Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD003644/F080356.dat-pride.xml.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD003644/F080352.dat-pride.xml.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD003644/F080357.dat-pride.xml.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD003644/F080349.dat-pride.xml.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD003644/F080354.dat-pride.xml.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD003644/F080350.dat-pride.xml.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD003644/F080358.dat-pride.xml.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD003644/F080346.dat-pride.xml.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD003644/F080359.dat-pride.xml.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD003644/F080355.dat-pride.xml.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD003644/F080351.dat-pride.xml.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD003644/11142014_CL2_45min.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD003644/11142014_IC2_45min.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD003644/02262015_VLL_MALDI_9_45min.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD003644/02262015_VLL_MALDI_7_45min.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD003644/11142014_CL1_45min.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD003644/11142014_IC1_45min.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD003644/11142014_CL3_45min.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD003644/11142014_IC3_45min.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD003644/11142014_IC4_45min.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD003644/02262015_VLL_MALDI_8_45min.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD003644/11142014_CL4_45min.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD003644/F080349.dat-pride.pride.mgf.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD003644/F080352.dat-pride.pride.mgf.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD003644/F080350.dat-pride.pride.mgf.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD003644/F080358.dat-pride.pride.mgf.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD003644/F080354.dat-pride.pride.mgf.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD003644/F080356.dat-pride.pride.mgf.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD003644/F080346.dat-pride.pride.mgf.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD003644/F080351.dat-pride.pride.mgf.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD003644/F080359.dat-pride.pride.mgf.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD003644/F080355.dat-pride.pride.mgf.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD003644/F080357.dat-pride.pride.mgf.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD003644/F080356.dat-pride.pride.mztab.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD003644/F080355.dat-pride.pride.mztab.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD003644/F080350.dat-pride.pride.mztab.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD003644/F080354.dat-pride.pride.mztab.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD003644/F080349.dat-pride.pride.mztab.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD003644/F080351.dat-pride.pride.mztab.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD003644/F080352.dat-pride.pride.mztab.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD003644/F080358.dat-pride.pride.mztab.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD003644/F080346.dat-pride.pride.mztab.