MassIVEapplication/xmlftp://massive-ftp.ucsd.edu/v03/MSV000087054/primaryOK2000000Laurence Florenshttps://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=3ca7d76fe31a4400be124e0d5cf02202laf@stowers.orgMassIVE3,115MOD:01047 - "A protein modification that effectively converts an L-lysine residue to a monohydroxylated lysine."MOD:00092 - "A protein modification that effectively converts an L-asparagine residue to L-asparagine amide."MOD:01024 - "A protein modification that effectively converts an L-proline residue to one of several monohydroxylated proline residues, including 3-hydroxy-L-proline and 4-hydroxy-L-proline."MOD:01060 - "A protein modification that effectively converts an L-cysteine residue to S-carboxamidomethyl-L-cysteine."MOD:00719 - "A protein modification that oxygenates an L-methionine residue to one of the diastereomeric L-methionine sulfoxide residues."ProteomicsQ Exactive PlusSchmidtea Mediterranea (ncbitaxon:79327)The Stowers Institute for Medical ResearchThe extracellular matrix (ECM) is a three-dimensional network of macromolecules that provides a microenvironment capable of supporting and regulating cell functions. However, only a few research organisms are available for the systematic dissection of the composition and functions of the ECM, particularly during regeneration. We utilized the free-living flatworm Schmidtea mediterranea to develop an integrative approach consisting of decellularization, proteomics, and RNAi to characterize and investigate ECM functions during tissue homeostasis and regeneration. ECM-enriched samples were isolated from planarians, and their proteomes were characterized by LC-MS/MS. The functions of identified ECM components were interrogated using RNA interference. Using this approach, we found that heparan sulfate proteoglycan is essential for tissue regeneration. Our strategy provides an experimental approach for identifying both known and novel ECM components involved in regeneration.Decellularization Enables Characterization and Functional Analysis of Extracellular Matrix in Planarian Regeneration.Sonpho Ekasit E, Mann Frederick G FG, Levy Michaella M, Ross Eric J EJ, Guerrero-Hernández Carlos C, Florens Laurence L, Saraf Anita A, Doddihal Viraj V, Ounjai Puey P, Sánchez Alvarado Alejandro ARegeneration, Regenerations, Matrix, Endogenous., assay, matrisome, Endogenous Regeneration, Extracellular Matrices, determination, Matrices, Extracellular, chemical analysissodium salt, l(4)16, Nalp1, nebb, Formol, ammonium formate, 13C-labeled, dmMOF, CG10718, ion, Lysine Hydrochloride, Keratin-Associated Proteins, mercapturic acid, magnesium formate, Apaf-1, N-glycosidase, zinc salt, heavy chain disease, Solvent, HOH, Lysine Acetate, Cultural, dMOF, B1, 2, 2-amino-3-mercaptopropanoic acid, WMS, L Lysine, C, SET, Dugesias, F, asparagina, C79325, hac-1, K, M, N, P, Oxomethane, klp38B, Keratin Associated Proteins, PROLINE, HPLC, Social, DmelCG4299, isolation and purification, LYS, Acn, D1, Chromatography, KLP 38B, nickel salt, SGS, DmCalm, N-glycanase activity, alpha-NAC, Zinc Cysteinate, cobalt (+2) salt, N-oligosaccharide glycopeptidase activity, anon-53Fa, Keratin, Longterm Effect, tio, alpha-amino carboxylic acids, predicted, ACMICD, sodium (4:1:1) salt, Cultures, magnesium salt, X1k, Methionin, Hmet, N-glycohydrolase F, dark/hac-1/dapaf-1, Cultural