Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/08/PXD011104/selectivitygastricmousestomachmaldi-tof.ibdftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/08/PXD011104/agingexperiment.imzMLftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/08/PXD011104/kidney-inhibconccurve.imzMLftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/08/PXD011104/GastricTumorModelMALDI-FT-ICR.tifftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/08/PXD011104/GastricTumorMouseModel-MALDI-TOF.tifftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/08/PXD011104/muscle-inhibconccurve.ibdftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/08/PXD011104/time-curve-dataset.tifftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/08/PXD011104/tcea-wash.ibdftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/08/PXD011104/TCEAwash.tifftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/08/PXD011104/kidney-inhibconccurve.ibdftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/08/PXD011104/Information-time-curve.docxftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/08/PXD011104/mousestomachmaldi-tof.ibdftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/08/PXD011104/agingexperiment.ibdftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/08/PXD011104/spleen-inhibconccurve.ibdftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/08/PXD011104/pancreas-inhibconccurve.ibdftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/08/PXD011104/liver-inhibconccurve.ibdftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/08/PXD011104/Aging.tifftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/08/PXD011104/GastricMouseTumorMALDIFT-ICR.ibdftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/08/PXD011104/Information-gastricmousemodel-MALDI-FT-ICR.docxftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/08/PXD011104/mousestomachmaldi-tof.imzMLftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/08/PXD011104/tcea-wash.imzMLftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/08/PXD011104/Information-TCEAwash.docxftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/08/PXD011104/time-curve-dataset.imzMLftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/08/PXD011104/Information-inhibitor-concentration-curvel-MALDI-TOF.docxftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/08/PXD011104/spleen-inhibconccurve.imzMLftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/08/PXD011104/selectivitygastricmousestomachmaldi-tof.imzMLftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/08/PXD011104/muscle-inhibconccurve.imzMLftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/08/PXD011104/Inhibitor-concentration-cruves.tifftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/08/PXD011104/Information-gastricmousemodel-MALDI-TOF.docxftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/08/PXD011104/time-curve-dataset.ibdftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/08/PXD011104/pancreas-inhibconccurve.imzMLftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/08/PXD011104/Information-TCEAaging.docxftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/08/PXD011104/GastricMouseTumorMALDIFT-ICR.imzMLftp://ftp.pride.ebi.ac.uk/pride/data/archive/2020/08/PXD011104/liver-inhibconccurve.imzMLprimaryOK200004390c.hopf@hs-mannheim.deKatrin ErichMass SpectrometryMass spectrometry imagingflexImagingProtease activityMaldi-msihttp://www.ebi.ac.uk/pride/archive/projects/PXD011104SpleenStomachLiverMusclePancreasKidneySample preparation for protease activity assessment by MALDI MSI For MALDI MSI analysis, frozen tissue was cut into 10 μm slices on a CM1950 cryostat (Leica Biosystems, Nussloch, Germany) at a temperature of -17 °C and thaw-mounted onto ITO-slides. In washing experiments, slices were rinsed with 2 x 1 mL 50 mM ammonium formiate (pH 7 and pH 3). The tissue was vacuum-dried for 15 min at 4 °C. To assess proteolytic in-situ activity, one “digest” slide and one “no digest” control slide were prepared per tissue type. For assessment of time-dependent endogenous protease activity on porcine tissue (5 time-points x 5 organs x 2 conditions, i.e. “digest” and “no digest” for a total of 50 MSI datasets), 4x1 μL protease inhibitor mix (PIM; 1x) consisting of 2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), 0.3 μM aprotinin, 116 μM bestatin hydrochloride, 14 μM E-64, 1 mM ethylenediaminetetraacetic acid (EDTA) and 1 μM leupeptin was applied to each tissue. Additionally, 1x1 μL ddH2O was pipetted on each tissue as solvent control. For studies of inhibitor concentration-dependent protease activity on porcine tissue, a dilution series (0x, 0.1x, 0.25x, 0.5x, 1x, 2.5x, 10x) of PIM was prepared in H2O, and one μl of each dilution was spotted onto tissue at arbitrarily chosen positions (n=2). Slides were kept on ice at all times. In experiments using mouse tumor samples, an additional tissue slice was covered with PIM, using the SunCollect (pump system, SunChrom, Friedrichsdorf) and the following conditions: 3x15 μL/min, Y-distance 25.5 mm, Speed in X= Low 4, Speed in Y=Medium 1. The final amount of PIM on tissue was 0.2 μL/mm2. After drying under vacuum for 3 min at room temperature (RT), porcine or mouse tissues on the “digest” slide (Fig. 1) were covered with the tracer protease substrate substance P (20 pmol/μL in ddH2O) using the SunCollect spray system as described for PIM above for a coating with 3.5 pmol/mm2 substance P. During the spray process, the slide was kept on a cool pad. After spraying, the slide was dried in desiccator for 3 min at RT. Both slides (“digest” and “no digest”) were incubated simultaneously at 37 °C and 95 % humidity in the SunDigest device (SunChrom) for 15 min, 30 min, 45 min, 60 min, 120 min or 360 min in time-course experiments and for 60 min in inhibitor-concentration-dependence experiments (porcine tissues). Mouse tissue was incubated for 30 min at 37 °C instead, as protease activity was apparently higher in snap-frozen mouse tissue than in slaughterhouse