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD003644/F080359.dat-pride.pride.mztab.gzftp://ftp.pride.ebi.ac.uk/pride/data/archive/2016/12/PXD003644/F080357.dat-pride.pride.mztab.gzprimaryOK200004920montaner.villalonga@gmail.comVíctor LlombartMass SpectrometryMass spectrometry imagingShotgun proteomicsMatrix Science Mascot 2.5.1Maldi imagingBiomarkersBrain ischemiaTmcaohttp://www.ebi.ac.uk/pride/archive/projects/PXD003644BrainBrain tissue processing, MALDI mass spectrometry and image analysis Brains (n=4) were isolated and immediately frozen in isopentane (Sigma, USA) at -40oC during 30 sec and stored at -80oC. Ten µm-thick coronal brain sections (+0.74 to +0.98 from bregma) were obtained using a criostat (Microm HM 505E, CBIS, USA) and mounted onto superconductive glass slides covered with ITO coating (Bruker Daltonics, Germany). Tissue preparations were dried with vacuum pump (Millipore, USA) at 600 mmHg for 15 minutes, washed for two minutes in 70% Ethanol and once in 96% Ethanol and then stored in a dessicator under vacuum. In order to be analyzed by MALDI TOF MS, samples werec overed with sinapinic acid (10mg/mL 60% acetonitrile and 0.2% trifluoroacetic acid (TFA)) as MALDI matrix sprayed using ImagePrep device (Bruker Daltonics). The optimal parameters of the device were set (dry time, incubation time and thickness). Finally the slides were mounted into MTP slide adapter (Bruker Daltonics) and transferred to the MALDI-TOF mass spectrometer. These four biologically independent samples were analyzed by MS. We acquired the IMS spectra in linear ion positive mode in an UltrafleXtreme MALDI-TOF/TOF mass spectrometer. MS data were averaged from 300 consecutive laser shots with a frequency of 2000 shot/s. The raster spatial resolution was 100 µm. After the MS analysis hematoxylin & eosin (H&E) staining was performed in each brain section and they were used as reference for colocalization to select the regions of interest (ROI). Laser Micro-Dissection and protein extracts We mounted consecutive sections of 12 µm thickness obtained from the brains analyzed by the MALDI imaging alternating PEN-membrane glass slides (Leica, Germany) and superfrost glass slides (Thermoscientific). From the samples mounted on PEN-membrane slides we cut sections of the infarct and the contralateral area by means of laser microdissection (Leica, Germany) and recovered them separately in 25 µL of 0.1% Rapigest SF surfactant (Waters). Samples mounted on glass slides were used for infarct core colocalization. Samples were sonicated at 80% amplitude for intervals of 10 sec for a total of 2 min at 4oC. Then, samples were centrifuged at 13,000g at 4ºC, the supernatants were recovered, supplemented with 4.5 µL of aprotinin (Sigma) and 10 µL of phenylmethylsulfonyl fluoride (Sigma) as protease inhibitors, and finally stored at -80oC until use. The obtained homogenates were fractionated on a 10-20% Tris-tricine gel (Biorad, USA) and 4 sections of each sample, infarct core and contralateral area, were cut from the gel. The gel bands, corresponding to the proteins with an apparent molecular weight bellow 25 kDa, were digested in-gel with trypsin and subsequently analyzed by MS/MS. Tissue homogenates and reverse phase HPLC For reverse phase HPLC separations, mechanical homogenates of infarct core and contralateral healthy areas of mouse brain were obtained in 0.2% of TFA and 60% of acetonitrile (ACN). Samples were sonicated using the previous parameters. Insoluble material was removed by centrifugation 13,000g at 4ºC, protease inhibitors were added into the recovered supernatant and samples were stored at -80oC until use. Proteins from the soluble material were separated using and Alliance HPLC system (Waters, USA) and a C4 widepore 3.6 um; 4.6x100 mm Aeris reverse phase (RP) column (Phenomenex). Solvent A was 0.1% (TFA) in water and solvent B was 0.1% TFA in acetonitrile. A linear gradient from 10 to 30% for 1 min, followed by a second step from 30 to 90% for 60 min was applied. Fractions were collected every 2 min, dried using SpeedVac (ThermoScientific, USA) and reconstituted in 30 µL of H2O 0.1 % TFA mixed at 1:1 volume ratio with sinapinic acid (SA) matrix (10 g/ml SA in 1:2 H2O:ACN, 0.2 % TFA). Samples were mounted onto a ground steel plate, let to cristalize and mass spectra were obtained by MALDI MS analysis (lineal mode, 24kV) to check for the presence of m/z of interest.PrideNot availableiodoacetamide derivatized residuemonohydroxylated residueData quantification and statistical MALDI-IMS data analysis After MALDI analysis, we used FlexImaging 3.0 (Bruker Daltonics, Germany) software to obtain ion density maps and to define the ROIs on each brain section. One ROI was defined on the infarct core at the cerebral cortex and another ROI on the contralateral healthy area based on H&E staining. Mass spectra from each