Beliefs, Amino acids, DI-2, I-2Dm, formate, 2-amino-3-carbamoylpropanoic acid, MCLDS, Glycopeptide N glycosidase, Peptide N Glycosidase, I-2PP1, Applications, nitrogen, Charges and Fees, Koerper, peptide:N-glycanase, ammonium (4:1) salt, NALP1, Liquimeth, L-pyrrolidine-2-carboxylic acid, multi-cellular organism, Peptidomics, Pierce, l(2)04329, 17alpha)-isomer, CG7826, N Oligosaccharide Glycopeptidase, agua, mass-to-charge ratio, Publication, isolation, H2-Qa1, CG7835, Gene Products, Qa-1(b), Lysine, aic, KLP38B, MAX, anatomical systems, T23d, Acinus, Hydrogen chloride, T23b, thallium (+1) salt, Longterm, rubidium salt, Open Source Software, 2pp2a, Cultural Relativisms, D-CaM, NEM2, ions, High-Performance Liquid Chromatographies, CG10574, Computer Software Application, acinusL, L-acetylcysteine, Enzyme, 2-amino-4-(methylthio)butanoic acid, Formalin, Glycopeptidase F, Peptide N-glycohydrolase F, acinusS, Neb, Glycopeptidase A, KX, dSet, FREE CYSTEINE, peptidos, NACA, UPLC, (2R)-2-amino-3-mercaptopropanoic acid, Associated Protein, PTHB1, dark/dapaf-1/hac-1, Nalp1a, lumen, 1-(14)C-labeled, body, prolina, mitotic nuclear envelope disassembly, CG3025, whole body, copper (+2) salt, Cultural Backgrounds, buffer, Smp1, mKIAA0670, APAF1, native protein, carbonyldiamide, Hac-1, C18, Acetate, cupric formate, chemical analysis, Long Term Effects, proline, NA, MFS1, anatomical spaces, aluminum formate, CG18069, AU020952, torn, ensemble, azote, ethanenitrile, 2-Amino-3-mercaptopropionic acid, Computer Programs, High Pressure, Klp38, Oxomethylene, Dm0332, Fees, glycopeptide N-glycosidase activity, E920, Relativism, CCM1, anon-EST:Posey59, Planarian, camKII, NCMe, DmelCG6829, classic hairy cell leukaemia, Peptide N-Glycanase, zinc formate, Amino acid, lead salt, protein, DEFCAP-L/S, peptide, L-Isomer Methionine, ammonium tetraformate, peptido, Cystein, Apaf1, l(4)ar, protein aggregate, prevention and control, Injectables, DmelCG18069, peptides, TAF-I, E927b, dMax, 2610036I19Rik, Open, hypoplasia, arc, mAPC, DEFCAP, ark, l(4)102EFb, T1, Asn, alpha-Keratin, IGAAD, CAMI, lithium salt, DmelCG10574, CAMC, ppm, C2orf34, deglycosylation, dihydrogen oxide, GPHYSD2, ammonium (2:1) salt, Long-Term Effects, CPVT4, KIF14, D-Apaf-1, Aminokarbonsaeure, CAMKII, 2-Amino-4-(methylthio)butyric acid, Dmel_CG7826, StF-IT-1, Th, Cys, fragmented, Source Softwares, dmax, lysine, Computer Applications Softwares, nickel (+2) salt, Dmel_CG7835, CLNMT, MNB, ammonium salt, cracked, cobaltous formate, CG4299, AW124434, (S)-pyrrolidine-2-carboxylic acid, L-Cystein, acqua, apaf-1, MOF, acetilcisteina, copper salt, Qed-1, Liquid, Glycopeptide N-glycosidase, Wasser, Peptide N-Glycosidase, 3909, Powers, i2pp2a, 1728, mitotic nuclear envelope catabolism, Cytokeratin, CaMK II, Cysteine Hydrochloride, Dapaf-1/HAC-1, Professional Power, (-)-(S)-proline, cesium salt, Aminocarbonsaeure, Peptide N Glycanase, N-Glycosidase A, CAMIII, alpha-amino acid, Computer, N-Glycosidase F, PHAPII, KLP-38B, protein levels., Hac1, polypeptide chain, Homo sapiens, Injection, ECTOL1, 14C-labeled, KMT, l(2)03909, tiny, Keratin Associated Protein, l(2)k00802, Pro-Mega, l(2)3909, ipp2a2, dapaf-1, 2-Pyrrolidinecarboxylic acid, dapaf, calcium formate, Charges, Xylella fastidiosa (strain Temecula1 / ATCC 700964), calcium chloride anhydrous, Camkii, Fee, 