectioned and consecutive slides are analysed by (A) histology;

(B) MALDI imaging mass spectrometry (MALDI–IMS). (C) Ion signals (m/z) are plotted with support of flexImage software to generate ion map images with ion mass (m/z),

ion spatial coordinates (x, y) and ion abundance. m/z: mass-to-charge ratio.

with Coomassie Brilliant Blue R250 (Serva, Heidelberg, Germany). 3. Results

After destaining (50% methanol, 12% acetic acid), the gel lanes were

cut in 20 pieces and subjected to in-gel tryptic protocol Histology of brain blocks A and B revealed chronic inactive mul-

as described in Maccarrone [20]. tiple sclerosis lesions with different extents of remyelination and

few foamy macrophages at lesion rims (data not shown). Block

2

A displayed the lesion 1 (51 mm ) constituting of two partially

2

remyelinated areas with 59% remyelination extent (28 mm and 2.8.2. LC–ESI–MSMS

2 2

2 mm ) and lesion 2 (5 mm ) which presented one small area

The protein and identification was performed using a

2

(1 mm ) with 17% remyelination extent (Supplementary Fig. 1A).

nano Eksigent-2D system (Eksigent, Dublin, CA) coupled via a nano

2

Block B presented lesion 1 (5 mm ) almost completely remyeli-

electrospray ion source (Thermo Fisher) to an LTQ-Orbitrap mass

2

nated (96% remyelination extent) and lesion 2 (91 mm ) with

spectrometer (Thermo Fisher Scientific, Bremen, Germany). The

2 2

2 remyelinated areas (27 mm and 5 mm ) equivalent to 35%

samples dissolved in 0.1% formic acid (eluent A) were loaded onto

remyelination extent (Supplementary Fig. 1B). In addition, clus-

an in house-packed nano column (75 ␮m × 15 cm) and eluted by

ters of erythrocytes were detected within lesion 2, indicating small

applying a 95% acetonitrile/0.1% formic acid (eluent B) gradient

blood vessels (Supplementary Fig. 1C).

from 2 to 45% for 40 min at a flow rate of 200 nl/min. The mass

To preserve the tissue morphology, the mass spectrometry anal-

spectrometer was operated in positive mode and data-dependent

ysis and the histological staining were performed on consecutive

scan mode. Full scans were recorded at the Orbitrap mass analyzer

brain tissue sections as depicted in Fig. 1A and B. The overlap of the

at a mass range of m/z 380–1600 and a resolution R = 60,000. The

LFB-stained and unstained images did not show any shifts in the

MS/MS analysis was performed on the five most intense peptide

morphological features (data not shown). The MALDI spectra were

ions and recorded in the LTQ mass analyzer.

first submitted to a multivariate statistical analysis, principal com-

ponent analysis and then to an unsupervised hierarchical clustering

analysis without taking in consideration histo-morphological fea-

2.8.3. Protein identification

tures and molecular targets.

The raw data generated by the mass spectrometer were

The unsupervised analysis of the mass spectra acquired within

searched against SwissProt 15.3 database, taxonomy human, using

brain slide block A generated sub-regions within the tissue area

Mascot search engine (www.matrixscience.com). The data derived

corresponding to normal-appearing white matter, grey matter and

from proteins/peptides extracted by 0.1% trifluoroacetic acid

lesions as depicted in Fig. 2A and Fig. 2C. In block B a phenotypic

were searched applying the following parameters: no enzyme,

composition difference in the area defined as grey matter by LFB-

methionine oxidation, N-terminal acetylation, Ser, Thr and Tyr

histochemistry was detected. In the grey matter area mainly three

phosphorylation as variable modifications. The raw data derived

molecular components have been determined, a group including

from the in-gel tryptic digestion samples were searched applying

two molecular components, cornflower-blue and yellow, consti-

the following parameters: trypsin as enzyme, 2 missed cleav-

tutes one of the grey matter areas and another group including the

ages, methionine oxidation, Ser, Thr and Tyr phosphorylation and

molecular components yellow and brown mainly defines the other

variable modifications, cystein carboxymethylation as static mod-

grey matter area (Fig. 2D,F). The immunostainings for CNPase and

ification. For protein database searches the mass accuracies for

PLP performed on the block B detected widespread subpial cortical

precursor and fragment ions were set to 20 ppm and 0.6 Da, respec- tively.