ROI were combined to a single dataset and exported to ClinProtools 3.0 (Bruker Daltonics, Germany). The analysis of the mass spectra was focused on the mass range 2000-24000 m/z. A 10% of minimal baseline width was applied as top hat baseline substraction and a factor of 2 was used for data reduction. For recalibration, a maximal peak shift of 2000 ppm was defined and a 20% match to calibrant peaks was set. Not recalibratable spectra were excluded from analysis. Signal to noise ratio for data acquisition was set to 5. An average spectrum from each ROI and the average peak list were obtained. To check the homogeneity of the spectra set we carried out principal components analysis (PCA) with the Matlab algorithm and used the 3 primary PCAs for the 3-D scaling representation. To select the most relevant m/z peaks from the average peak list we used the loading plot coupled to each PCA graphic. We also obtained the area under the curve (AUC) to assess the discrimination of contralateral (CL) and infact (IC) of each m/z peak. Bottom-up nano LC-MS/MS The samples obtained after laser microdissection and resolved by tricine gels, as well as fractions from RP-HPLC that contained m/z of interest were digested with trypsin. Peptides obtained from in-gel and in-solution digestions were subsequently analyzed on a LTQ Velos-Orbitrap mass spectrometer (ThermoFisher Scientific, Bremen, Germany) coupled to a nano-HPLC system (Proxeon, Odense, Denmark). Briefly, peptide mixtures were initially concentrated on an EASY-column, 2 cm long, 100 μm internal diameter (id), and packed with ReproSil C18, 5 μm particle size (Proxeon, Denmark), and subsequently loaded onto an EASY-column, 75 μm id, 10 cm long, and packed with ReproSil-Pur C18-AQ, 3 μm particle size (Proxeon, Denmark). An ACN gradient (5− 35% ACN/0.1% formic acid in water, in 90 min, flow rate of 300 nL/min) was used to elute the peptides through a stainless steel nanobore emitter (Proxeon, Denmark) onto the nanospray ionization source of the LTQ Velos-Orbitrap mass spectrometer. MS/MS fragmentation spectra (200 ms, 100− 2800 m/z) of 20 of the most intense ions, as detected from a 500 ms MS survey scan (300−1500 m/z), were acquired using a dynamic exclusion time of 20 s for precursor selection and excluding single-charged ions. Precursor scans were acquired in the Orbitrap analyzer at a mass resolution of 30,000. MS/MS spectra were acquired at the LTQ Velos analyzer using a relative collision energy of 35. An intensity threshold of 1000 counts was set for precursor selection. Bioinformatics for protein identification In order to match the relevant identified m/z peaks after MALDI IMS with the molecular masses of the proteins identified by bottom-up analysis, we used the PeptideMass tool from ExPasy (http://web.expasy.org/peptide_mass/)[23]. This tool allows the generation of the theoretical peptide masses of the input proteins. By selecting the “no cutting” option as enzyme all the putative protein fragments resulting after their processing were displayed (i.e. pro-form, signal sequences, transit peptides, active peptide, etc). All known post-translational modifications and variants of the proteins were considered and all other parameters were used as default. The identified MALDI-IMS m/z peaks were matched with the masses of proteins and/or peptides retrieved by PeptideMass allowing a mass shift of 2000 ppm.ProteomicsJoan MontanerLTQ Orbitrap VelosCOMPLETEvall hebron institute of researchMus Musculus (mouse)27888142 Llombart V, Trejo SA, Bronsoms S, Morancho A, Feifei M, Faura J, García-Berrocoso T, Simats A, Rosell A, Canals F, Hernández-Guillamón M, Montaner J. Profiling and identification of new proteins involved in brain ischemia using MALDI-imaging-mass-spectrometry. J Proteomics. 