MassIVEapplication/xmlftp://massive-ftp.ucsd.edu/v07/MSV000094148/primary200OKProteomicsElizabeth VierlingOrbitrap FusionArabidopsis Thaliana (ncbitaxon:3702)https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=2e6417fab8de4080a73524c6dd048da6vierling@umass.eduUniversity of Massachusetts AmherstMassIVE13MS:1002864 - No post-translational-modifications are included in the identified peptides of this datasetsodium salt, Water, ammonium formate, 13C-labeled, Ribulose Diphosphate Carboxylase, PhrB photolyase activity, mannite, ion, E-421, Powder, CASP-1, Liquid Chromatography Mass Spectrometry, H(2)O, Keratin-Associated Proteins, SDH, magnesium formate, Ultra Performance Liquid Chromatography-Mass Spectrometry, Capillary, IGLON3, CycEI, Apaf-1, zinc salt, Steels, heavy chain disease, Kap-alpha2, Osmofundin, WATER, Circulatory Collapse, HOH, dmTAF[[II]]230, SDS, HPLC-MS, mercurio, Beds, B1, 1, 2, Ribulosebiphosphate, 3, AGM4, CaDTPA, 4, 5, rounded, epithelium, WMS, N-ethyl O-isopropyl thiocarbamate, Octoxynol-9, Ultra Performance Liquid Chromatography Mass Spectrometry, hac-1, cel, N, Dimp-alpha2, Pentetates, Sinusoidal Bed, Keratin Associated Proteins, HPLC, SUPPRESSOR OF AUXIN RESISTANCE 3, salt, Stainless Steels, Propanon, scientific observation, D1, Chromatography, s, Biphosphate Carboxylase-Oxygenase, neocuproine monohydrochloride, DC-LAMP, nickel salt, SGS, Transjugular Intrahepatic Portasystemic Shunt, dTAF[[II]]230, l(1)LB9, Caspase-1 subunit p10, Zinc-DTPA, cobalt (+2) salt, mannitol, land plants, alphaKap2, anon-53Fa, Keratin, Longterm Effect, Ribulose, Sinusoids, ug/ml, not genetically inherited, DETAPAC, DmelCG7788, ACMICD, 5 Biphosphate Carboxylase, Small-Subunit, common salt, sodium (4:1:1) salt, Pyroacetic ether, magnesium salt, Calcium Trisodium, l(2)05206, Penthanil, anon-WO0140519.258, dark/hac-1/dapaf-1, E 421, Small Subunit, LY57, formate, Shwachman-Diamond type metaphyseal dysplasia, TIPSS, oho31, 5 Biphosphate Carboxylase Oxygenase, 5 Bisphosphate Carboxylase Small Subunit, Taf250, ammonium (4:1) salt, Ribulose 1, performic acid, Drying, 5430428I19, DrICE, Pierce, CG4799, pen, precursor, CG7826, Hg, agua, mass-to-charge ratio, Ly57, dimethylcetone, Gene Products, CG7835, Cdi7, Capillary Bed, alpha2, dTAF[[II]]250, Longterm, Hydrogen chloride, thallium (+1) salt, cell, PCE-2, rubidium salt, 4733401P19Rik, Spectrometries, ions, alpha-(4-(1, High-Performance Liquid Chromatographies, Hypovolemic Shock, dTAF250, Caspase-1 subunit p20, OX-2, Kpna2, peptidos, UPLC, 2.1, Pentetate Zinc Trisodium, Associated Protein, Lam, 4-(2-hydroxyethyl)-, PTHB1, measuring, dark/dapaf-1/hac-1, Ribulose Diphosphate, 1-(14)C-labeled, copper (+2) salt, Rnt, buffer, dTAF230, APAF1, l(2)k02514, native protein, DmCycE, Hac-1, carbonyldiamide, Lyophilization, C18, cupric formate, chemical analysis, Long Term Effects, MFS1, Ribulosebiphosphate Carboxylase, NaCl, aluminum formate, Sinusoidal, MES, DC LAMP, Diethylenetriamine Pentaacetic Acid, AU020952, Taf[[II]]250, SWDS, Carboxylase-Oxygenase, ethanenitrile, Interleukin-1 beta-converting enzyme, p. pigmentosa retinae, Collapse, High Pressure, Capillary Beds, lamp, deoxyribonucleate pyrimidine dimer lyase (photosensitive), LAMP, 5-Bisphosphate Carboxylase Small-Subunit, Triton X-45, WAP3, Meth, NCMe, 5-Bisphosphate, 80Hg, DmelCG6829, leg, Triton X-305, classic hairy cell leukaemia, zinc formate, CG7788, High Performance Liquid Chromatography Mass Spectrometry, crice, HEPES Monosodium Salt, lead