G. Maccarrone et al. / J. Chromatogr. B 1047 (2017) 131–140 135

Fig. 2. Optical images of LFB-stained brain tissue slides. Hierarchical clustering dendrograms and respective images generated by MALDI imaging mass spectrometry

(MALDI–IMS) data (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).

Block A, brain slide: (A) LFB-stained image, lesion 1 (L1), lesion 2 (L2), white matter (WM) and grey matter (GM) regions are indicated. (B) Dendrogram of MALDI spectra

acquired across block B slide. (C) The reconstruction of the top five dendrogram branches reflects white matter (green and violet), lesion 1 (sky-blue), grey matter (brown)

and a sub-area (pink) within lesion 2 (green).

Block B, brain slide: (D) LFB-stained image, lesion 1 (L1), lesion 2 (L2), white matter (WM) and grey matter (GM) regions are indicated. (E) Dendrogram of MALDI spectra

acquired across block A slide. (F) MALDI-IMS image is generated from the reconstruction of the top nodes of the dendrogram branches. Two sub-areas within lesion 2 (violet

and green), two components within lesion 1 (cornflower-blue and pink) are identified. The white matter (WM) mainly consists of one component (pink). The grey matter

area is constituted of three molecular components, a group of two components (cornflower-blue and yellow) defined one area, another group of two components (yellow

and brown) mainly identified the other grey matter area.

The construction of the dendrogram branches and the corresponding ion mass images are displayed using a colour-coded system. The image pixels are coloured according

to the colour of the respective dendrogram node.

Fig. 3. LFB-staining, PLP and CNPase immunohistochemical images. Optical images of block B brain slide, (A) LFB-staining, (B) PLP, (C) CNPase. Cortical lesions (CL, GM*) and

white matter lesions (L), normal appearing white matter (WM) and grey matter (GM) are indicated.

demyelination (cortical lesions) in the grey matter area. The cortical Fig. 2. The molecular masses m/z 15205 and 15940 are specifi-

lesions are mainly located in the areas labelled GM* (Fig. 3A–C). cally distributed within the BV area (Fig. 4A). We hypothesized

To determine the molecular composition within the brain the presence of human haemoglobin isoforms. This finding was

lesions and the sub-areas detected by the unsupervised analy- confirmed and validated by hematoxylin and eosin staining which

sis, regions of interest based on the tissue histo-morphology were revealed erythrocyte clusters in the BV area indicating the pres-

defined. In block A lesion 1, lesion 2, BV and ROI3, a region at the ence of blood vessels (Supplementary Fig. 1C). The mass m/z 18,608,

rim of lesion 1, areas were defined (Fig. 4A). The analysis revealed which marked the white matter area and part of lesion 2 (Fig. 4A),

molecular masses smaller than 6 kDa in lesion 1 area (17% remyeli- corresponds to isoform 5 of Myelin Basic Protein (18591 Da).

nation), while within lesion 2 (59% remyelination) compounds with In block slide B, lesion 1, lesion 2, normal-appearing white mat-

molecular masses greater than 10 kDa were detected (Table 2). ter, grey matter and GM* (cortical lesions) areas were defined

The ion density maps are depicted in Fig. 4A and Supplementary (Fig. 4B). The supervised statistical analysis of MALDI-IMS spectra

136 G. Maccarrone et al. / J. Chromatogr. B 1047 (2017) 131–140

Fig. 4. Images of ion density maps generated by MALDI imaging mass spectrometry (MALDI–IMS). (A) LFB-stained block A slide lesion 1 (L1), lesion 2 (L2), white matter

(WM) and grey matter (GM) are indicated. The BV area is delimited with black dotted lines. MALDI-IMS ion density maps of mass signals (m/z) expressed within the WM,

BV, L1 and L2 areas are shown. (B) LFB-stained block B, white matter (WM), grey matter (GM), lesion 1 (L1), lesion 2 (L2) and GM* (CL: cortical lesions) areas are indicated.