2016 Nov 22;152:243-253 10.1016/j.jprot.2016.11.014llombs@hotmail.comBiomedicalVall Hebrón Institute of Research10.6019/PXD003644The identification of proteins involved in brain ischemia might allow the discovery of putative biomarkers or therapeutic targets for ischemic stroke. Our aim is to study the distribution of proteins within mouse brain after an ischemic insult using MALDI imaging-mass-spectrometry and to identify relevant proteins involved in brain damage. We occluded the middle cerebral artery of C57BL/6J mice. Brain slices were analyzed by MALDI-TOF and infarct (IC) and contralateral (CL) regions were compared using ClinProTools. The ion distribution maps of relevant m/z values were obtained by FlexImagin3.0. Protein identification was conducted through a bottom-up approach consisting on complementary sample fractionation methods. Some identifications were confirmed by immunohistochemistry and western blot. We identified 102 m/z values with different abundances between IC and CL (p<0.05), from which 21 m/z peaks were selected as more relevant. Thirteen of them were found increased in the infarct region and 4 m/z values showed AUC>90% between IC and CL. Identification analyses confirmed altered expressions of ATP5i, COX6C and UMP-CMP kinase in IC compared to CL.<h4>Biological significance</h4>Using MALDI-IMS we identified for the first time new proteins that might be involved in brain ischemia representing potential diagnostic biomarkers or target molecules for neurological recovery.Profiling and identification of new proteins involved in brain ischemia using MALDI-imaging-mass-spectrometry.Llombart Víctor V, Trejo Sebastián Alejandro SA, Bronsoms Sílvia S, Morancho Anna A, Feifei Ma M, Faura Júlia J, García-Berrocoso Teresa T, Simats Alba A, Rosell Anna A, Canals Francesc F, Hernández-Guillamón Mar M, Montaner Joan Jl(2)k04204, Bovine Kunitz Pancreatic Trypsin Inhibitor, Water, MeCN, cinnamic acid, ion, Molecular Weights, FEZL, CG1685, BOUND WATER, monosomy 2q32, oxidane, Steels, Kap-alpha2, Solvent, Long Term, thomson., WATER, HOH, 5730420M11Rik, Trifluoroacetate, Antilysin, protein polypeptide chains, Trypsin Inhibitor, Kontrykal, type I, TSPAN-33, Protease Inhibitor, CH3-C#N, GRP1, Microdissections, Grp1, SEC, 3, 4, Analysis, myofibromatosis, 7, 9, WMS, ASD2, insoluble, SET, HA-8, Divorced, Grain, C79325, Analyses, rya-r44F, Molecular, phosphatase 2A inhibitor I2PP2A, PTPSTEP, sec, b-SAAS, Tissue, Su(Mir)1, Dimp-alpha2, Fluoride, Hemotoxylin, Weights, proteins, Divorces, suprasegmental levels of nervous system, Shots, 1810043K16Rik, HPLC, Rya-R44F, DmelCG4299, AI047805, Benzenemethanesulfonyl Fluoride, set, Dehydrated ethanol, PEN19, Peptidohydrolase Inhibitor, dehydrated, CG10844, column, g/ml, ACN, 3-(4-hydroxy-3, sample, Acn, PEN20, 10(6H)-pentol, CT, monosomy 2q32-q33, s, Prx3, Histological Labeling, GPH, SGS, spiritus vini, Ito, suprasegmental structures, alphaKap2, Ct, Longterm Effect, Incontinentia pigmenti type 1, integral to membrane, Kunitz, Slc39a1, Bovine Pancreatic Trypsin Inhibitor, Spectrum Analysis, not genetically inherited, IMPalpha2, IA-4, Absolute Alcohol, ACMICD, H2O, Dm1, soluble, SATB2-associated syndrome, Weight, Hydrolase Inhibitor, Del(2)(q32), Xsl, 11b-dihydro-, simple tissue, Pancreatic, [OEtH], Pcm, Hydrogen Oxide, anon-WO0140519.258, Acid, 2q32q33 microdeletion syndrome, multicentric myofibromatosis, HLA-DR-associated protein II, instrument, DI-2, Grv, Antagonists, CG11628, I-2Dm, proportionality, Spectrometry, rate, Striatum-enriched protein-tyrosine phosphatase, inhibitor of granzyme A-activated DNase, oho31, 2-d)pyran-3, beta Trypsin, alcohol etilico, Methylcarbinol, I-2PP1, Speed, TAF-IBETA, grv, 2-methylbutane, TAF-Ibeta, Mouse, sinapic acid, methyl cyanide, Endopeptidase, Peptide Peptidohydrolase Inhibitor, 5-dimethoxy-, (E)-sinapic acid, PEN, Pen, 42/4, OG12X, l(1)VE614, GRP1/cytohesin 1, hypomelanosis of Ito, Effects, Endopeptidase Inhibitors, etanol, Hematoxiline, number, infantile hemangiopericytoma, CG4799, IMAGE, pen, protein-containing complex, Chalk, body system, CG7826, importin alpha2/pendulin, agua, mass-to-charge ratio, l(2)k05821, CG11633, period, SATB2 syndrome, l(2)k15606, TOF, Pulmin, l-SAAS, CG7835, Gene Products, CG42273, l(2)k14401, Kallikrein-Trypsin, system, Big PEN-LEN, Peptide Hydrolase, millimeters of mercury, alpha2, ZNF312, T6K12_14, Cesium Trifluoroacetate, Ito hypomelanosis, proportion, anatomical systems, Acinus, Longterm, ethanol, PUF6, beta-Trypsin, Hydroxybrasilin, 2pp2a, Long-Term, ions, kf, Antagonist, CG10574, Basic Pancreatic Trypsin Inhibitor, acinusL, C2H5OH, 2PP2A, Protease Antagonists, acinusS, Kpna2, dSET, dSet, Peptide