tissue, but otherwise treated as porcine tissue. Homogeneity of the substance P coating on “no digest” slides was evaluated, and experiment with inhomogeneous coatings were rejected. MALDI-TOF and MALDI-FT-ICR mass spectrometry imaging MALDI-TOF MSI with DHB matrix was performed as outlined(14). Porcine tissues were analyzed using an ultrafleXtreme MALDI-TOF/TOF instrument (Bruker Daltonik) in reflector positive mode and a mass range of m/z 500-2500. Tissues were measured at a lateral resolution of 200 μm, summing up 300 shots per pixel. External quadratic calibration using peptide standard II was performed for all MS instruments. Data was acquired and processed using flexControl V3.4 and flexImaging V4.1 software (Bruker Daltonik). For mouse samples, a rapifleX MALDI-TOF instrument (and flexImaging V5.1 (Bruker Daltonik) was used at a lateral resolution of 50 μm (20 μm for the replicate experiment), summing up 400 shots per pixel. After MALDI-TOF data acquisition, the same slides were analyzed using a 7T solariX XR MALDI FT-ICR (Bruker Daltonik). For imaging, the instrument was used in positive ion mode in an m/z range of m/z 100-3000 with a raster width of 20 μm and 15 laser shots/pixel. The following parameters were used: Ion transfer (Funnel 1 150 V, Skimmer 1 15 V, Funnel RF Amplitude 70 Vpp); Octopole (Frequency 5 MHz, RF Amplitude 350 Vpp); Collision Cell (RF Frequency 2 MHz, RF Amplitude 1200 Vpp); Transfer Optics (Time of Flight 1.5 ms, Frequency 4 MHz, FR Amplitude 350 Vpp); Quadrupole (Q1 Mass 350 m/z); Excitation Mode (Sweep Excitation, Sweep Step time 15 μs). Spectra of mouse tumor stomach were recorded using 512k (FID 0.2447 s) data point transient, corresponding to an estimated resolving power of 33,000 at m/z 400 resulting in a manageable data size of 22 GB. The average detected mass resolution at m/z 1347.73 was 23,000. Internal calibration was done using a lock mass of m/z 780.551 [PC(34:2)+Na]+. Data acquisition was performed using ftmsControl (Bruker Daltonik); ion images were obtained from flexImaging 4.1. For verification of substance P and its peptide products, CID fragmentation was used. Data analysis was performed using Biotools 3.2 and Sequence Editor 3.2 software (Bruker Daltonik).PrideNot availableNo PTMs are included in the datasetProcessing of mass spectra and statistical analysis To export mean intensities, we manually drew regions-of-interest (ROI) on tissue around spots with inhibitor (+inhibitor) or without inhibitor (-inhibitor) on “digest”- and “no digest” control slides, respectively. Mean spectra of those ROIs were exported into mMass (V5.5.0(20)) for baseline-subtraction and peak picking (S/N > 5). Intensities of the m/zvalues of interest were exported into Excel (Microsoft). Non-linear regression analysis was performed in Prism 5.0 software (Graphpad Software). For statistical analysis of the mouse tumor tissue, TOF data sets were converted into imzML file format(21) using the flexImaging converter (resampled to 80,000 data points), while the centroided FT-ICR datasets were read directly by an in-house conversion tool. Subsequently, the datasets were imported into R 3.3.1 (R Foundation for Statistical Computing, Vienna, Austria)(22, 23) using MALDIquant and MALDIquantForeign packages(24). The segmentation was performed using spatially aware shrunken centroid clustering(25) (r=2,k=2,s=3) as implemented in Cardinal(26). The variable r=2 to distinguish between tumor and non-tumor. The segmentation was performed based on the m/z segment of 798.54 ± 5 and 798.54 ± 0.05 for the TOF and FT-ICR datasets, respectively. In that mass range, the lipid PC(34:1)+K+ with m/z 798.54, known to be enriched in gastric tumor in this mouse model, is present(14). No pre-processing was performed on the imported raw data. Intensities for Substance P and its peptide cleavage products were exported and grouped into tumorous- and non-tumorous segment.ProteomicsProf. Dr. Carsten HopfultraflexBruker Daltonics solarix seriesPARTIALCenter for Mass Spectrometry and Optical Spectroscopy (CeMOS), Mannheim University of Applied Sciences, Paul-Wittsack Str. 10, 68163 Mannheim, GermanyBos Taurus (bovine)Mus Musculus (mouse)katrin.erich89@gmail.com30293968 Erich K, Reinle K, Müller T, Munteanu B, Sammour DA, Hinsenkamp I, Gutting T, Burgermeister E, Findeisen P, Ebert MP, Krijgsveld J, Hopf C. Spatial Distribution of Endogenous Tissue Protease Activity in Gastric Carcinoma Mapped by MALDI Mass Spectrometry Imaging. Mol Cell Proteomics. 2019 18(1):151-161 10.1074/mcp.ra118.000980TechnicalBiomedicalCeMOS HS MannheimGermanyAberrant protease activity has been implicated in the etiology of various prevalent diseases including neurodegeneration and cancer, in particular metastasis. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) has recently been established as a key technology for bioanalysis of multiple biomolecular classes such as proteins, lipids, and glycans. However, it has not yet been systematically explored for investigation of a tissue's endogenous protease activity. In this study, we demonstrate that different tissues, spray-coated with substance P as a tracer, digest this peptide with different time-course profiles. Furthermore, we reveal that distinct cleavage products originating from substance P are generated transiently and that proteolysis can be attenuated by protease inhibitors in a concentration-dependent manner. To show the translational potential of the method, we analyzed protease activity of gastric carcinoma in mice. Our MSI and quantitative proteomics results reveal differential distribution of protease activity - with strongest activity being observed in mouse tumor tissue, suggesting the general applicability of the workflow in animal pharmacology and clinical studies.Spatial Distribution of Endogenous Tissue Protease Activity in Gastric Carcinoma Mapped by MALDI Mass Spectrometry Imaging.Erich Katrin K, Reinle Kevin K, Müller Torsten T, Munteanu Bogdan B, Sammour Denis A DA, Hinsenkamp Isabel I, Gutting