MALDI–IMS ion density maps of mass signals (m/z) expressed within WM, GM* (CL), L1 and L2 areas are shown.

The image resolution was set to 200 ␮m. The ion abundances are shown based on a colour-coded gradient system.

acquired across lesion 1, lesion 2 and GM* (cortical lesions) areas tary Fig. 4. To identify the molecular masses, the protein extracts

resulted in a list of compounds whose mass values are listed in from the block B lesion 2 and cortical lesions (GM*) were sub-

Table 2. Lesion 1 (96% remyelination) and the normal-appearing jected to proteomics workflow based on LC–ESI–MSMS analysis.

white matter regions are defined mostly by high molecular mass The mass m/z 5000, mapped by MALDI–IMS within block B lesion

compounds m/z 16,473 and m/z 18,650 as depicted by the respec- 2 core and rim (Fig. 4B), was identified as thymosin beta-4 (m/z

tive ion density maps shown in Fig. 4B and Supplementary Fig. 3. 5000, phosphorylated form) and confirmed by anti-thymosin beta-

The lesion 2 area (35% remyelination) is described by two groups 4 immunohistochemical staining (Fig. 5A). In block A the mass m/z

of molecular compounds, demyelinated core of lesion 2 (m/z 3547, 5000 was detected by MALDI-IMS mainly in lesion 1 and partially

3779, 3805, 3822, 4054, 4873, 5000, 5103, 5210) and lesion 2 rim in grey matter (Fig. 4A). The immunohistochemistry analysis con-

(m/z 3340, 5000, 5233, 17364, 17574). The ion density maps of firmed the presence of thymosin beta-4 in block A lesion 1 and

the two groups are shown in Fig. 4B and Supplementary Fig. 3. In in grey matter areas (Fig. 5B a). To validate the identification of

the GM* (cortical lesions) low molecular weight compounds (m/z thymosin beta-4, 12 brain sections from distinct multiple sclerosis

2225, 2569, 2622, 2964, 3340, 3805, 7524, 8475) were found; the patients were analysed by MALDI-IMS. The thymosin beta-4 mass

respective ion density maps are depicted in Fig. 4B and Supplemen- was detected in 12 patients. For validation of anti-thymosin beta-4

G. Maccarrone et al. / J. Chromatogr. B 1047 (2017) 131–140 137

Fig. 5. Thymosin beta-4 (Tb4) identification by MALDI Imaging mass spectrometry (MALDI–IMS) and validation by immunohistochemical analysis. (A) Identification. The

workflow includes thymosin beta-4 mass detection by MALDI–IMS, identification by LC–ESI–MSMS proteomics analysis and assessment by immunohistochemistry. (B)

Validation. MALDI–IMS ion maps, LFB-stained and anti-thymosin beta-4 immuno histochemical images of six out of eight distinct brain slides are reported. One sample

group includes demyelinated and/or active lesions, the second group includes remyelinated lesions. The results of the histopathologic analysis of the brain slides are: (a) Two

demyelinated/only partially remyelinated lesions; (b) Slightly hypocellular, at the border hypercellular lesion with myelin preservation; (c) Inflamed vessel in the centre of

the slight; hypercellular lesions with myelin preservation; (d) Black line: remyelinated lesion; red line: very small demyelination; (e) Completely remyelinated lesion; (f)

Completely remyelinated lesion. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6. Immunohistochemical stained human brain tissue slide of multiple sclerosis patient. Thymosin beta-4 (brown dots) is mostly expressed in (A) and (B)

microglia/macrophages (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).

138 G. Maccarrone et al. / J. Chromatogr. B 1047 (2017) 131–140

Table 2

Mass values (m/z) of ion species identified by MALDI imaging mass spectrometry in the multiple sclerosis human brain lesions.