Peptidohydrolase Inhibitors, DmelCG10844, 2.1, Kallikrein Trypsin Inactivator, Zymofren, DRR, 1-(14)C-labeled, DRY, Proteins, Dilmintal, CG18076, Haematoxylon, stepk, Stainings, acetonitrile, AI848336, encephalon, mKIAA0670, Proprotein convertase subtilisin|kexin type 1 inhibitor, Incontinentia pigmenti type 1 (formerly), DmelCG4799, native protein, I-2PP2A, dry, chemical analysis, Dm I-2, Long Term Effects, alcool ethylique, MFS1, Mass Spectrum Analyses, T6K12.14, AU020952, l(2)k00424, Contrical, XTP5, 5-dimethoxycinnamic acid, Separations, Kap alpha2, kak, Benz(b)indeno(1, ethanenitrile, Childhood, Peptidohydrolase Inhibitors, Ol5, SAAS CT(1-49), OG12, Longterm Effects, DmelCG11628, ME-IV, Gene Proteins, ZFP312, Peptidase, l(2)k04913, Antiprotease, MTP1, qip2, quotient, Peptide Hydrolase Inhibitors, quantitative, Og12x, Contrykal, [OH2], Benzenemethanesulfonyl, formerly, IPP2A2, Endopeptidase Inhibitor, Grain Alcohol, Meth, NCMe, determination, OHO31, Peptide Hydrolase Inhibitor, Imaging, 6330543G17Rik, protein, Xkl-1, srp1alpha, 1-hydroxyethane, alcohol, Cut, Ethanol, SAASCT, pen-2, Traskolan, BANF, l(1)7Ba, l(1)7Bb, IREG1, Alkohol, Phenylmethylsulfonyl, Min, Kontrikal, Inhibitors, synganglion, KEP, infantile myofibromatosis, protein aggregate, impalpha2, Hydrolase Inhibitors, monosomy 2q32q33, Effect, lLEN, DmelCG42273, Mass Spectrum Analysis, portion of tissue, l(2)144/1, kDa, TAF-I, TACHD, Ethyl, 2610036I19Rik, membrane region, 2610510L13Rik, min, HLA-HA8, mAPC, Aethanol, [CH2Me(OH)], CG11387, imp-alpha2, IGAAD, fSAP152, chromosome 2q32-q33 deletion syndrome, Slc11a3, Big SAAS, DmelCG10574, Ice, l(2)k08110, trifluoro-, dya, b-PEN-LEN, PEN., dihydridooxygen, l(2)CA4, house mouse, synapitic acid, dihydrogen oxide, GPHYSD2, Long-Term Effects, Iniprol, bLEN, ratio, phapii, Dissections, oho-31, bs29g06.y1, Alcohol, Hydroxybrazilin, N-(2-hydroxy-1, hi syndrome, Peptidase Inhibitor, mmHg, rch1, mouse, aqua, Dmel_CG7826, StF-IT-1, Th, Labeling and Staining, Ethyl alcohol, membranous organ component, Peptide Peptidohydrolase, shortstop/kakapo, Spectroscopy, glass, Immunization, DmelCG1685, proSAAS, Ionen, DmelCG18076, 3.1.3.48, tissue portion, BcDNA:GH10590, ETHANOL, Acetic acid, Dmel_CG7835, Crystal, Mnb, MNB, membrane of organ, EtOH, RyR, Step, mice, Encephalon, Trifluoroacetic, hydrogen hydroxide, 5-dimethoxyphenyl)-, Del(2)(q32q33), CG4299, MASS, AW124434, SHOT, surfactant, acqua, organ system, Dusg, Peptidase Inhibitors, Labelings, Cesium, RYR, Ryr, hPUF-A, STEP, 6a, trans-sinapinic acid, surface active agent, Antiproteases, Wasser, Big LEN, i2pp2a, Inhibitor, time, Rch1, Oho31, the brain, PEN-19, ryr, Dalpha2, 5-dimethoxy-4-hydroxycinnamic acid, Histological Labelings, nucleocapsid, (E)-, surfactants, l-LEN, FBN, Gene, Protease, Spectrum Analyses, ACINUS, Xsl-1, Xsl-2, presence, PHAPII, cytohesin/GRP1, Aethylalkohol, SAAS, template-activating factor I, polypeptide chain, glass syndrome, pigmentary mosaicism, cis-(+)-, DPend, Phenylmethanesulfonyl Fluoride, ECTOL1, 2-propenoic acid, integral component of membrane, Tina, RyRs, Mass, Histological, pro-SAAS, Protease Antagonist, Separated, Mass Spectroscopy, Shot, steel, Glass, l(2)k10821, Staining, Incontinentia pigmenti achromians, Absolute, iones, Selb, ipp2a2, imp alpha2, l(2)SH2 0323, eau, alpha2A-Kap, BPTI, OCTD, Kallikrein-Trypsin Inactivator, Benzenemethanesulfonyl fluoride, Neural-specific protein-tyrosine phosphatase, 10^[-6], Imp-alpha2, DmelCG11387, taf-ibeta, b-LEN, region of membrane, Pan3, Long-Term Effect, FEZ, 2q32-q33 microdeletion syndrome, membrane, data, DYRK1, Inactivator, MTP, 2q32q33 microdeletion syndromes, 3H-labeled, protein complex, kop, igaad, CG18637, 4-hydroxy-3, Labeling, Peptide, light amplification by the stimulated emission of radiation, count in organism, secret agent, natural protein, SAAS CT(25-40), Mus, water, IMS, Protein, I2PP2A, whole membrane, core, Dyrk1, gram per millilitre, Vacuums, Phenylmethanesulfonyl, connected anatomical system, Little SAAS, Ito type, l(2)SH0323, ACETONITRILE, cyanomethane, 1-bis(hydroxymethyl)ethyl)glycine, WMS2, Ethyl Alcohol, CYH1, Proprotein convertase 1 inhibitor, Basic, Mass Spectrum, VSD1, l(2)k05434, Separation, SAS, transmembrane, dRyR, Fpn1, l(2)k03010, Little