Article pubs.acs.org/jpr
New Analysis Workflow for MALDI Imaging Mass Spectrometry: Application to the Discovery and Identification of Potential Markers of Childhood Absence Epilepsy Mélanie Lagarrigue,† Theodore Alexandrov,‡,# Gabriel Dieuset,§,∥ Aline Perrin,⊥ Régis Lavigne,† Stéphanie Baulac,⊥ Herbert Thiele,# Benoit Martin,§,∥ and Charles Pineau*,† †
Inserm U1085, IRSET, Proteomics Core Facility Biogenouest, Campus de Beaulieu, F-35042 Rennes, France Center for Industrial Mathematics, University of Bremen, 28359 Bremen, Germany § INSERM U1099, F-35000 Rennes, France ∥ Université de Rennes 1, LTSI, F-35000 Rennes, France ⊥ Inserm UMR S975/CNRS UMR 7225, Centre de Recherche de l’Institut du Cerveau et de la Moelle Épinière, Université Pierre et Marie Curie, Hôpital de la Pitié-Salpêtrière, F-75013 Paris, France # Steinbeis Innovation Center SCiLS, Richard-Dehmel-Str. 69 D, 28211 Bremen, Germany ‡
S Supporting Information *
ABSTRACT: Childhood absence epilepsy is a prototypic form of generalized nonconvulsive epilepsy characterized by short impairments of consciousness concomitant with synchronous and bilateral spike-and-wave discharges in the electroencephalogram. For scientists in this field, the BS/Orl and BR/Orl mouse lines, derived from a genetic selection, constitute an original mouse model “in mirror” of absence epilepsy. The potential of MALDI imaging mass spectrometry (IMS) for the discovery of potential biomarkers is increasingly recognized. Interestingly, statistical analysis tools specifically adapted to IMS data sets and methods for the identification of detected proteins play an essential role. In this study, a new cross-classification comparative design using a combined discrete wavelet transformation-support vector machine classification was developed to discriminate spectra of brain sections of BS/Orl and BR/Orl mice. Nineteen m/z ratios were thus highlighted as potential markers with very high recognition rates (87−99%). Seven of these potential markers were identified using a top-down approach, in particular a fragment of Synapsin-I. This protein is yet suspected to be involved in epilepsy. Immunohistochemistry and Western Blot experiments confirmed the differential expression of Synapsin-I observed by IMS, thus tending to validate our approach. Functional assays are being performed to confirm the involvement of Synapsin-I in the mechanisms underlying childhood absence epilepsy. KEYWORDS: imaging mass spectrometry, absence epilepsy, MALDI-time-of-flight mass spectrometry, protein markers, classification method
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the basis of 8 inbred strains4) and present opposite epileptic characteristics. BS/Orl strain displays spontaneous and recurrent spike-wave discharges (SWD) (8 ± 1 Hz) in both the cortex and ventrolateral thalamus concomitant with twitching of the vibrissae and an interruption of ongoing behavior. These SWD are suppressed by the antiabsence seizure drugs ethosuccimide and sodium valproate and they are exacerbated by the antiseizure drug, carbamazepine, known to aggravate CAE. Remarkably, BR/Orl strain does not display any SWD either spontaneously or after systemic injection of drugs known to induce SWD. These findings suggest that the BR/Orl strain, beyond its absence of spontaneous SWD activity, possesses a CAE-resistant genome
INTRODUCTION Childhood absence epilepsy (CAE) is a prototypic form of generalized nonconvulsive epilepsy characterized by short impairments of consciousness concomitant with synchronous and bilateral spike-and-wave discharges (SWD) in the electroencephalogram (EEG).1 It has been demonstrated that CAE is linked to a number of cognitive, behavioral and emotional disorders.2 A genetic origin of this form of epilepsy has been clearly identified. In particular, several mutations in GABA-A receptor subunit genes but also in genes related to Na+, K+, Ca2+ channels have been reported in families with CAE.3 Among the different existing models of CAE, the BS/Orl and BR/Orl strains constitute an original mouse model “in mirror”. These two strains are the product of a genetic bidirectional selection derived from an eight-way cross (a genetic pool created on © 2012 American Chemical Society
Received: July 27, 2012 Published: September 20, 2012 5453
dx.doi.org/10.1021/pr3006974 | J. Proteome Res. 2012, 11, 5453−5463
Journal of Proteome Research
Article
France). The MALDI matrix (sinapinic acid, SA) was obtained from Bruker Daltonik GmbH (Bremen, Germany).
whereas the BS/Orl strain represents a mouse model of absence epilepsy. The potential of MALDI imaging mass spectrometry (IMS) for the discovery of disease markers that can help for elucidation of disease pathways or early diagnosis is now well accepted.5−8 Indeed, IMS has the particular advantage of preserving information on the spatial localization of molecules of interest in the tissue that is lost when techniques based on the analysis of biological fluids or tissue homogenates are used. Contrary to most conventional imaging techniques, IMS allows the simultaneous localization of hundreds of molecular species directly from a tissue section without labeling or a priori knowledge of targeted compounds.9 Moreover, IMS can detect a wide range of biomolecules such as proteins, peptides, lipids, sugars, as well as drugs and their metabolites.10 Although MALDI imaging mass spectrometry has proven its potential for the discovery of disease markers, important developments are still required. Recent technological advances performed to reach a routine lateral resolution of 20 μm11 have to be pursued to improve resolution near the single cell level (∼5 μm). The routinely detectable mass range, which is currently limited to low-mass proteins (
