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Mattias LarssonUrinary Bladder Smooth MuscleSkinThe collagen gel networks were digested with RIPA buffer. The samples were run on a SDS-PAGE gel. SDS–PAGE gel lanes corresponding to the collagen molecular weight were sliced into pieces and proteins were reduced/alkylated before overnight Trypsin (Promega) digestion47. Resulting peptides were extracted and dried by vacuum centrifugation. Afterwards, peptides were desalted on C18 StageTips48, and dried again by vacuum centrifugation prior to mass spectrometric analysis. For LC MS/MS analysis, peptides were resuspended and separated by reversed-phase chromatography on a Dionex Ultimate 3000 RSLC nanoUPLC system in-line connected with an Orbitrap Q Exactive mass spectrometer (Thermo Fischer Scientific).PrideHans Mattias LarssonCOMPLETEInstitute of bioengineering, School of Life science, EPFL, Switzerland30793047 Zielinski MS, Vardar E, Vythilingam G, Engelhardt EM, Hubbell JA, Frey P, Larsson HM. Quantitative intrinsic auto-cathodoluminescence can resolve spectral signatures of tissue-isolated collagen extracellular matrix. Commun Biol. 2019 2:69 10.1038/s42003-019-0313-xEPFLmattiashans.larsson@epfl.chMass SpectrometryGel-based experimentCathodoluminescence-semExtracellular matrixhttps://www.ebi.ac.uk/pride/archive/projects/PXD012407Not availableDatabase search was performed using MS-Amanda49, and SEQUEST in Proteome Discoverer v.1.4 against a home-made database containing the Bos Taurus reference proteome (Uniprot release 2017_05), the rattus norvegicus reference proteome (Uniprot release 2017_05), and the human reference proteome (UniProt release 2017_05). All searches were performed with trypsin cleavage specificity, with up to 2 missed cleavages allowed and ion mass tolerance of 20 ppm for the precursor and 0.05 Da for the fragments. Carbamidomethylation was set as a fixed modification, whereas oxidation (M), acetylation (Protein N-term), phosphorylation (STY), pyroglutamic acid (peptide N-term Q) were considered as variable modifications.ProteomicsRattus Norvegicus (rat)Homo Sapiens (human)Bos Taurus (bovine)mattiashans.larsson@epfl.chTechnicalBiologicalSwitzerland10.6019/PXD012407By analyzing isolated collagen gel samples, we demonstrated in situ detection of spectrally deconvoluted auto-cathodoluminescence signatures of specific molecular content with precise spatial localization over a maximum field of view of 300 µm. Correlation of the secondary electron and the hyperspectral images proved ~40 nm resolution in the optical channel, obtained due to a short carrier diffusion length, suppressed by fibril dimensions and poor electrical conductivity specific to their organic composition. By correlating spectrally analyzed auto-cathodoluminescence with mass spectroscopy data, we differentiated spectral signatures of two extracellular matrices, namely human fibrin complex and rat tail collagen isolate, and uncovered differences in protein distributions of isolated extracellular matrix networks of heterogeneous populations. Furthermore, we