LEN, CC1, Tripcellim, sample population, incised, Protein Gene Products, Trypure, Incontinentia pigmenti, dSET/TAF-Ibeta, hydroxyethane, ipoa1, HMI, 2610030F17Rik, MALDI, PEN-20, Ion, Kunitz Pancreatic Trypsin Inhibitor, SSKS, assay, AA407739, 1300010A20Rik, SCH, IPA, Trasylol, trans-sinapic acid, Rya-r76CD, presence or absence in organismischemic disease of brain, Brain, ischemic encephalopathy, Ischemic, Ischemias, Spectrum., cerebrovascular ischemia, Proteins, Spectrometry, FBN, Gene, Ischemia, proteins, Encephalopathy, Imaging, MASS, Ischemic Encephalopathy, Protein Gene Products, ACMICD, OCTD, Spectroscopy, Gene Proteins, Brain Ischemias, ischemia cerebrovascular, brain ischemia, Disruption of blood oxygen supply to brain, MALDI, Cerebral Ischemias, ECTOL1, Protein, SSKS, Gene Products, ischaemic encephalopathy, brain ischemic disease, MFS1, Analysis, Cerebral Ischemia, Cerebrovascular ischemia, Brain ischemia, GPHYSD2, Cerebral, WMS, Ischemic Encephalopathies, WMS2, SGSsodium salt, Forms, ammonium formate, Size, 13C-labeled, cadmium salt, ion, Particle, magnesium formate, zinc salt, Steels, Long Term, caudal communicating segment, 5730420M11Rik, Polypeptides, protein polypeptide chains, cortical plate (areas), cobalt(II) formate dihydrate, Microdissections, B1, Software Engineering, Analysis, myofibromatosis, Faroe Islands, Archipallium, WMS, Benson syndrome, average, SET, C79325, Analyses, Cortex Cerebrus, phosphatase 2A inhibitor I2PP2A, proteins, suprasegmental levels of nervous system, HPLC, DmelCG4299, AI047805, set, Stainless Steels, density, column, ACN, D1, Acn, n, Stainless steel, Histological Labeling, SGS, nickel salt, sleeping sickness, NK/GPI, LC-MS2, Gpi, elp3, suprasegmental structures, Data Set, Chm, cobalt (+2) salt, African sleeping sickness, Pdnp1, Paleocortex, Pollution, arteria cerebri communicans posterior, Longterm Effect, Normalcy, not genetically inherited, Software Tools, ACMICD, cortical plate (CTXpl), Computer Applications, 4833416E15Rik, sodium (4:1:1) salt, magnesium salt, Dm1, Algorithm, Computer Applications Software, PCA, Pca, Sizes, NK|GPI, Hydrogen Oxide, PCS, multicentric myofibromatosis, Software Applications, HLA-DR-associated protein II, DI-2, Source Software, I-2Dm, Archipalliums, proportionality, formate, rate, cromium (+3), inhibitor of granzyme A-activated DNase, beta Trypsin, I-2PP1, Applications, TAF-IBETA, ELP3, DmelCG5229, liquid chromatography-tandem mass spectroscopy, ammonium (4:1) salt, TAF-Ibeta, Allocortex, lead (+2) salt, Bglap-rs1, Computer Software Applications, Particle Sizes, breadth, nickel formate dihydrate, lead formate, Effects, aluminum salt, infantile hemangiopericytoma, precursor, thomson, protein-containing complex, body system, African trypanosomiasis, CG7826, mass-to-charge ratio, period, LC-MSMS, CG7835, Gene Products, CG42273, system, Pgi, HAT1, Application, Normalities, Open Source Softwares, proportion, anatomical systems, Acinus, thallium (+1) salt, Longterm, Gpi-1r, Software Application, Gpi-1s, Phi, beta-Trypsin, rubidium salt, Gpi-1t, Open Source Software, 2pp2a, Long-Term, dmHAG408, methanoic acid, ions, dmHAG404, CG10574, Solution, Computer Software Application, acinusL, Faeroe Islands, 2PP2A, Tools, acinusS, Steel, dSET, dSet, MFSD7B, peptidos, PTHB1, Cortical Plates, Proteins, Stainings, potassium formate, copper (+2) salt, MF, Amf, encephalon, mKIAA0670, Tool, polypeptide, M6S1, Cortex, native protein, I-2PP2A, African Trypanosomiasis, C18, cupric formate, chemical analysis, Dm I-2, Long Term Effects, MFS1, aluminum formate, NK, AU020952, lithium formate, elp-3, choanal atresia, posterior communicating segment of the basilar artery, Longterm Effects, ME-IV, Computer Programs, Gene Proteins, Applications Softwares, Periallocortex, quotient, liquid chromatography tandem mass spectrometry, ttw, Data Analyses, Cortical Plate, liquid chromatography tandem mass spectroscopy, IPP2A2, determination, selection process, zinc formate, lead salt, protein, D-elp3, peptide, ammonium tetraformate, peptido, ClvPrd, BANF, Min, synganglion, infantile myofibromatosis, protein aggregate, Gpi-1, cortex cerebralis, Effect, Computer Program, Paleocortices, DmelCG42273, Pca-1, LCMSMS, peptides, TAF-I, Nlk, long, Open, 