Tobias T, Burgermeister Elke E, Findeisen Peter P, Ebert Matthias P MP, Krijgsveld Jeroen J, Hopf Carsten CBovine Kunitz Pancreatic Trypsin Inhibitor, Water, projections, l(4)17, l(4)13, Gli, dalpha-SNAP, IL1BC, ion, PNT-P1, Cid[Mel], Ci[D], Ethylenediaminetetraacetic acid, CASP-1, FEZL, A4, BOUND WATER, EY3-1, Tumor, oxidane, Solvent, CENP-A, Long Term, (ethylenedinitrilo)tetraacetic acid, DmelCG5052, WATER, Slaughterhouse, CENP-C, HOH, Antilysin, dmTAF[[II]]230, Polypeptides, Trypsin Inhibitor, hydrogen chloride, Kontrykal, type I, azanium, GRP1, PH 3, Grp1, 2, Software Engineering, L-Leucinamide, Analysis, D-ets-2, 3520, Il1bc, PADI5, dJ1068F16.2, WMS, ASD2, average, Houses, BcDNA:RE21270, Hypothalamic, 10x single cell library construction, TFIID TAF250, Analyses, cel, N, PTPSTEP, Tissue, Nonmigrant, AI844555, Fluoride, Shots, Transient, D17Mit170, IL-1BC, SNAP-25, AI047805, pim-1, anterior intestine, Wasserstoffchlorid, CI, N-acetyl-L-leucyl-N-(4-((aminoiminomethyl)amino)-1-formylbutyl)-, leupeptin, CT, PAD, phosphatidylcholine(34:2), Ammonium(1+), acidum edeticum, SGS, GPH, CiD, Ce, Ito, dTAF[[II]]230, Ci, anatomical protrusion, Caspase-1 subunit p10, Ct, Longterm Effect, 2'', pad, TAF200, Incontinentia pigmenti type 1, Kunitz, Neurokinin A, Euler Gaddum Substance P, Normalcy, Tl3, Bovine Pancreatic Trypsin Inhibitor, Tl2, not genetically inherited, collisionally activated dissociation, Software Tools, CID, ciD, Cid, DmelCG7788, ACMICD, Computer Applications, H2O, Stomachs, Dm1, peptidase activity, pmol, Computer Applications Software, shelf, Chlorwasserstoff, ci-D, simple tissue, Pancreatic, Migrants and Transients, Software Applications, pntP2, Pointed-P1, instrument, CENP-A/Cid, CENP-A/CID, CG11628, Source Software, CENPA, Ets94F, Hypothalamic Substance P, Striatum-enriched protein-tyrosine phosphatase, l(2)31Df, N'-1, Euler-Gaddum, projection, ridge, Applications, Slaughterhouses, Taf250, Drice, Neuropeptide K, RIC4, Migrants, Mouse, chlorane, DRICE, Ci-155, Occlusive Peripheral Arterial Disease, TAF230, Computer Software Applications, breadth, 0998/12, l(1)VE614, GRP1/cytohesin 1, hypomelanosis of Ito, Effects, chlorure d'hydrogene, lamellae, DrIce, DrICE, C-terminal-flanking peptide, acetyl-L-leucyl-L-leucyl-L-arginal, thomson, body system, process of organ, combined T and B cell immunodeficiency, ventriculus, CG7826, EDTA tetraanion, agua, MA2, mass-to-charge ratio, CG11633, period, lamella, CenpA, ammonium cation, DMPOINT1A, peripheral arterial disease, TOF, CG10309, Pulmin, CG7835, CG42273, CG6625, Kallikrein-Trypsin, system, MAM, Siah, ZNF312, Application, Normalities, Open Source Softwares, Ito hypomelanosis, DmelCG7615, p85SPR, anatomical systems, 2'''-(ethane-1, dTAF[[II]]250, 2-Ethane diylbis-(N-(carboxymethyl)glycine), pointed-RC, Longterm, Hydrogen chloride, ethylenediamine tetraacetic acid tetraanion, cell, Slaughter Houses, Software Application, PCE-2, frag2, Open Source Software, Long-Term, dependence, Worker, ions, kf, EK3-2, Basic Pancreatic Trypsin Inhibitor, Substance K, Computer Software Application, dTAF250, l(3)07825, (S)-isomer, peripheral artery disease, Tools, Caspase-1 subunit p20, transient structure, Humidities, CG2125, peptidos, CG17077, PIM, hydrolase, Kallikrein Trypsin Inactivator, laminae, Zymofren, PIX, inhibitors, DmelCG6625, Ets, cou, caspase 3, anatomical process, Dilmintal, NH4+, pim, stepk, chloridohydrogen, BG:DS00004.13, CE-2, Cell, Tool, dTAF230, polypeptide, Incontinentia pigmenti type 1 (formerly), Substance P, Lr, Experiment, Temperatures, ci[D], chemical analysis, Long Term Effects, p85Cool1, TAF[[II]]250/230, MFS1, MIXL, peripheral arterial disorder, pnt-P1, pnt-P2, organ process, AU020952, Taf[[II]]250, Contrical, DmelCG2125, ci155, CenH3, DMDA1, IL-1 beta-converting enzyme, Childhood, Interleukin-1 beta-converting enzyme, PDI4, primary structure of sequence macromolecule, Longterm Effects, DmelCG11628, ME-IV, Computer Programs, processes, ZFP312, Applications Softwares, cool-1, Nomads, Bra, CG7615, mesenteron, Contrykal, [OH2], CenpA/CID, General activity, hydrochloric acid, POINT, Data Analyses, formerly, ethylenediaminetetraacetate tetraanion, CG8705, PAD4, Activity, determination, SEC9, DmelCG17077, instrument configuration, CG7788, crice, 116, Imaging, Hydrogenchlorid, Pnt, combined T cell and B cell immunodeficiency, 10X, bA416N4.2, peptide, Cut, NKA, PDI5, ClvPrd, peptido, png, Traskolan, l(1)7Ba, stomach chamber, l(1)7Bb, Min, Kontrikal, 2', 10x, Effect, acting on D-amino acid peptides, alpha-SNAP, Computer Program, DmelCG42273, MIX, Edetic acid, PIST, DmelCG10309, Pnt-P1, CG13329, portion of tissue, peripheral arterial diseases, 2-diyldinitrilo)tetraacetate, me75, Migrant Worker, reference sample, ion(4-), peptides, TACHD, Open, min, Computer Programs and Programming, mAPC, PPT, Neuromedin L, Estimated, CG11387, T1, DEPP, peptide hydrolase activity, ICE, papilla, l(2)k08110, Fseg, Cenp-A, Squatters, dihydridooxygen, (ethane-1, house mouse, dihydrogen oxide, ICR, GPHYSD2, Slaughter House, Long-Term Effects, Iniprol, betaPix, ethylenediaminetetraacetate, PLATEST, hi syndrome, CG5052, ice, iCE, drice, lateral, aqua, mouse, lamina, Dmel_CG7826, MM2, flanges, Th, D-Ets-2, {[-(BIS-CARBOXYMETHYL-AMINO)-ETHYL]-CARBOXYMETHYL-AMINO}-ACETIC ACID, Euler-Gaddum Substance P, ets94F, 10x sequencing, 0123/09, TAFII-250, TAF250/230, Source Softwares, Programs, Immunization, Program, NH4(+), Fasting-induced gene protein, TAFII250, Ionen, drIce, drICE, Neuropeptide gamma, Migrant, 3.1.3.48, Computer Applications Softwares, tissue portion, 2410016C14Rik, Workers, Softwares, BcDNA:GH10590, GOPC1, Dmel_CG7835, Slaughter, cenpA, Mnb, MNB, MILD1, NPK, Ci/Gli, Ci/GLI, CenH3/CID, Step, mice, shelves, HCl, P45, hydrogen hydroxide, RGAG2, alphaSnap, MASS, AW124434, CG17603, TAF[[II]], dotted, acqua, organ system, congenital combined immunodeficiency, ptd, PntP2, Health, DMDA, stippled, spine, STEP, SR3-5, Computer Software Applications., PntP1, Peripheral Arterial