Block A Block A Block A Block A Block B Block B Block B

Lesion 1 (17%) Lesion 2 (59%) BV ROI3 Lesion 1 (96%) Lesion 2 (35%) GM*

(Cortical lesion)

3549 10680 15205 2230 2225 3340 2225

3780 10886 15940 2572 2424 3547 2424

3823 14084 2626 2622 3779 2569

4054 16445 2967 16474 3805 2622

4092 17345 18650 3822 2964

5000 17542 4054 3340

5212 18608 4873 3805

5231 18813 5001 7524

5103 8475

5210 17364

5233 17574 17364

17574

Ion masses generated by the supervised statistical analysis of MALDI-IMS data obtained from the analysis of block A and B brain slides. The value of remyelination extents

are reported in percentage.

by immunohistochemistry serial brain slides from eight patients which were not detectable by routine histology, but were con-

were used (Fig. 5B). Thymosin beta-4 was detected by immuno- firmed by immunostaining with antibodies against CNPase and PLP

histochemistry at the rim of demyelinated and/or chronic active (Fig. 3B,C). These results highlight the potential of the unsupervised

lesions, in grey matter and highly abundant around an inflamed approach for hypothesis-free MALDI-IMS data analysis to identify

blood vessel in one of the brain slides (Fig. 5B). Thymosin beta-4 abnormalities that are not detectable by routine histopathology.

is not present in fully remyelinated lesion as depicted in Fig. 5B The concordance of MALDI-IMS ion mass images with histopathol-

(remyelinated lesions). In-depth analysis of lesions revealed that ogy images was demonstrated by the discovery of the sub-area BV

thymosin beta-4 is mainly expressed in macrophages and activated within the block A slide, mapped by the compounds, m/z 15,205

microglia (Fig. 6). In the normal-appearing tissue, thymosin beta-4 and m/z 15,940 characteristic for haemoglobin. Indeed, the hema-

was mainly detected in the grey matter, but not in oligodendro- toxylin and eosin stain revealed in the discrete area BV the presence

cytes. of erythrocytes in the tissue (Supplementary Fig. 1C).

The goal of our study was to determine proteins and peptides

4. Discussion specifically expressed in the normal-appearing white matter, grey

matter and in the lesions. To achieve this aim we interrogated the

In this study, for the first time the MALDI imaging technology is MALDI imaging spectra of white matter lesions, cortical lesions and

applied to map proteins and peptides expressed in human multiple normal-appearing white matter area and compared those using

sclerosis brain lesions. The determination of molecular masses and statistical tools.

spatial coordinates of proteins specifically expressed in the lesions The statistical analysis resulted in protein and peptide mass sig-

leads to the identification and localization of potential biomarkers nals whose pattern profiles distinguished the areas with different

involved in the tissue degradation or regeneration. These find- myelin content. Indeed, the molecular compositions were specifi-

ings can advance the knowledge in understanding mechanisms cally correlated with the histological features and allowed unbiased

involved lesion formation or regeneration. discrimination between the different partially remyelinated lesions

Recently, proteomics studies performed on active multiple scle- in both brain slides. The large overlap between the molecular com-

rosis lesions revealed targets for new therapies [14]. However, pounds present in the lesions of the two brain slides validated the

since the proteomics approach was based on the disruption of the link between analytes and tissue anatomical features. The lesions

tissue, these data lacked the proteins’ spatial distribution which with low extents of remyelination showed a marked overlap of

is important information for assessing the correlation between seven low molecular weight compounds (Table 2). As expected,

anatomical features, progress and development of the disease. The insufficiently remyelinated plaques were composed of low molec-

MALDI-IMS approach used in the present study enables the simul- ular weight components, possibly generated from degradation

taneous detection of molecular masses, their spatial distributions processes or caused by a lack of myelin proteins. In block B lesion

and abundances derived from multiple analytes without destroy- 1 with 96% remyelination extent, low molecular weight specimens

ing the tissue morphology. The method is unbiased and allows to were detected in addition to high molecular mass components. We

discover and to evaluate in parallel multiple compounds within the hypothesize that they are derived from protein breakdown occur-

same tissue section. This is in contrast to immunohistochemistry ring during the lesion formation. The block A lesion 2 with 59%

which requires target-specific reagents and multiple tissue sam- remyelination consisted of components with molecular weights

ples. The MALDI-IMS data nicely complement results obtained with of 10–18 kDa, which indicates that the remyelination process was

immunohistochemistry. incomplete or in progress. Interestingly, the protein profiles of cor-