2610036I19Rik, 2610510L13Rik, Computer Programs and Programming, min, mAPC, analyzer, twy, copper, SCAN, CG5229, IGAAD, fSAP152, lithium salt, DmelCG10574, ppm, Plate, GPHYSD2, HAT, ammonium (2:1) salt, Long-Term Effects, ratio, phapii, Noise, atresia of nares, N-(2-hydroxy-1, Dmel/ELP3, LC-MS/MS, Dmel_CG7826, Maps, StF-IT-1, Th, Labeling and Staining, Source Softwares, Programs, Program, C76301, mOC-X, Ionen, Computer Applications Softwares, POSTERIOR, Softwares, nickel (+2) salt, AXPC1, Dmel_CG7835, Mnb, MNB, parent ion, ammonium salt, ORG, Org, Encephalon, cobaltous formate, Gpi1-r, Gpi1-s, MASS, CG4299, AW124434, Gpi1-t, Stainless, organ system, nanospray, Health, Labelings, precursor ion, copper salt, PC-1, Noise Pollution, Polypeptide, pallium of the brain, cortex cerebri, i2pp2a, Ly-41, time, Gpi1s, the brain, CD203c, NPP1, AI461847, nucleocapsid, Histological Labelings, cesium salt, FBN, Gene, Computer, ACINUS, PHAPII, LC-MS-MS, formic acid, potassium salt, template-activating factor I, polypeptide chain, ECTOL1, 14C-labeled, Histological, CG15433, Cerebral, Npps, FLVCR, cortex of cerebral hemisphere, Allocortices, Staining, iones, Cortical, biparietal Alzheimer disease, ipp2a2, ppm., calcium formate, OCTD, 10^[-6], taf-ibeta, posterior choanal atresia, cromium (+3) salt, Long-Term Effect, Cortex Cerebri, sodium formate, Applications Software, Open Source, data, DYRK1, Computer Software, nickel formate, protein complex, Cerebral Cortices, igaad, strontium formate, DmelELP3, strontium salt, Labeling, Peptide, light amplification by the stimulated emission of radiation, Software Tool, LC/MS/MS, imperforate nares, natural protein, AI428932, IMS, Protein, I2PP2A, core, Africam sleeping sickness, Dyrk1, connected anatomical system, Software, human African trypanosomiasis, WMS2, 1-bis(hydroxymethyl)ethyl)glycine, brain cortex, DmelCG15433, 10^[-9], Normality, CC1, Engineering, Tripcellim, chromic formate, Noises, Protein Gene Products, Trypure, dSET/TAF-Ibeta, 2610030F17Rik, MALDI, Ion, Data, PCARP, SSKS, Periallocortices, calcium salt, Peptid, assay, E-NPP1, AA407739, SCHBiological Markers, Viral Marker, Surrogate Endpoints, ion, Ischemic, HSN1E, determination, Laboratory, colony forming unit granulocyte, cerebrovascular ischemia, Mus domesticus, FEZL, Biochemical, Ischemia, Endpoint, Imaging, CASP-14, protein, Serum, Technic, House Mouse, Long Term, caudal communicating segment, Brain Ischemias, brain ischemia, Laboratory Markers, protein polypeptide chains, Techniques, diseases, Immunogold-Silver Techniques, macrophage, Cerebral Ischemias, Biological, Method, Immunogold-Silver Technique, brain ischemic disease, diseases and disorders, synganglion, Kinase, Analysis, myofibromatosis, Brain ischemia, infantile myofibromatosis, protein aggregate, present in fewer numbers in organism, Effect, WMS, Benson syndrome, ASD2, portion of tissue, increased, human 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Technics, Immunolabeling Techniques, COVIc, assay, E-NPP1, ttw, Laboratory Mouse, CLEC2C, methodology, Immune MarkersCVA (cerebral vascular accident), undetermined stroke, Biological Markers, Viral Marker, Apoplexy, Cryptogenic Ischemic Strokes, Surrogate Endpoints, ion, Ischemic, Strokes, HSN1E, Laboratory, CVA (Cerebrovascular Accident), cerebrovascular ischemia, Mus domesticus, Biochemical, Ischemia, Endpoint, Imaging, CASP-14, protein, neutral molecular compounds, Serum, Technic, Ischaemic Strokes, House Mouse, Long Term, Brain Ischemias, brain ischemia, Acute Stroke, Laboratory Markers, protein polypeptide chains, Techniques, Immunogold-Silver Techniques, Cerebral Ischemias, Biological, Method, Immunogold-Silver Technique, cerebrovascular accident, CVA, brain ischemic disease, Vascular Accidents, synganglion, cerebral infarction, Analysis, Brain ischemia, protein aggregate, Effect, SYNDROME, molecule, WMS, Cryptogenic Embolism, increased, molecules, ATP5K, activation inducer 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Immunohistocytochemistry, Acute Ischemic Strokes, Ischemic Encephalopathy, MCMT, C57BL6J, encephalon, Cryptogenic Embolism Strokes, Immune Marker, BL-AC/P26, Cerebrovascular Apoplexya, native protein, natural protein, Mus, Surrogate