10^[-6], CG8472, ECM, Ultra Performance Liquid Chromatography, Long-Term Effect, 10X sequencing, TCEP, Controlled, High-Performance Liquid Chromatography, small, Applications Software, Open Source, DYRK1, Controlling, Acetylcysteine, Computer Software, DL-Methionine, protein complex, igaad, CAMKIIalpha, enzyme activity, strontium salt, dCaMKII, group, AI875693, [HCl], L-alpha-acetamido-beta-mercaptopropionic acid, calcium/calmodulin-dependent protein kinase II, AI461935, Metazoa, CAM, I2PP2A, Dugesia, Dyrk1, Dark, (3alpha, ACETONITRILE, Data Base, l(2)k07614, (-)-2-pyrrolidinecarboxylic acid, PRSS, CC1, Tripcellim, L-2-Amino-3-mercaptopropionic acid, CaMK-II, dApaf-1/DARK/HAC-1, chromic formate, sample population, Protein Gene Products, dSET/TAF-Ibeta, 2610030F17Rik, (R)-mercapturic acid, concentration, AA960152, SSKS, calcium salt, alpha-amino-gamma-methylmercaptobutyric acid, Qa-1, L-(-)-proline, MMRN, dAPAF-1, 3-OHECN, Qa1, cadmium salt, MeCN, SLEV1, calm1, BOUND WATER, Basodexan, prevention, Long Term, DmelCG9648, 5730420M11Rik, Polypeptides, Personal, protein polypeptide chains, hydrogen chloride, cobalt(II) formate dihydrate, Methanal, CH3-C#N, High Performance Liquid Chromatography, L-Proline, Cal49A, Software Engineering, N Glycosidase F, N Glycosidase A, Animalia, DL-Asparagine, ethnicity, N Glycanase, amino acids, L-Asparagine, AI327027, CaCl2, proteins, (2S)-pyrrolidine-2-carboxylic acid, AI047805, set, Wasserstoffchlorid, N-Glycosidase, GPIa*, column, T18c(37), ACN, Lysin, sample, High Speed Liquid, Half Cystine, Social Power, XKR1, CLR17.1, Calcium chloride anhydrous, Power, Genus Mediterranea, dCaM, preventive therapy, DL-Proline, animalia, N-Oligosaccharide, Trypsin/K, CaM KII, Liquid Chromatography, l(2)SH0173, CaM II, Software Tools, bHLHd7, bHLHd6, bHLHd9, DmelCG8759, bHLHd8, Computer Applications, H2O, Cultural Background, bHLHd5, bHLHd4, Stickstoff, Dm1, alphaNAC, Computer Applications Software, Chlorwasserstoff, jack-bean glycopeptidase, N-Oligosaccharide Glycopeptidase, (2R)-2-amino-3-sulfanylpropanoic acid, Hydroxylations, CG6829, (2R)-2-acetylamino-3-sulfanylpropanoic acid, Hydrogen Oxide, Peptide N glycohydrolase F, Acid, CAMKIId, Software Applications, whole organism, HLA-DR-associated protein II, Source Software, cromium (+3), alpha, (-)-proline, beta Trypsin, human, NEB177D, TAF-IBETA, CARD7, NAC, TAF-Ibeta, Acids, methyl cyanide, chlorane, lead (+2) salt, Peptide-N-Glycanase, humans, Computer Software Applications, Psychological Power, 2-amino-4-(methylsulfanyl)butanoic acid, 38B.12, nickel formate dihydrate, 38B.10, lead formate, Effects, False, chlorure d'hydrogene, Biocatalysts, fractured, DmKlp38B, aluminum salt, L-Zystein, Hasp, Carbamide, thomson, protein-containing complex, DmelCG10718, Human, N-Glycanase, DmelCG3025, pyrrolidine-2-carboxylic acid, CG42273, CaM KMT, Half-Cystine, Carmol, Man, Application, Backgrounds, Open Source Softwares, Hpro, L Cysteine, Software Application, E-920, beta-Trypsin, uL/min, Hcys, Long-Term, methanoic acid, Solution, Injectable, Melting, max, 2PP2A, Tools, CYSTEINE, N-acetyl-L-(+)-cysteine, (R)-2-acetylamino-3-mercaptopropanoic acid, glycopeptidase activity, L Proline, dSET, Customs, PHKD, AI256814, metionina, VAMAS1, Gm14, Gm15, dApaf-1, Proteins, split, chloridohydrogen, potassium formate, alpha-amino