Disease, E(E2F)3D, p45, PNTP2, Wasser, Polypeptide, time, PNTP1, Platelets, mKIAA0142, Ammonium, d230, dalphaSNAP, Pak3bp, ETS2, spotted, Ets2, FBN, dTAFII250, ethylenediaminetetraacetic acid, Computer, EfW1, 2210402P09Rik, [NH4](+), TYPE, Migrant Workers, SP(1-11), protrusion, DAGA4, cytohesin/GRP1, l(4)102ABc, pntegfr, 0608/07, pigmentary mosaicism, House, dmTAF1, ECTOL1, Taf230, Acide ethylenediaminetetracetique, l(3)77ABa, l(3)77ABc, Low, RIC-4, SCG3, cloruro de hidrogeno, acide edetique, TAF250, CenH3[Cid], CenH3[CID], Taf200, PC 34:2, Incontinentia pigmenti achromians, Cool, iones, ammonium ion, Transients, inhibiteur, l(2)SH2 0323, DmelCG13329, Nonmigrants, Taf1p, ridges, eau, BPTI, OCTD, Squatter, X-linked combined immunodeficiency, edta, Kallikrein-Trypsin Inactivator, 2-diyldinitrilo)tetraacetic acid, inhibidor, Neural-specific protein-tyrosine phosphatase, DmelCG11387, monoacetate, acting on L-amino acid peptides, EDTA, Long-Term Effect, acting on peptide bonds, Abattoir, 10X sequencing, TAF, FEZ, l(3)j1B7, Controlled, combined immunodeficiency, Applications Software, Decidual protein induced by progesterone, protease activity, Open Source, DYRK1, Controlling, Inactivator, data, TAF[[II]]250, dSNAP, Computer Software, DDX40, inhibitor, l(3)84Ab, cenH3, dsnap, Peptide, acido edetico, light amplification by the stimulated emission of radiation, LGMD2C, [HCl], Software Tool, CAD, Mus, Ci155, p230, water, CAL, Hydrochloride, Nomad, sequence, TFIID, PC(34:2), Dyrk1, Vacuums, CES2A1, connected anatomical system, Ito type, Software, l(2)SH0323, flange, l(3)s118306, WMS2, CYH1, Basic, VSD1, TAF[[II]]230, ammonium, GPCho(34:2), Normality, CC1, H4edta, Engineering, TAF[II]250, dJ94G16.2, betaPix-b, betaPix-c, incised, Interleukin-1 beta convertase, FID, process, Incontinentia pigmenti, HMI, FIG, Ets58AB, MALDI, DmelCG17603, proteinase activity, Ion, concentration, Data, velocity, SNAP, SCARMD2, Kunitz Pancreatic Trypsin Inhibitor, SSKS, 3.4.22.36, Calibrations, ARG147, processus, Peptid, edetic acid, assay, 10x Genomics, gaster, IPA, Trasylol, snap, TAF1, CID/CENP-Aportion of tissue, protease activity, gastric (stomach) cancer, pattern, distribution, MALDI., Tissue, cancer of the stomach, fundus of stomach cancer, cancer of fundus of stomach, gastric carcinoma, malignant fundus of stomach neoplasm, peptide hydrolase activity, gastric cancer, Ca fundus - stomach, proteinase activity, peptidase activity, malignant neoplasm of fundus of stomach, acting on L-amino acid peptides, malignant tumour of fundus of stomach, cancer of stomach, tissue portion, NOS, simple tissue, malignant tumor of fundus of stomach, stomach cancer, stomach carcinoma, acting on peptide bonds, carcinoma of the stomach, hydrolase, gastric fundus cancer, acting on D-amino acid peptides, carcinoma of stomachCPD photolyase activity, Bru, PhrB photolyase activity, Raw, FEZL, NDPP, cleavage, Tumor, Foundation, pigmented epithelium, Stomach Neoplasms, NEOPL STOMACH, peptide, Polypeptides, NKA, serial element, ClvPrd, peptido, GPCho(34:1), 3, Software Engineering, rectum adenocarcinoma, NUP96, Analysis, outer pigmented layer of retina, tumor of the stomach, gastric neoplasm, epithelium, WMS, Computer Program, ASD2, Svc, portion of tissue, Hypothalamic, reference sample, peptides, Analyses, TACHD, Open, Tissue, pigmented retina, Computer Programs and Programming, PPT, present in organism, Neuromedin L, DNA cyclobutane dipyrimidine photolyase activity, SUPPRESSOR OF AUXIN RESISTANCE 3, pigment epithelium of retina, tumorous, pigmented retina epithelium, house mouse, Cancer of the Stomach, GPHYSD2, ICR, SGS, PRE, deoxyribonucleic cyclobutane dipyrimidine photolyase activity, mouse, Th, Neurokinin A, Euler-Gaddum Substance P, Euler Gaddum Substance P, segment, neoplasm of the stomach, pigmented retinal epithelium, Source Softwares, STOMACH NEOPL, Software Tools, ACMICD, Programs, shrunken, Program, Computer Applications, retinal pigment, Neuropeptide gamma, Statistical Regression, Computer Applications Software, Lipid, Computer Applications Softwares, tissue portion, dipyrimidine photolyase (photosensitive), adenocarcinoma - rectum, simple tissue, Gastric Neoplasms, Softwares, retinal pigment layer, Gm92, Stomach Cancer, Statistical Regressions, Diagnostics, Software Applications, NPK, stomach neoplasm (disease), Source Software, mice, retinal pigmented epithelium, NDPP1, Hypothalamic Substance P, MASS, Euler-Gaddum, Stomach, Applications, phr A photolyase activity, DNA-photoreactivating enzyme, neoplasm of stomach, Neuropeptide K, tumor of stomach, Mouse, Polypeptide, Computer Software Applications, lipids, Neoplasms, C-terminal-flanking peptide, CARDINAL, FBN, rectal adenocarcinoma, Computer, photoreactivating enzyme activity, thomson, deoxyribodipyrimidine photolyase activity, stratum pigmentosum retinae, SP(1-11), mass-to-charge ratio, stomach neoplasm, TOF, ECTOL1, CARD-inhibitor of NF-kappa-B-activating ligand, stratum pigmentosa retinae, Regression Analyses, Del(8)44H, Application, ZNF312, stomach tumor, Open Source Softwares, MOS3, PC 34:1, deoxyribocyclobutadipyrimidine pyrimidine-lyase activity, TUCAN, PRECOCIOUS, adenocarcinoma of rectum, Software Application, adenocarcinoma of the rectum, inhibiteur, Open Source Software, F23A5.3, Gastric Cancer, Substance K, Computer Software Application, OCTD, inhibidor, Tools, Cancer of Stomach, peptidos, FEZ, statistical analysis, Apoptotic protein NDPP1, F23A5_3, Controlled, Cancer, Applications Software, inhibitors, Open Source, Tumor up-regulated CARD-containing antagonist of CASP9, Controlling, data, Computer Software, Regressions, inhibitor, PC(34:1), phosphatidylcholine(34:1), gastric tumor, Peptide, Tool, Stomach Cancers, polypeptide, read, stratum pigmentosum (retina), Software Tool, MODIFIER OF SNC1, Substance P, Mus, PRISM, deoxyribonucleic photolyase activity, Neoplasm, MFS1, segment., Software, Gastric, WMS2, VSD1, DAKAR, RPE, Col4a-1, DACAR, photolyase activity, Regression Diagnostics, Engineering, Gastric Cancers, Cancers, Gastric