End Point, Protein, Long Term Effects, ischaemic encephalopathy, (CVA), sequence, MFS1, techniques, ISCHSTR, Acute Ischemic Stroke, WMS2, Biologic Markers, Immunolabeling, CXXC9, Serum Marker, Immunocytochemistry, Stroke NOS, Surrogate, increased number, CVA - Cerebrovascular accident unspecified, Endpoints, COX-VIc, House Mice, Cerebrovascular Accident, Atp5k, cox6c.2, Atp5i, Surrogate Marker, Cerebral Strokes, sample population, primary structure of sequence macromolecule, Longterm Effects, Laboratory Mice, Protein Gene Products, Gene Proteins, present in greater numbers in organism, Disruption of blood oxygen supply to brain, Acute Strokes, Acute, MALDI, GP32/28, Ion, stroke syndrome, Stroke and cerebrovascular accident unspecified (disorder), SSKS, Immunogold-Silver Technics, Immunolabeling Techniques, COVIc, Cryptogenic Ischemic Stroke, Laboratory Mouse, CLEC2C, CEREBROVASCULAR ACCIDENT, methodology, Immune MarkersBrain, Biological Markers, Viral Marker, ischemic encephalopathy, Viral, Surrogate Endpoints, Surrogate Endpoint, Ischemic, Ischemias, Clinical Markers, Laboratory, protein complex, Clinical Marker, cerebrovascular ischemia, Serum Markers, Biochemical, End Point, FBN, Endpoint, Ischemia, Biochemical Markers, Imaging, protein, Ischemic Encephalopathy, Serum, protein-containing complex, Biologic Marker, Immune Marker, Surrogate End Points, ACMICD, Spectroscopy, Brain Ischemias, Surrogate Markers, brain ischemia, protein polypeptide chains, Laboratory Markers, native protein, natural protein, polypeptide chain, Marker, Biological, Surrogate End Point, Cerebral Ischemias, ECTOL1, Protein, ischaemic encephalopathy, brain ischemic disease, MFS1, Analysis, protein aggregate, Cerebrovascular ischemia, Brain ischemia, Cerebral, WMS, Ischemic Encephalopathies, WMS2, Biologic Markers, ischemic disease of brain, Biomarker, Serum Marker, Clinical, End Points, Surrogate, Spectrum., Biological Marker, Endpoints, Spectrometry, proteins, Encephalopathy, Immunologic, MASS, Laboratory Marker, Surrogate Marker, OCTD, ischemia cerebrovascular, Disruption of blood oxygen supply to brain, Immunologic Markers, Immune, MALDI, Markers, Biochemical Marker, Viral Markers, SSKS, Cerebral Ischemia, GPHYSD2, Immunologic Marker, Biologic, SGS, Immune Markers492trueProtein profiling and identification of new biomarkers of brain ischemia by MALDI-imaging-mass-spectrometryIntroduction The identification of proteins involved in brain ischemia might allow the discovery of new putative biomarkers or potential therapeutic target of one of the most important neurological disorders. MALDI Imaging-Mass-Spectrometry (IMS) is a powerful technique that allows the visualization of protein distribution along a tissue without labeling. Our aim is to study the distribution of proteins along mouse brain after an ischemic insult and to identify relevant proteins involved in brain ischemia. Materials and methods We occluded the middle cerebral artery (60min) of C57BL/6J mice (n=4) with 24h of reperfusion. Brain slices were analyzed by MALDI-TOF and mass spectra from infarct (IC) and contralateral (CL) regions were compared using ClinProTools. Relevant m/z were selected after PCA analysis and their ion distribution maps were analyzed by FlexImagin3.0. Protein identification was conducted on mouse brain homogenates through a bottom-up approach consisting on complementary fractionations based on tricine gels and RP-HPLC. The identifications were confirmed by immunohistochemistry. Results We identified 102 m/z with different abundances between IC and CL (p<0.05), from which 21 m/z were selected by PCA as more relevant. Thirteen of them were found increased in the infarct region and 4 m/z showed a discrimination higher than 90% between IC and CL. After bottom-up identification, immunohistochemistry analysis confirmed increased ATP5i and COX6C expressions, and decreased UMP-CMP kinase in IC compared to CL. Conclusions We identified for the first time by MALDI-IMS several m/z peaks with different abundances between IC and CL. These proteins involved in brain ischemia might represent potential diagnostic biomarkers or target molecules for neurological 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