acids, acetonitrile, L-Isomer, EMILIN4, Tool, polypeptide, l(2)03552, I-2PP2A, DEFCAP-L|S, Glycopeptidase, Dm I-2, hemorrhaged, 5alpha, L-Lysine, Met, lumen space, lithium formate, underdeveloped, prophylaxis, CamKIIalpha, Dark/Dapaf-1/HAC1, (R)-2-amino-3-mercaptopropanoic acid, 4-diamino-4-oxobutanoic acid, primary structure of sequence macromolecule, Longterm Effects, ME-IV, Gene Proteins, Applications Softwares, control, Power (Psychology), Relativisms, [OH2], Cultural Relativism, Enisyl, hydrochloric acid, Ca2+/calmodulin-dependent protein kinase II, Hac-1/Dark, IPP2A2, E 920, CALML2, determination, DD132, N-acetylcysteine, Aminosaeure, CG8759, Hydrogenchlorid, Prolin, Alkylations, Background, Tier, Methionine, BANF, sl(2)ry, Keratin Associated, Min, animal, ARK, Effect, Computer Program, DmelCG42273, KIAA0926, Psychological, 3-hydroxy-5-estrane-17-carbonitrile, CDPK1, PNGase F, Man (Taxonomy), reference sample, enzymes, cisteina, Formaldehyd, 2610510L13Rik, min, PNGase A, epsilon-diaminocaproic acid, Computer Programs and Programming, purification, copper, microlitres per minute, SCAN, ASN, Calm, preventive measures, fSAP152, High-Performance, T18c, CaM kinase, CIDED, dihydridooxygen, High-Performance Liquid, dApaf1, Racemethionine, Cam1, 37b, 37c, NACalpha, phapii, Modern, aqua, CG9648, 10x sequencing, Psychological Powers, classic hairy cell leukemia, Thermo Scientific, ur, Programs, Program, organism, carbamide, N-linked-glycopeptide-(N-acetyl-beta-D-glucosaminyl)-L-asparagine amidohydrolase activity, HCD, Ionen, mitotic nuclear envelope degradation, Softwares, Mnb, Amino Acid, CHARGES, prolinum, Xylella fastidiosa str. Temecula1, HCl, hydrogen hydroxide, L-Cysteine, hac1, MASS, alpha Keratin, chloracetamide, dapaf-1S, H2NC(O)NH2, Mof, dapaf-1L, Charge, Biocatalyst, Mot, Polypeptide, 7N, human being, L-alpha-pyrrolidinecarboxylic acid, L Isomer, FBN, Gene, ACINUS, formic acid, Buffer, Dark/Hac-1/dApaf1, Karbamid, hydroxylation, potassium salt, Dark/Hac-1/dApaf-1, reduced, Harnstoff, AL024000, Klp38B, L-Methionine, Animal, Disulfide, PNGase, cloruro de hidrogeno, Pedameth, CamKII, iones, metazoa, dark, eau, OCTD, Asparagin, (S)-2-pyrrolidinecarboxylic acid, Nlrp1, DARK, taf-ibeta, liquid, cromium (+3) salt, culture, (S)-2-carboxypyrrolidine, dArk, Dapaf-1, sodium formate, nickel formate, 3H-labeled, space, CaM, strontium formate, apaf1, Peptide, Software Tool, natural protein, DCK, Protein, Cam, caM, Calcium/calmodulin-dependent protein kinase, Cal, Hydrochloride, sequence, FORMALIN, 3200001F09Rik, Professional, Software, Dark/Apaf-I, Zystein, cyanomethane, WMS2, DmelCG8472, L-Prolin, uree, 2-amino-3-sulfanylpropanoic acid, cam, Engineering, Glycopeptide, Amino, Methylene oxide, PP1044, nitrogeno, Trypure, Personal Power, H-2T23, Peptide-N(4)-(acetyl-beta-glucosaminyl)Asparagine Amidase, Ion, Modern Man, [CaCl2], Peptid, High Performance Liquid, assay, acetylcysteinum, sl(2)ry3, SCH, AA407739, 6-diaminohexanoic acidliquid chromatography tandem mass spectroscopy, LC-MS2, LC/MS/MS, LCMSMS, GPIa*, liquid chromatography-tandem mass spectroscopy, LC-MSMS, ECM, LC-MS/MS, liquid chromatography tandem mass spectrometry, MMRN, EMILIN4., LC-MS-MSliquid chromatography tandem mass spectroscopy, Proteoglycan, post-transcriptional gene silencing by RNA, Planarian, d230, Activity, Sulfate