Neoplasm, retinal pigment epithelium, p. pigmentosa retinae, Computer Programs, ZFP312, Applications Softwares, Regression, NEOPL GASTRIC, SSKS, Peptid, deoxyribonucleate pyrimidine dimer lyase (photosensitive), Statistical, variable, GASTRIC NEOPLmalignant Growth, mode of action, Endopeptidase Inhibitor, pharmacodynamics, Activity, Laboratory, Mus domesticus, cancer of the stomach, cleavage, Peptide Hydrolase Inhibitor, Glykan, CASP-14, neoplasm metastasis, Tumor, House Mouse, Long Term, malignant fundus of stomach neoplasm, cancer metastasis, peptide, Polypeptides, polisacarido, NKA, Tier, ClvPrd, peptido, diseases, malignant neoplasm of fundus of stomach, Protease Inhibitor, pathogenesis, diseases and disorders, Inhibitors, Hydrolase Inhibitors, Work Flow, gastric fundus cancer, animal, Effect, acting on D-amino acid peptides, peptidolysis, portion of tissue, human disease, Hypothalamic, peptides, ATP-dependent proteolysis, neoplasm (disease), Tissue, Glycane, PPT, Swiss Mice, proteins, Neuromedin L, Protein Degradation, CA, peptide hydrolase activity, Peptidohydrolase Inhibitor, Ongoing loss of nerve cells, malignant neoplasm, malignant tumour of fundus of stomach, Glycan, Homo sapiens disease, carcinoma of the stomach, house mouse, Long-Term Effects, Neuro-degenerative disease, malignancy, Degradations, aberrant, Peptidase Inhibitor, animalia, Arts, mouse, Longterm Effect, Neurokinin A, Euler-Gaddum Substance P, Euler Gaddum Substance P, Peptide Peptidohydrolase, results, Glykane, organism, Workflows, peptidase activity, Digestion, Neuropeptide gamma, Lipid, Mini-ICE, Diseases, organ system cancer, Hydrolase Inhibitor, tissue portion, Progressive neurodegenerative disorder, NOS, simple tissue, mechanism of action., Mus musculus, Industrial, gastric (stomach) cancer, Industrial Arts, NPK, whole organism, Caspase-14 subunit p10, Antagonists, mice, Protein Digestions, Swiss Mouse, Hypothalamic Substance P, Caspase-14 subunit p19, Neurodegenerative disease, Euler-Gaddum, MICE, gastric carcinoma, domesticus, polysaccharides, disease, Peptidase Inhibitors, Neuropeptide K, Koerper, Antiproteases, stomach cancer, Mouse, Polypeptide, Endopeptidase, Peptide Peptidohydrolase Inhibitor, cancer, Inhibitor, time, Work Flows, lipids, 3.4.22.-, atypia, other disease, multi-cellular organism, Effects, Peptidomics, Endopeptidase Inhibitors, cell type cancer, C-terminal-flanking peptide, Degradation, number, glycans, Gene, Protease, mini-ICE, presence, SP(1-11), period, method, Ca fundus - stomach, House, cancer of stomach, method used in an experiment, Gene Products, disease or disorder, Mus musculus domesticus, Protease Antagonist, malignant tumor of fundus of stomach, stomach carcinoma, atypical, Peptide Hydrolase, neoplasm, attenuated, Mice, malignant tumour, Polysaccharide, study, tumor cell migration, pattern, Longterm, Swiss, distribution, metastasis, metazoa, causes, Long-Term, Protein Degradations, Antagonist, Substance K, non-neoplastic, gastric cancer, mechanism of action, Protease Antagonists, acting on L-amino acid peptides, causality, disorder, Peptide Peptidohydrolase Inhibitors, peptidos, Long-Term Effect, acting on peptide bonds, hydrolase, Pharmacologies, tumor metastasis, protease activity, body, malignant, Proteins, disorders, whole body, defective, medical condition, Protein Digestion, fundus of stomach cancer, Digestions, cancer of fundus of stomach, Peptide, polypeptide, count in organism, Substance P, MT, Proteolyses, Mus, malignant neoplasm (disease), Protein, Long Term Effects, condition, polisacaridos, primary cancer, distinct, House Mice, Peptidohydrolase Inhibitors, malignant tumor, Longterm Effects, Laboratory Mice, Protein Gene Products, plan specification, Gene Proteins, Peptidase, MALDI, pharmacologic action, proteinase activity, Antiprotease, malignant neoplastic disease, concentration, Peptid, Peptide Hydrolase Inhibitors, quantitative, Laboratory Mouse, General activity, carcinoma of stomach, presence or absence in organismmalignant Growth, mode of action, Endopeptidase Inhibitor, pharmacodynamics, Activity, Laboratory, Mus domesticus, cancer of the stomach, cleavage, Peptide Hydrolase Inhibitor, Glykan, CASP-14, neoplasm metastasis, Tumor, House Mouse, Long Term, malignant fundus of stomach neoplasm, cancer metastasis, peptide, Polypeptides, polisacarido, NKA, Tier, ClvPrd, peptido, diseases, malignant neoplasm of fundus of stomach, Protease Inhibitor, pathogenesis, diseases and disorders, Inhibitors, Hydrolase Inhibitors, Work Flow, gastric fundus cancer, animal, Effect, acting on D-amino acid peptides, peptidolysis, portion of tissue, human disease, Hypothalamic, peptides, ATP-dependent proteolysis, neoplasm (disease), Tissue, Glycane, PPT, Swiss Mice, proteins, Neuromedin L, Protein Degradation, CA, peptide hydrolase activity, Peptidohydrolase Inhibitor, Ongoing loss of nerve cells, malignant neoplasm, malignant tumour of fundus of stomach, Glycan, Homo sapiens disease, carcinoma of the stomach, house mouse, Long-Term Effects, Neuro-degenerative disease, malignancy, Degradations, aberrant, Peptidase Inhibitor, animalia, Arts, mouse, Longterm Effect, Neurokinin A, Euler-Gaddum Substance P, Euler Gaddum Substance P, Peptide Peptidohydrolase, results, Glykane, organism, Workflows, peptidase activity, Digestion, Neuropeptide gamma, Lipid, Mini-ICE, Diseases, organ system cancer, Hydrolase Inhibitor, tissue portion, Progressive neurodegenerative disorder, NOS, simple tissue, mechanism of action., Mus musculus, Industrial, gastric (stomach) cancer, Industrial Arts, NPK, whole organism, Caspase-14 subunit p10, Antagonists, mice, Protein Digestions, Swiss Mouse, Hypothalamic Substance P, Caspase-14 subunit p19, Neurodegenerative disease, Euler-Gaddum, MICE, gastric carcinoma, domesticus, polysaccharides, disease, Peptidase Inhibitors, Neuropeptide K, Koerper, Antiproteases, stomach cancer, Mouse, Polypeptide, Endopeptidase, Peptide Peptidohydrolase Inhibitor, cancer, Inhibitor, time, Work Flows, lipids, 3.4.22.