demonstrated that cathodoluminescence can monitor the progress of a human cell-mediated remodeling process, where human collagenous matrix was deposited within a rat collagenous matrix. The revealed change of the heterogeneous biological composition was confirmed by mass spectroscopy.Quantitative intrinsic auto-cathodoluminescence can resolve spectral signatures of tissue-isolated collagen extracellular matrix.Zielinski Marcin S MS, Vardar Elif E, Vythilingam Ganesh G, Engelhardt Eva-Maria EM, Hubbell Jeffrey A JA, Frey Peter P, Larsson Hans M HMRat, Collagens, human being, norvegicus, Laboratory, alpha-Collagen, Matrix, Norway Rat, Gene, protein, Spectrum Analyses, protein-containing complex, Matrices, composed of, Norway Rats, Human, Fibrins, protrusion, organ field, rat, Rattus sp. strain Wistar, Human cell, Microfibril, Rats, Homo sapiens, Dermodress, Mammals, Collastat, Genetic heterogeneity, establishment and maintenance of position, Mass, Gene Products, field, Norway rat, establishment and maintenance of substrate location, Analysis, Rattus rattiscus, Zyderm, Extracellular Matrices, protein aggregate, Man, Mass Spectroscopy, Mass Spectrum Analysis, brown rat, matrisome, Rattus norvegicus, Man (Taxonomy), tail fin, Analyses, developmental field, Microfibril Collagen Hemostat, Avitene, stubby, Electrical Conductivity, composition, Diffusions, alpha Collagen, Laboratory Rat, tail, collagen, Conductivity, Collagen Fleece, Extracellular, Collagen, Collagenfleece, Rattus norvegicus8, establishment and maintenance of substance location, anatomical protrusion, protein complex, Modern, Proteins, future organ, Rattus, compositionality, Spectrum Analysis, Spectroscopy, shortened, single organism localization, Mus norvegicus, Rattus norwegicus, native protein, Laboratory Rats, Protein, Collagen Hemostat, fibril, establishment and maintenance of localization, NICR1066, Antithrombin I, Mass Spectrum Analyses, Pangen, Mass Spectrum, caudal subdivision, content, Tails, other collagen, Heterogeneity, Spectrometry, Avicon, single-organism localization, Electrical, rats, human, Protein Gene Products, extracellular, Gene Proteins, localisation, Electric, spine, Human 细胞, Modern Man, structure, Mass., Collagen Felt, carrier, Norway, short, establishment and maintenance of cellular component location, Gunn ratsTaurine, dairy cow, Rat, IPP2A2, ion, Laboratory, Taurus Cattle, cleavage, Norway Rat, protein, STY, Sty, Dairy Cows, Norway Rats, phosphorylation, 5730420M11Rik, peptide, Polypeptides, rat, Drug Tolerance, variable., peptido, DmelCG1921, Norway rat, protein aggregate, sty, WMS, Yak, SET, Cows, Man (Taxonomy), peptides, TAF-I, Domestic Cow, Dairy Cow, Bos bovis, CLK|STY, Domestic, arg, Beef, Laboratory Rat, DmelCG4299, dSPRY, Bos Tauurus, IGAAD, set, oxen, DmelCG10574, ppm, SIAT7C, domestic cow, Zebus, GPHYSD2, drug tolerance, SGS, phapii, Beef Cows, Taurus Cattles, immune system tolerance, Modern, acetylation, acid, StF-IT-1, Cattle, Rattus, Indicine, Bos indicus, CG1921, ACMICD, Mus norvegicus, Rattus norwegicus, Ionen, Bos