Proteoglycans, experimental, Laboratory, Peptidomics, Epican, Matrix, dTAFII250, PGP-1, hyaluronate receptor, Matrices, EfW1, LC-MS-MS, dmTAF[[II]]230, Extracellular matrix receptor III, dmTAF1, Taf230, LC-MSMS, freshwater planarian, Post Transcriptional, Extracellular Matrices, Research Activity, Laboratory Research, PGP-I, TAF250, Priorities, Taf200, matrisome, Dugesias, epican, methods, dTAF[[II]]250, LCMSMS, tissue maintenance, TFIID TAF250, pre-mortem, cel, Research, cell, experimental section, phagocytic glycoprotein I, Taf1p, Platyhelminth, LHR, quelling, Cosuppression, free, heparan sulfate proteoglycan, dTAF250, Flatworms, Gene Silencings, HSPG, Phagocytic glycoprotein I, GPIa*, phagocytic glycoprotein 1, Extracellular, ECM, Heparan Sulfate Proteoglycan, MDU2, MDU3, Development and Research, Research Priority, TAF, Heparan Sulfate, Hyaluronate receptor, LC-MS2, Dissections, RNA, dTAF[[II]]230, TAF[[II]]250, Phagocytic glycoprotein 1, posttranscriptional gene silencing by siRNA, LC-MS/MS, TAF200, Endogenous, l(3)84Ab, Research Priorities, CD44 antigen, BG:DS00004.13, TAFII-250, TAF250/230, cosuppression, Cell, hermes antigen, EMILIN4, Proteoheparan Sulfate, Heparan sulfate proteoglycan, dTAF230, GP90 lymphocyte homing|adhesion receptor, RNAi, Proteoglycans, Hermes antigen, TAFII250, LC/MS/MS, Priority, Interference, ly-24, p230, Research Activities, TAF[[II]]250/230, TFIID, Dugesia, RNA Silencing, Silencing, Post-Transcriptional Gene, CD44, Research and Development, Post-Transcriptional Gene Silencings, Taf[[II]]250, Regenerations, HUTCH-I, TAF[[II]]230, MIC4, Posttranscriptional, Posttranscriptional Gene Silencing, extracellular matrix receptor III, flatworms, TAF[II]250, CG17603, Co Suppression, TAF[[II]], Post Transcriptional Gene Silencing, experimental procedures, Activities, Dugesia mediterranea, lymphocyte antigen 24, living, Heparan, Post-Transcriptional, DmelCG17603, Endogenous Regeneration, CDw44, Gene Silencing, Taf250, SR3-5, liquid chromatography-tandem mass spectroscopy, ECMR-III, Regeneration, Dugesia (Schmidtea) mediterranea, liquid chromatography tandem mass spectrometry, Endogenous., flatworm, MMRN, Co-Suppression, Post-Transcriptional Gene Silencing, Proteomes, TAF230, GP90 lymphocyte homing/adhesion receptor, TAF1, Flatworm0PXD024753falseLC/MS/MS Analyses of enriched ECM-enriched preparations from planariaECM isolation - Whole-mount planarian decellularization has been optimized based on a previous publication (Sonpho et al. 2020). We optimized three protocols for decellularization, which we refer to as No Pretreatment ECM (NP-ECM), Formaldehyde ECM (FA-ECM), and N-Acetyl Cysteine pretreatment (NAC-ECM). All worms were collected from static culture. All three protocols were designed for 20 planarians. Proteomics analysis - All decellularized samples were centrifuged at 16,900 g at 4C to recover the ECM pellets. Non-decellularized planarians (whole animal, WA) were prepared for comparison by freezing them in liquid nitrogen. ECM and WA pellets were resuspended in 120 uL of 100 mM Tris-HCl, pH 8.5, and 8 M Urea. The samples were vortexed vigorously to dissolve pellets. To reduce disulfide bonds, Tris(2-carboxyethyl)phosphine hydrochloride (TCEP, Pierce) was added to 5 mM and incubated at RT for 30 min. Reduced cysteines were alkylated by adding 2.4 uL of a freshly made 0.5 M