-, atypia, other disease, multi-cellular organism, Effects, Peptidomics, Endopeptidase Inhibitors, cell type cancer, C-terminal-flanking peptide, Degradation, number, glycans, Gene, Protease, mini-ICE, presence, SP(1-11), period, method, Ca fundus - stomach, House, cancer of stomach, method used in an experiment, Gene Products, disease or disorder, Mus musculus domesticus, Protease Antagonist, malignant tumor of fundus of stomach, stomach carcinoma, atypical, Peptide Hydrolase, neoplasm, attenuated, Mice, malignant tumour, Polysaccharide, study, tumor cell migration, pattern, Longterm, Swiss, distribution, metastasis, metazoa, causes, Long-Term, Protein Degradations, Antagonist, Substance K, non-neoplastic, gastric cancer, Protease Antagonists, acting on L-amino acid peptides, causality, disorder, Peptide Peptidohydrolase Inhibitors, peptidos, Long-Term Effect, acting on peptide bonds, hydrolase, Pharmacologies, tumor metastasis, protease activity, body, malignant, Proteins, disorders, whole body, defective, medical condition, Protein Digestion, fundus of stomach cancer, Digestions, cancer of fundus of stomach, Peptide, polypeptide, count in organism, Substance P, MT, Proteolyses, Mus, malignant neoplasm (disease), Protein, Long Term Effects, condition, polisacaridos, primary cancer, distinct, House Mice, Peptidohydrolase Inhibitors, malignant tumor, Longterm Effects, Laboratory Mice, Protein Gene Products, plan specification, Gene Proteins, Peptidase, MALDI, pharmacologic action, proteinase activity, Antiprotease, malignant neoplastic disease, concentration, Peptid, Peptide Hydrolase Inhibitors, quantitative, Laboratory Mouse, General activity, carcinoma of stomach, presence or absence in organismportion of tissue, protease activity, proteinase activity, peptidase activity, acting on L-amino acid peptides, MALDI., tissue portion, Tissue, simple tissue, acting on peptide bonds, hydrolase, acting on D-amino acid peptides, peptide hydrolase activity439trueTissue protease activity with MALDI MSIAberrant protease activity has been implicated in the etiology of various prevalent diseases including neurodegeneration and cancer, in particular metastasis. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) has recently been established as a key technology for bioanalysis of multiple biomolecular classes such as proteins, lipids, and glycans. However, it has not yet been systematically explored for investigation of a tissue’s endogenous protease activity. In this study, we demonstrate that different tissues, spray-coated with substance P as a tracer, digest this peptide with different time-course profiles. Furthermore, we reveal that distinct cleavage products originating from substance P are generated transiently and that proteolysis can be attenuated by protease inhibitors in a concentration-dependent manner. To show the translational potential of the method, we analyzed protease activity of gastric carcinoma in mice. Our MSI and quantitative proteomics results reveal differential distribution of protease activity – with strongest activity being observed in mouse tumor tissue, suggesting the general applicability of the workflow in animal pharmacology and clinical studies.Aberrant protease activity has been implicated in the etiology of various prevalent diseases including neurodegeneration and cancer, in particular metastasis. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) has recently been established as a key technology for bioanalysis of multiple biomolecular classes such as proteins, lipids, and glycans. However, it has not yet been systematically explored for investigation of a tissue’s endogenous protease activity. In this study, we demonstrate that different tissues, spray-coated with substance P as a tracer, digest this peptide with different time-course profiles. Furthermore, we reveal that distinct cleavage products originating from substance P are generated transiently and that proteolysis can be attenuated by protease inhibitors in a concentration-dependent manner. To show the translational potential of the method, we analyzed protease activity of gastric carcinoma in mice. Our MSI and quantitative proteomics results reveal differential distribution of protease activity – with strongest activity being observed in mouse tumor tissue, suggesting the general applicability of the workflow in animal pharmacology and clinical studies.2020-08-062020-08-06PXD01110428065516460991804982953585111454224942024527872842188090368093270429330845811363336351524260119834300662758385NCBITaxon:128068888561896111430118726355557114526236493461364031952065126957366649274670738538836411354615821582310665688954762721123294422291180992323122586365458333460792519365914320597129386558195851149315242592637371250232NCBITaxon:12721119577481866148858431043002451358672217554374081691804542668342953194530586126683514982250581496367445215855452811320814795641003923751463694204943320896420896344597413797913871272838440066793529367156759388036478434716052165994048321659721659545501315282693042165915627161696833218321729204277944762376454317203094980197525556978159509439843948840479NCBITaxon:2157525338551531051067439899037122857759515205968762262311210884555588845587077918178081736309431794571698936160488409859NCBITaxon:37162104782679401505907148364320421061320491381693630355543204663263331199456545351102541110321745921612928867641002642036945694101676341021076127735152486713165823925472793342814613584301417298174633457744491675476048932128735675684030135062191726518130194111929439367110411310239320255598632582788396591047593474951604901652998355256631758543980110243260866310245980875741755518320196447145268045796631367019083233115611415535510421932555217634981812359871069955529NCBITaxon:4751850298335476113615741465477180066556484337677299767982510216998238976484074137351155711089725482444474153NCBITaxon:5055789969844100036465349334147147493548019467512991143597412764731370863265478765698353221599838192011106775772710348198523487804794640986099586157187176