salt, Pentetate, protein, A230106A15Rik, Poly(oxy-1, High Performance Liquid Chromatography-Mass Spectrometry, Head, quicksilver, ammonium tetraformate, pen-2, peptido, Liquid Chromatography-Mass, congenital, Apaf1, protein aggregate, impalpha2, prevention and control, Injectables, methyl ketone, HEPES Monosodium, l(2)144/1, peptides, E927b, 5R)-Hexane-1, azogue, hypoplasia, arc, mAPC, Heads, Dryings, Ribulose-Bisphosphate, ark, Ribulose Biphosphate Carboxylase, manna sugar, T1, Ribulose Bisphosphate, imp-alpha2, alpha-Keratin, Inflorescence, lithium salt, 1-Piperazineethanesulfonic acid, ICE, Run, Trapping, Shwachman-Bodian syndrome, MKS, Lyw-57, blood capillary, CD208, dihydrogen oxide, GPHYSD2, Long-Term Effects, ammonium (2:1) salt, Sinusoid, bs29g06.y1, iCE, D-Apaf-1, drice, dimethylketone, deoxyribonucleic cyclobutane dipyrimidine photolyase activity, Dmel_CG7826, AI836084, Th, TAFII-250, TAF250/230, Osmitrol, Triton X-100, 6-hexol, glass, SLP65, TAFII250, retinal pigment, Ribulose-Bisphosphate Carboxylase Large Subunit, drIce, 9-dimethyl-o-phenanthroline, Inflorescence Type, nickel (+2) salt, Dmel_CG7835, Ptc, Crystal, MNB, SLP-65, ammonium salt, Rubisco Small Subunit, Fishing, MS/MS, P45, cobaltous formate, AW124434, CG17603, TAF[[II]], Stainless, acqua, apaf-1, DNA-photoreactivating enzyme, precipited material, copper salt, p45, Liquid, Wasser, HEPES, 1728, Ribulose-Bisphosphate Carboxylase Small Subunit, Oho31, Cytokeratin, MPF, CG1849, Dapaf-1/HAC-1, cesium salt, dTAFII250, cycE, Failure, TIPS, CYCLE, Hac1, polypeptide chain, dmTAF1, Octylphenoxy, Injection, ECTOL1, 14C-labeled, tiny, Keratin Associated Protein, ethylcarbamothioic acid, Pro-Mega, MRC, TAF250, Ultra-Performance Liquid Chromatography-Mass Spectrometry, MOS3, photolysis, Glass, CYCE, PRECOCIOUS, 6-hexaol, DmelCG3938, MS2, Runt, CyclE, D-Mannitol, dapaf-1, imp alpha2, 3938, dapaf, calcium formate, alpha2A-Kap, Freeze, Xylella fastidiosa (strain Temecula1 / ATCC 700964), l(2)k05007, dm-cycE, Octylphenoxypolyethoxyethanols, Ultra Performance Liquid Chromatography, Type, Long-Term Effect, Azeton, TAF, BLNK-S, Controlled, High-Performance Liquid Chromatography, small, DYRK1, Controlling, TAF[[II]]250, 2q32q33 microdeletion syndromes, protein complex, 5R)-hexane-1, Diethylenetriamine, l(3)84Ab, Transjugular Intrahepatic Portosystemic, strontium salt, [HCl], p230, water, Dyrk1, CES2A1, Dark, ACETONITRILE, tandem MS, TAF[[II]]230, PRSS, waterbath, Triton X 45, CC1, Tripcellim, l(1)19Ea, MOX2, CGI-97, dApaf-1/DARK/HAC-1, sample population, chromic formate, MOX1, Ribulose Biphosphate Carboxylase-Oxygenase, Protein Gene Products, Interleukin-1 beta convertase, Flower, DmelCG17603, D930023J12Rik, Carboxydismutase, SSKS, 3.4.22.36, calcium salt, Tassel, Peptide., dAPAF-1, TAF1, IL1BC, cadmium salt, MeCN, Tandem, fond, D-(-)-Mannitol, Octoxinols, Triton X305, BOUND WATER, LAMP-3, Basodexan, oxidane, Long Term, prevention, Bca, l(1)AA33, 2-Propanone, Polypeptides, protein polypeptide chains, hydrogen chloride, cobalt(II) formate dihydrate, CH3-C#N, High Performance Liquid Chromatography, NUP96, Carboxylase Small-Subunit, Il1bc, MMTS, bca, Ribulose Biphosphate, Tassels, TFIID TAF250, Glycine, 5-phenanthroline, Salt, proteins, IL-1BC, AI047805, Bed, Indium-DTPA, Wasserstoffchlorid, column, l(1)B2/13.1, sample, methyl methanesulfonothioate, High Speed Liquid, Stainless steel, Octylphenoxy Polyethoxyethanol, 9-dimethyl-1, Types, 2G9, preventive therapy, (2R, Freeze Dryings, Liquid