chloroacetamide stock solution (Sigma) and incubating at RT in the dark for 30 min. Samples were deglycosylated with PNGase F by adding 5ul of a solution consisting of 18uL of Glycobuffer 2 (10x) and 27uL of PNGaseF (NEB, P0708S). Endoproteinase Lys-C (Promega), 5 uL at 1 ug/uL, was added and samples were incubated at 37C overnight while shaking. Solutions were diluted to 2 M urea by adding 360 uL of 100 mM Tris-HCl and 2 uL of CaCl2. For trypsin digestion, 10 uL of Trypsin (Promega) at 0.1 ug/uL was added and samples were incubated at 37C overnight while shaking. Reactions were quenched by adding 90% formic acid to a final concentration of 5%. Peptides were diluted with Buffer A (5% acetonitrile (ACN), 0.1% formic acid (FA)). Then, samples were injected on an in-house packed 50 um inner diameter microcapillary column packed with 10 cm of 1.9 um Aqua C18 resin (Dr. Maisch GmbH). The samples were injected in 100% Buffer A and subsequently eluted using an Ultimate 3000 UPLC (Dionex) at a flow rate of 0.120 uL/min for 240 min from 10-40% Buffer B (80% ACN, 0.1% FA) before ramping to 95% B in 25 min. The high organic concentration was maintained for 15 min before re-equilibration and the reinjection with the next sample. Peptides were ionized by the application of a 2.5 kV positive voltage applied distally to the peptides eluting into a Q-Exactive Plus (QE+) mass spectrometer (Thermo Scientific, Waltham, MA). Full MS spectra were recorded on the eluting peptides over a 350 to 1700 m/z range at 70,000 resolving power. The AGC (automated gain control) target was 1x106 with a max injection time set to 50 ms. The top 15 peptides with charges 2-5 were fragmented by High energy Collision Dissociation (HCD) at 27% normalized collision energy (NCE) and the MS/MS fragmentation was collected at 17,500 resolving power, with an AGC target of 1x105 and a maximum injection time set to 150 ms. Dynamic exclusion was enabled for 30 seconds. Mass spectrometer scan functions and HPLC solvent gradients were controlled by the XCalibur data system (Thermo Scientific, Waltham, MA). MS/MS spectra were first searched using ProLuCID v. 1.3.3 with a peptide mass tolerance of 10 ppm and 25 ppm for peptide and fragment ions, respectively. Trypsin specificity was imposed on both ends of candidate peptides during the search against a protein database containing 30,863 S. mediterranea predicted protein sequences as well as 483 usual contaminants such as human keratins, IgGs and proteolytic enzymes. To estimate false discovery rates (FDR), each protein sequence was randomized (keeping the same amino acid composition and length) and the resulting "shuffled" sequences were added to the database, for a total search space of 62,564 amino acid sequences. A mass shift of 57.0215 Da was statically added to cysteine residues to account for alkylation by CAM, mass shifts of +15.9949 was differentially added to methionine, lysine, and proline residues to account for oxidation and hydroxylation, and -0.98401 Da was differentially added to asparagine residues to account for deglycosylation by PNGaseF. DTASelect v.1.9 was used in combination with an in-house software, swallow v1.0 to select and sort peptide/spectrum matches (PSMs) to FDRs of less than 1% at the peptide and protein levels. Mon Mar 15 16:09:00 GMT 2021MSV00008705434416386