685991915729513722117110129292107687834974198602028091488406334249234235152335985988655974649987039202111072943311711397215987419085037117082711708284182350241451139692931902197822474636046147123749402375939723287581722532566489050794526591972211915853101774208588596NCBITaxon:615750521028307989515586411051989972375059576131720002634196102641088878176179212190659407121906772275885815689964975151950582873757244NCBITaxon:22023717NCBITaxon:17036122019998570506050617476792745062724915961533355418400524643213686150596487844612286662941786661347515363291018194301086731018510184NCBITaxon:1763573039635586331492862876705801321650852718481865368595105135553482260710185687294296110091416333NCBITaxon:619184023210007348824123363630710470058127187878645159812894555663577553470951451225786260704NCBITaxon:13356262607052607076584574054163677483167233150561007133705654195NCBITaxon:305550445791399878168119011928752829126806315327341478281055524NCBITaxon:9606135858469008111167815022830519302836728593008529005NCBITaxon:569382688306401794699151011849943525815159014482428381113944260419370475927237086042328167728503515819901195195326738637013702492405360883858162248818615681756261583558371533376057267119350199808611935021678791085791371137127240719065037147591726298157125627457572709030903113335503728142433747924910078711082NCBITaxon:96157243359463318586260799141262200298492670NCBITaxon:1263719281249212528303856838569591169266100658137352375612750454266625515197881356638375064422336009412934979054632245352472140125751511649908515151438992374537474454464152432359376059202228870515805955365550881579327160293851578114978633892894535936479437NCBITaxon:685441275422511715901090134167387158955210129661030612967158515845158493765939343271591002263767338899046142345128768959119833296646704146479293657616428034116022164133159928038280353690910891768814989462181214108034817939228788929502711313329528282944568617493468191126526997227321890382107601247190400727159736997828114311402064119810871355477436581424507229533796982042321242231291632180334708714537209164573321535815754699287770202060328114319338293550833291292211487252649997283035739087183087267271037908550717629970767892683704155737292140532941281607839563924314262654898227377561155962073220865958827355516936053307972551811354914321371597495350261159556143213840607258781414471509493349194963401614334728005936046316461046603815184547448500356709747777195124017484185560133575554792042741580536076994688103762014887100594120037582890931643542826885007123038399001389464940838283594144242322197857742171598921409212205853939621157781154756929793244704749906604162103591905158114452749907312987779913112273170187546991103589915NCBITaxon:37296103609833418710491130798133679531640729946710340157865826453033373306383011306413992530642363650591169654788546981121162034317652616103372861299404458620734011535711195296526117455311147161338654131316634305521323120179836025323731276125523299351299385057316317642619794011513458445772678728623219334151346186208621160791392227983516811961255228149539322323116761757312108077213487999948861856962814084341033517787996321849620994887321392488209711533820961153392095330439375569927612686266447NCBITaxon:269704933986243694110312195883205092029061013068599975864445882864324995252972464260667411583432127178942786378942614774052567378639123343551419983493179986865540553435291486545147124546688925104298635712467915164135825763288401937334508771823803912903912952971974869317978911028688999513373105487220341712516321389850762554701193806222523256318865713034431121308516558658862238NCBITaxon:44664946454636493NCBITaxon:1107114280062421241873032883564913743751415167845881087439145943854331116781077854314408043933452703134NCBITaxon:110829534623226939773821899318226900351283871459531168853399669527869595418694200308954486972443662066563954530055862335518042643086922003022848124264281504751499105102991029847147241445238626290319541832902512089557497431170089306218644193061575584131016538235230805655772238235939946360106601623601043994839947179748254440423761021279056881337751823635572116229629817677187093128112147861050238245185579669465431863205182406672829744173944932351245012885318388919867948853191113782559103716473516601261557228331349390NCBITaxon:101142845031631297801985738595239693934323795602608712134639598427529595266437334323223544314275242243081433619593133456582609432749592560291699631122233605201216979525283869728529046436801218707334747279010132342956031278161119219408188297603818124988563122034018843973312744141639107243139855858885872520493692955464803834369122432640817212419355955217493991281614055920425465519458228860918382727592127442340817019332982972275985955712744201274426235455552857120729432841399616601876254829647187614378681490247480119915095648188229725431761012148015412045514816723847384818829451906951503034166336036165984048498505693101510528353528352693735294512637534052066987418649729722405212585941097677451804812310061911079NCBITaxon:813478009412694322710279434472568484792911463870333849917226175610946161435120325812051365218505235259447320549423349373473864527000446644991759189158258038804333037668624480461206781122026049910806124619629022246197320637522991867051186705750906812193879118794718680239800718680157230721545146835741258140818611757150847691838858032903116086113888691521677811472767212711036839978445972475129513987043512624696928123977133339952261261034304813817818219813188268210802281561005476241122926571632957553501922221979111196866693819791212471246148545132838828139510802302949110401459541619341390363120416221106658903264460711117022721041170612744321085956210283169412831666943694559729949514106588827114113161879010105841330879478447517415080817053942065423174471270709964153292428141119466984114545869537932920101981264690410661550537145755139251923239232014081576401316973160621213392830312383911283128224260512803395228110108328252535315285128112853353153498257419481478753623142410135674295105242619145481NCBInr 