In this study we focused on evaluating the ability of MALDI- tical lesions showed a similar pattern of compounds as the white

IMS to determine multiple analytes linked to brain tissue areas matter lesions with low myelin content (Table 2). Although corti-

with different myelin contents. Initially, we performed a hierarchi- cal lesions have been previously shown to differ from white matter

cal clustering analysis and classification of the MALDI-IMS dataset lesions by lower inflammation and mild astrogliosis, the MALDI-

without prior knowledge of the histological image. The unsuper- IMS results suggest that similar degradation processes may occur

vised analysis resulted in classes of compounds which presented within multiple sclerosis lesions of the white matter and grey mat-

a large correlation to white matter, grey matter and lesion areas. ter [21,22]. These findings are consistent with the hypothesis that

Interestingly, the in-depth interrogation of the unsupervised hier- cortical lesions are caused by similar autoimmune processes as

archical clustered MALDI dataset led us to discover cortical lesions white matter lesions [23].

G. Maccarrone et al. / J. Chromatogr. B 1047 (2017) 131–140 139

Another objective of this study was to identify peptides and multiple sclerosis lesions is demonstrated. Our data suggest a neu-

proteins expressed in the white matter and cortical lesions. For roprotective and neurorestorative role of thymosin beta-4 in the

this purpose we applied a proteomics workflow including liquid remodeling and remyelination processes occurring in the central

chromatography and electrospray ionisation tandem mass spec- nervous system of multiple sclerosis patients. However, to explore

trometry [24,25]. The proteomic analysis led to the identification the underlying mechanisms of remyelination in multiple sclerosis

of thymosin beta-4 whose mass (m/z 5000) was previously detected further studies on the identification of the molecular compounds

by the supervised statistical analysis within white matter partially in-situ are warranted.

remyelinated lesions (Fig. 4A,B). Immunohistochemistry of eight

multiple sclerosis cohort brain sections assessed that thymosin Funding

beta-4 is mainly expressed in macrophages and activated microglia

located at the rim of chronic active and/or demyelinated lesions This work was supported by the Max Planck Society (www.

(and in the grey matter) (Figs. 5 B, 6). In remyelinated lesions, mpg.de). This work was partly founded by Novartis Pharma GmbH,

thymosin beta-4 is detected neither by MALDI-IMS nor by immuno- Germany.

histochemistry as depicted in Fig. 5B (remyelinated lesions).

Thymosin beta-4 is a peptide that plays an important role in Acknowledgments

cell migration, in recruitment of different progenitor cells and in

the recovery processes in different tissues. Its main function is the The authors acknowledge the UK Multiple Sclerosis Tissue Bank,

promotion of cell migration by chemotactic effects and modula- Division of Neuroscience and Mental Health Imperial College, Lon-

tion of the cytoskeleton by sequestration of G monomers don for providing the brain tissue, Dr. Michael Becker (Bruker

[26]. As thymosin beta-4 is expressed in different immune cells, Daltonik GmbH) for supporting the imaging data analysis and Clau-

a role of the peptide in immune reactions has been postulated dia Kühne (Max-Planck Institute of Psychiatry) for recording the

[27]. During oxidative stress thymosin beta-4 has been shown to close-up images of the brain lesions and the assistent student

exhibit inhibitory effects on inflammation [26]. In our study thy- Korbinian Ehrmann (Max-Planck Insitute of Psychiatry) for sup-

mosin beta-4 is expressed in the grey matter. This observation porting in sample preparation for protein identification.

is similar to the results reported on human central nervous sys-

tem tissue not affected by multiple sclerosis [28]. The presence

Appendix A. Supplementary data

of thymosin beta-4 at the rim of not yet completely remyelinated

multiple sclerosis lesions may hint to a thymosin beta-4 role in

Supplementary data associated with this article can be found,

restorative effects in the neighborhood of normal appearing white

in the online version, at http://dx.doi.org/10.1016/j.jchromb.2016.

matter. In multiple sclerosis mouse model it was observed that thy- 07.001.

mosin beta-4 treatment have a protective role for the inhibition of

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