taurus, Immunologic Tolerance, Yaks, Holstein, parent ion, Acid, HLA-DR-associated protein II, DI-2, acide, I-2Dm, acids, MASS, CG4299, acido, rats, beta Trypsin, human, I-2PP1, Self Tolerance, TAF-IBETA, Tolerance, precursor ion, Taurus, Zebu, TAF-Ibeta, Polypeptide, Bos indicus Cattle, bovine, i2pp2a, Proteomes, CLK, human being, norvegicus, FBN, Gene, precursor, protein-containing complex, PHAPII, Human, Rattus sp. strain Wistar, l(3)rQ526, Rats, Homo sapiens, cattle, ECTOL1, Gene Products, UNQ2787/PRO7177, Indicine Cattles, Rattus rattiscus, l(3)j5E11, Man, Domestic Cows, brown rat, Rattus norvegicus, iones, ipp2a2, beta-Trypsin, spry, Acetylations, Argos, 2pp2a, CG4531, Dairy, Taurine Cattle, ions, CG10574, OCTD, Beef Cow, 2PP2A, 10^[-6], Cow, taf-ibeta, DmAos, dSET, dSet, peptidos, rat <Rattus norvegicus>, rats <Rattus norvegicus>, Saeure, rlt, Rattus norvegicus8, CS3-3, cow, Bos indicus Cattles, protein complex, Holstein Cow, Proteins, igaad, Phosphorylations, total expressed protein, Taurine Cattles, Peptide, group, CLK/STY, polypeptide, Spry, l(3)rJ812, Bos grunniens, Immune Tolerance, native protein, I-2PP2A, Bos primigenius taurus, Laboratory Rats, Dm I-2, Protein, I2PP2A, MFS1, domestic cattle, l(3)05959, Cattles, Data Base, WMS2, PRSS, ensemble, gil, Saeuren, Tripcellim, Sry, l(3)05845, Protein Gene Products, DmelCG4531, Trypure, Gene Proteins, dSET/TAF-Ibeta, Aos, 2610030F17Rik, ox, Ion, Modern Man, SSKS, l(3)rJ472, Indicine Cattle, Peptid, Norway, AA407739, Gunn rats, Immunological Tolerancematrisome, Collagens, other collagen, Microfibril Collagen Hemostat, Tissue, Avitene, alpha-Collagen, Matrix, Avicon, Matrices, alpha Collagen, Microfibril, collagen, Dermodress, Collagen Fleece, Collastat, Extracellular, Extracellular., Collagen Hemostat, Collagen Felt, simple tissue, Collagen, Zyderm, Extracellular Matrices, Collagenfleece, Pangensmall, F10B6_15, Collagens, Molecular Weights, determination, Proteins, alpha-Collagen, AI836084, FBN, Gene, SDH, backward, buffer, Peptide, F10B6.15, Buffer, ACMICD, SDS, Polypeptides, Microfibril, peptido, reduced, Dermodress, congenital, C18, ECTOL1, Collastat, Weight, Protein, chemical analysis, B1, Collagen Hemostat, Gene Products, MFS1, tiny, Vacuums, Zyderm, Separated, WMS, Pangen, WMS2, Pro-Mega, Separation, Divorced, PRSS, peptides, underdeveloped, Molecular, other collagen, Separations, Microfibril Collagen Hemostat, beta-Trypsin, Avitene, Shwachman-Diamond type metaphyseal dysplasia, hypoplasia, Tripcellim, 4733401P19Rik, SWDS, Weights, Avicon, Shwachman-Bodian-Diamond syndrome, Divorces, Shwachman syndrome, MASS, CGI-97, beta Trypsin, alpha Collagen, lipomatosis of pancreas, Protein Gene Products, OCTD, Gene Proteins, Trypure, collagen, SOLO DANCERS, Pancreatic insufficiency and bone marrow dysfunction, Collagen Fleece, congenital lipomatosis of pancreas, D1, SSKS, Collagen Felt, Peptid, peptidos, Shwachman-Bodian syndrome, Polypeptide, assay, Collagen, anatomical systems., GPHYSD2, Chromatographies, Collagenfleece, PTHB1, SGS, reversedRat, d230, Collagens, human