20130519115062129459706473129282996339413469597538631211705193516265390021343980313098006978103690122020718853225660173654856615664162425103695118702348334824688221187056386321219690226186129925016468606885656512623217219565810102865606165606442838656717136041602358700349415048022160100298823578613252911187002157641809699756655666452467134887937965223926291613633666336721751117645406560103789059232146429314344870933114767468342808NCBITaxon:405592359093226465236526212633749821787074982163472565690569156925693872249821165448720569734507699300166690510894491089448NCBITaxon:2759661410NCBITaxon:4932653587155689520710894441000589655087322875351352481263730069303926659581143988725121177687268723872487291178665498727134976724223130554417725784541581419198822445446NCBITaxon:120661174673104105909882497473446899601677674468587529600NCBITaxon:581111822637082484119165578457845848402119443955307158059656636164034624136810893161001428099612NCBITaxon:548681NCBITaxon:582054641439450330775797552283637379196627290581526009605960687594467087562270862665964960432325957050933548330902982938783481062675672675664306151279365431947448385449239470472112298411915231917016108961548313969169287827573640782112931457075298352061892577712850248052701262834854189854871427867158779627300251563041126736125994952869646964396444911462472746128746011345069638526385263996563024097639245264110909467469416348112386935174635231NCBITaxon:565995576784119562330591736582257313269796990346134434636064404213451124951533043479096109760616194032257329246200966927294227294351338968583554767894314696891280175576009681118696932011167807811869414361833303811869817194759321718118691443906NCBITaxon:10298NCBITaxon:7771257118387271566338404330618596948364190802703847664568703257310568708618218678661833568851350NCBITaxon:7803906451515NCBITaxon:15029903164515169431297029820762115861218451210725730919307910944609137494400209285201319747078529507619212638546146663915383379NCBITaxon:10239NCBITaxon:714173613095705470448300641751585923890037159084128108406972282334575412NCBITaxon:1024528104NCBITaxon:10243392017481097797067135622583313618550992428670652779658334240906200492910263494951034809NCBITaxon:6312406741307670703766191009408707487613810804329032176023993592672947474376032768917812126719285321773846451772177437272557104905615348119922512153239576769014NCBITaxon:722715697472664709131704725852092026971303276978596285963411476130913081307126387121203533264837315321469338323411479398158370131413134937608333413118333357047111497039655397283332101841833312146874550451080187492002627241530091076620637373505411490280148517501303713444143983NCBITaxon:115523988139927515622435623593117928662623168390614404211945995677716032932853422NCBITaxon:5759345089998103365786185336583517431275457428316273121536024271831627713528086913519948875058NCBITaxon:10407316275242715502779167971820795413496600271306274154967550278029159541264432392915866002557021272113558867551234916129658793094520312410515192843435815398580156269811825902471491331678456099069027256027256122142725633544228410133562634166392867572279017904NCBITaxon:5796537457574057411123352356237854835552222104577512621613109157559691542931013641396224213921390185732327148962239230741164027812881048961601067574458024025122968335294413936658140366576257634432206672400540296543NCBITaxon:1170914082742823177298184318344322622469765755499229575757592845935561822845907066714367334442403531849223112105421152763910969762261111795752981882633184319229438152764098666134312416265281044214438679626782531751327804267608565050176946996NCBITaxon:131317125079533894279558801198732519795962141140822414082232645614219710141228518676318543152076773767774219253066129454458057041744726724042649329NCBITaxon:177320889320889520755910117664553152316633020043674510116132842665672265872139269635889542301953188291581966558839194366534112336813135904498224911550554382641310337998666940068845421576711435353115104298315468911129859356253232273075660469136165105612149165238976336316812149159704126308213494091328881885513613NCBITaxon:1865381889083264241916073NCBITaxon:186536621017366041185650315750393765573111623427306855743680863772163157491529526689535862048867NCBITaxon:13886296230595975528400319729176160899429170300273105231100911009040541009264653224323276056834062178606106453947781619821597397574998024199474275775804227779195524913136623940731300146407662532041044111170869374515479221198561198572992014625944072NCBITaxon:5920165636713926767162481450134408440877111977810740162699412791314781967949679540814082380394528135340381659668872292612351786162432312432301359923561271087671797854096409797882103529305243277627912924911557484090978040924093447135120253163761629838633178313625152002238865856944155401813397174168709796628773239669202979362899315634301922344713763433872227954866343629916628233456433169173623125924858611093667421386388420493211354724847947314473116968111290255161398128481317333672214407721316931555778122152250255183610036NCBITaxon:11266NCBITaxon:439488113132318679448485145494137499010041715916922596345910659213334991065901249615334542NCBITaxon:6622432754427124327421340112390110049716571678496410289688333295491249668425282652158109434326521577160197127908571622562237NCBITaxon:103572702NCBITaxon:1035811948816543605076011415784435590NCBITaxon:103591707212726348008185934783413223438022113445826276261890364196947139501982528634955983964271191364527262127262327262411607191177189123899312141024605231218948403211821314269704940324803113180803049031417981305212483431114778749093238481437113153688918413684174940804980405788225931943427149182561364911543734131567131641631594030210097146297728235256750601895192952492914505111524964922274849249673146051951876626020115334858002153818625622691232801360327561423142227511903041434011911022145052049321017552759142849342664179906914174609111020594082511211143919042824582762167539131463977851866139077527142735071061981353008559292148691719825214511577281414448340732036962232834959NCBITaxon:11676122962116343423754513678303086784952371094679895580158023627302939680000-0002-9945-7670