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 multiple sclerosis 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 central nervous system 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 peptides 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 myelin 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 thymosin 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) staining (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