being, norvegicus, Product, Biologic Pharmaceuticals, Laboratory, alpha-Collagen, Matrix, Norway Rat, Gene, dTAFII250, protein, Spectrum Analyses, Matrices, protein-containing complex, EfW1, Norway Rats, Human, Fibrins, protrusion, dmTAF[[II]]230, School-Age, organ field, rat, Rattus sp. strain Wistar, Microfibril, Rats, Homo sapiens, dmTAF1, Dermodress, Taf230, Biological, Mammals, Collastat, establishment and maintenance of position, Mass, Gene Products, DGS, field, Norway rat, establishment and maintenance of substrate location, Analysis, Rattus rattiscus, Zyderm, Extracellular Matrices, Biological Product, protein aggregate, Man, Mass Spectroscopy, TAF250, Biologicals, Mass Spectrum Analysis, brown rat, Drugs, School-Age Populations, Taf200, matrisome, Rattus norvegicus, dTAF[[II]]250, Man (Taxonomy), tail fin, Biologic Drugs, TFIID TAF250, Analyses, cel, cell, Natural, developmental field, gel, Microfibril Collagen Hemostat, Natural Product, Avitene, Electrical Conductivity, Taf1p, Population, Biological Drugs, Diffusions, alpha Collagen, Laboratory Rat, dTAF250, tail, Biological Medicine, collagen, Conductivity, DmelCG1106, Collagen Fleece, Extracellular, Medicine, Medicines, Collagen, School Age Population, TAF, Collagenfleece, Biologic Drug, Biologic, Rattus norvegicus8, Pharmaceuticals, establishment and maintenance of substance location, Products, dTAF[[II]]230, TAF[[II]]250, anatomical protrusion, Biologic Products, protein complex, Biologic Medicines, Modern, Proteins, TAF200, l(3)84Ab, future organ, Rattus, BG:DS00004.13, TAFII-250, TAF250/230, Spectrum Analysis, Cell, dTAF230, Spectroscopy, single organism localization, Mus norvegicus, TAFII250, Rattus norwegicus, native protein, School Age, p230, Biopharmaceuticals, Laboratory Rats, Protein, Biopharmaceutical, Collagen Hemostat, TAF[[II]]250/230, fibril, TFIID, Biologic Product, establishment and maintenance of localization, Antithrombin I, Mass Spectrum Analyses, Pangen, Taf[[II]]250, Mass Spectrum, Populations, TAF[[II]]230, Biological Medicines, caudal subdivision, Tails, Biological Drug, other collagen, Spectrometry, Avicon, TAF[II]250, Biologics, single-organism localization, CG17603, Electrical, rats, TAF[[II]], School Age Populations, human, CG1106, Protein Gene Products, Drug, Gene Proteins, DmelCG17603, localisation, Electric, Taf250, spine, SR3-5, Modern Man, Mass., Collagen Felt, carrier, Natural Products, Norway, School-Age Population, TAF230, establishment and maintenance of cellular component location, Gunn rats, TAF1matrisome, Collagens, other collagen, Microfibril Collagen Hemostat, number, Tissue, Avitene, alpha-Collagen, Matrix, Avicon, Matrices, presence, alpha Collagen, count in organism, Microfibril, collagen, Dermodress, Collagen Fleece, Collastat, Extracellular, Extracellular., Collagen Hemostat, Collagen Felt, simple tissue, Collagen, Zyderm, Extracellular Matrices, Collagenfleece, Pangen, IntrinsicfalseQuantitative intrinsic auto-cathodoluminescence can resolve spectral signatures of tissue-isolated collagen extracellular matrixBy analyzing isolated collagen gel samples, we demonstrated in situ detection of spectrally deconvoluted auto-cathodoluminescence signatures of specific molecular content with precise spatial localization over a maximum field of view of 300 µm. Correlation of the secondary electron and the hyperspectral images proved ~40 nm resolution in the optical channel, obtained due to a short carrier diffusion length, suppressed by fibril dimensions and poor electrical conductivity specific to their organic composition. By correlating spectrally analyzed auto-cathodoluminescence with mass spectroscopy data, we differentiated spectral signatures of two extracellular matrices, namely human fibrin complex and rat tail collagen isolate, and uncovered differences in protein distributions of isolated extracellular matrix networks of heterogeneous populations. Furthermore, we demonstrated that cathodoluminescence can monitor the progress of a human cell-mediated remodeling process, where human collagenous matrix was deposited within a rat collagenous matrix. The revealed change of the heterogeneous biological composition was confirmed by mass spectroscopy.2019-02-192019-01-17PXD012407NEWT:6945NEWT:184922NEWT:6703NEWT:3555NEWT:2NEWT:157546NEWT:35554NEWT:38942NEWT:307972NEWT:32046NEWT:544496NEWT:2102NEWT:2042546NEWT:45351NEWT:43179NEWT:4513NEWT:5722NEWT:1247NEWT:1129NEWT:376741NEWT:55153NCBITaxon:10407NEWT:1736309NEWT:309800NEWT:281395NEWT:10360NEWT:1211601NEWT:876138NEWT:47664NEWT:557436NEWT:317548NEWT:3654NEWT:237561NEWT:5833NEWT:6928NEWT:10036NEWT:36745NEWT:498019NEWT:1351NEWT:1438992NEWT:1352NEWT:2649997NEWT:272563NEWT:79220NEWT:224326NCBITaxon:79857NEWT:1096976NEWT:82688NEWT:95648NEWT:3885NEWT:3888NEWT:5821NEWT:1589NEWT:135622NCBITaxon:4896NEWT:6915NEWT:3649NEWT:101510NEWT:28903NEWT:3880NEWT:272559NEWT:28909NEWT:515849NEWT:3641NEWT:383379NEWT:466585NEWT:10029NEWT:913645NEWT:1000589NEWT:85963NEWT:85962NEWT:143361NEWT:317447NEWT:7955NEWT:7959NEWT:2261NEWT:31156NEWT:398580NEWT:4565NEWT:1264690NEWT:515619NEWT:31155NEWT:192875NEWT:34305NEWT:59729NCBITaxon:183674NEWT:224308NEWT:84645NEWT:626528NEWT:3347NEWT:139927NEWT:4558NEWT:209285NEWT:5888NEWT:211586NEWT:747078NEWT:1283NEWT:931281NEWT:4550NEWT:1000561NEWT:294381NEWT:197NEWT:1390363NEWT:77133NEWT:288705NEWT:29176NCBITaxon:79824NEWT:4787NCBITaxon:4563NEWT:5755NEWT:44689NEWT:3218NEWT:5759NEWT:1736231NEWT:556182NEWT:1270NEWT:374990NEWT:498217NEWT:156471NEWT:2242NEWT:4784NEWT:11320NEWT:360106NEWT:156476NEWT:286NEWT:391619NEWT:360104NEWT:287NEWT:246197NEWT:10117NEWT:10239NEWT:10116NEWT:1280NEWT:1735272NEWT:83334NEWT:83332NEWT:44685NEWT:317513NEWT:1148NEWT:580240NEWT:5508NEWT:294128NEWT:11676NEWT:55571NEWT:35500NEWT:1140NEWT:100226NEWT:4530NEWT:1143NEWT:4896NEWT:75058NEWT:13616NEWT:1390NEWT:1094343NEWT:1336795NEWT:172NEWT:296543NEWT:316435NEWT:1773NEWT:1895NEWT:1182590NEWT:3712NEWT:82380NEWT:105023NEWT:866628NEWT:935293NEWT:64152NEWT:4924NEWT:749200NEWT:375146NEWT:990346NEWT:145953NEWT:257309NEWT:100816NEWT:263NEWT:230741NEWT:52283NEWT:284812NEWT:8175NCBITaxon:1313NEWT:43330NEWT:980415NEWT:1603293NEWT:408169NEWT:44544NEWT:47946NEWT:4911NEWT:645463NEWT:3702NEWT:129249NEWT:243277NEWT:990119NEWT:2850NEWT:408172NEWT:227321NEWT:408170NEWT:493760NEWT:106590NEWT:260710NEWT:257313NEWT:400772NEWT:3708NEWT:128161NEWT:332648NEWT:106592NEWT:536231NEWT:46704NEWT:1436733NEWT:460519NEWT:1187947NEWT:1432138NEWT:269796NEWT:10312NEWT:1424507NCBITaxon:1773NEWT:9598NEWT:8030NEWT:9483NEWT:1639NEWT:188229NEWT:3818NEWT:480NEWT:4909NEWT:67767NEWT:759272NEWT:432359NEWT:46835NEWT:1182263NEWT:109757NEWT:943146NEWT:2711NEWT:300852NEWT:1502NEWT:376686NEWT:95486NEWT:9103NEWT:1883446NEWT:29159NEWT:253NEWT:10306NCBITaxon:2759NEWT:1233435NEWT:93061NEWT:8022NEWT:145943NCBITaxon:4932NEWT:595536NEWT:240906NEWT:593117NEWT:89920NEWT:3635NEWT:5811NEWT:235443NEWT:180923NEWT:108458NEWT:272623NEWT:272624NEWT:2719099NEWT:411483NEWT:884019NEWT:198215NEWT:411490NEWT:983964NEWT:118499NEWT:169963NEWT:32644NEWT:527796NEWT:225117NEWT:499175NEWT:109779NEWT:43665NEWT:1266464NEWT:476272NEWT:3747NEWT:195051NEWT:367830NEWT:1255228NEWT:178616NEWT:649908NEWT:410289NEWT:373153NEWT:375451NEWT:352472NEWT:357NEWT:1071661NEWT:360094NEWT:470NEWT:41364NEWT:1313NEWT:411469NEWT:84023NEWT:559292NEWT:39491NCBITaxon:5811NEWT:29491NEWT:411464NEWT:411460NEWT:2014887NEWT:2762NEWT:1174673NEWT:1328426NEWT:562NEWT:411470NEWT:33952NEWT:2094720NCBITaxon:2697049NEWT:571256NEWT:40483NEWT:28038NEWT:1663NEWT:1423NEWT:4932NEWT:3603NEWT:2759NEWT:3847NEWT:38293NEWT:1428NEWT:327159NEWT:178876NEWT:1660NEWT:327160NEWT:573NEWT:9031NEWT:1872122NEWT:7091NEWT:108931NEWT:241368NEWT:42528NEWT:190802NEWT:9778NEWT:150475NEWT:303NEWT:9417NEWT:7111NEWT:347515NEWT:1216979NEWT:7237NEWT:5180NEWT:256737NEWT:9541NEWT:115104NEWT:1121114NEWT:663NEWT:1081927NEWT:1238993NEWT:67825NEWT:185579NEWT:941442NEWT:220668NEWT:13076NEWT:1821314NEWT:1249668NEWT:7108NEWT:317NEWT:1808362NEWT:5286NEWT:7227NEWT:7469NEWT:885318NEWT:9402NEWT:9644NEWT:415540NEWT:550NEWT:675060NEWT:4081NEWT:334542NEWT:554NEWT:27592NEWT:98334NEWT:426428NEWT:63459NEWT:1276815NEWT:36185NEWT:588858NEWT:9639NCBITaxon:11071NEWT:242231NEWT:7574NEWT:1715256NEWT:7215NEWT:575412NEWT:929793NEWT:29204NEWT:28112NEWT:2172103NEWT:6494NEWT:7460NEWT:6491NEWT:507601NEWT:643680NCBITaxon:6157NEWT:42752NEWT:746360NEWT:6239NEWT:470150NEWT:162425NEWT:216257NEWT:102169NEWT:543734NEWT:9986NEWT:4054NEWT:73239NEWT:226186NEWT:1268063NEWT:8782NEWT:1263854NEWT:118503NEWT:435590NEWT:1902NEWT:160488NEWT:28104NEWT:1908NEWT:13164NEWT:216129NCBITaxon:2NEWT:985076NEWT:1215323NEWT:2664179NEWT:623NEWT:986NEWT:52641NEWT:7038NEWT:6192NEWT:28532NCBITaxon:38727NEWT:198618NEWT:353152NEWT:2829NEWT:366581NEWT:216599NEWT:216595NEWT:1194669NEWT:51329NEWT:243230NEWT:8355NEWT:633NEWT:9685NEWT:7029NEWT:1080772NEWT:8479NEWT:1283300NEWT:632NEWT:6183NEWT:6063NEWT:630NEWT:334747NEWT:61235NEWT:15368NEWT:6289NEWT:436486NEWT:6287NEWT:300641NEWT:727NEWT:9796NEWT:725NEWT:170187NEWT:469008NEWT:260707NEWT:256318NCBITaxon:6191NEWT:1836NEWT:185431NEWT:2925NEWT:29760NEWT:260704NEWT:1719NEWT:703612NEWT:260705NEWT:80863NEWT:2697049NEWT:105231NEWT:1216981NEWT:52638NEWT:4097NCBITaxon:6073NEWT:884204NEWT:978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