Chromatography, TAF200, l(2)SH0173, Circulatory Failure, Lyophilizations, Flower Head, Cyc E, Triton X100, IMPalpha2, Mn-Dtpa, H2O, BG:DS07108.3, Dm1, soluble, SATB2-associated syndrome, Chlorwasserstoff, dipyrimidine photolyase (photosensitive), Pentaacetic Acid, Ribulose Bisphosphate Carboxylase, CG6829, Hydrogen Oxide, hydrargyrum, Indium DTPA, High Pressure Liquid Chromatography Mass Spectrometry, Spectrometry, methyl methanethiolsulfonate, cromium (+3), Shwachman syndrome, Inflorescence Types, beta Trypsin, Speed, higher plants, Drice, 2018, 3-tetramethylbutyl)phenyl)-omega-hydroxy-, methyl cyanide, chlorane, DRICE, lead (+2) salt, Polyethoxyethanol, TAF230, B130007O04Rik, F10B6_15, Transjugular Intrahepatic, nickel formate dihydrate, cycline, DCLAMP, lead formate, Effects, chlorure d'hydrogene, DrIce, DmcyclinE, aluminum salt, TSC403, Carbamide, protein-containing complex, thomson, Chalk, 2-ethanediyl), F10B6.15, importin alpha2/pendulin, 5-Biphosphate Carboxylase, cdi7, SATB2 syndrome, propanone, cyclinE, Peat, Triton X 305, CDI7, CG42273, l(2)k14401, Carmol, plantae, UPLC-MS, Sinusoidal Beds, ligand, Monosodium Salt, beta-Trypsin, F23A5.3, Long-Term, (L)-Mannitol, methanoic acid, DmcycE, ESI, Pentacine, Injectable, Triton X45, congenital lipomatosis of pancreas, Steel, iglon3, Kochsalz, Ptc1, Dimethyl ketone, N-bis(2-(bis(carboxymethyl)amino)ethyl)-, LB5, caspase 3, 10-phenanthroline, dApaf-1, Proteins, Octoxinol, Pentetate Calcium Trisodium, BG:DS00004.13, chloridohydrogen, potassium formate, High Pressure Liquid Chromatography-Mass Spectrometry, CE-2, acetonitrile, Cell, lLB5, Triton X 100, DmelCG4799, Natriumchlorid, Ptch, TAF[[II]]250/230, O-(1-methylethyl) ester, Calcium Trisodium Pentetate, Mes, Transjugular Intrahepatic Portasystemic, RPE, lithium formate, l(2)k02602, underdeveloped, prophylaxis, Ribulose Biphosphate Carboxylase Oxygenase, Kap alpha2, Dark/Dapaf-1/HAC1, Shwachman-Bodian-Diamond syndrome, VIaL, IL-1 beta-converting enzyme, Mass Spectrometry-Mass Spectrometry, mes, Lamp, Longterm Effects, ME-IV, Gene Proteins, D-CycE, control, Pancreatic insufficiency and bone marrow dysfunction, Z 200, LC-MS, earth, [OH2], hydrochloric acid, Hac-1/Dark, determination, Pflanze, OHO31, Ca-DTPA, Hydrogenchlorid, pigmented epithelium, Mass Spectrometry Mass Spectrometry, extracted material, AW046674, viridiplantae, Alkylations, halite, Ccne, mercure, 3-Phospho-D-glycerate carboxy-lyase (dimerizing), Keratin Associated, Octoxynol 9, Min, Effect, ARK, DmelCG42273, Shock, Cd208, reference sample, pigmented retina, min, plants, Mass Spectrometry, copper, DNA cyclobutane dipyrimidine photolyase activity, lipomatosis of pancreas, WFDC14, Photodegradation, preventive measures, cloruro sodico, dulcite, Sn-DTPA, High-Performance, SOLO DANCERS, Ice, RUN, Liquid Chromatography-Mass Spectrometries, dihydridooxygen, High-Performance Liquid, 1200002D17Rik, dApaf1, neocuproine hydrochloride, 3R, PRE, oho-31, 5-Biphosphate Carboxylase-Oxygenase, rock salt, ice, aqua, classic hairy cell leukemia, Carboxylase, Pentacin, br37, Thermo Scientific, Aceton, ur, 4R, AA33, DTPA, Ly-57, carbamide, Shunt, HCD, Ionen, drICE, Dow Z-200, Inflorescences, BBS13, retinal pigment layer, Mnb, parent ion, BASH, Xylella fastidiosa str. Temecula1, HCl, Plant, hydrogen hydroxide, hac1, MASS, natrii chloridum, beta-Ketopropane, liquid silver, 5-Bisphosphate Carboxylase, alpha Keratin, dapaf-1S, phr A photolyase activity, H2NC(O)NH2, dapaf-1L, l35Dd, Diphosphate Carboxylase, precursor ion, SR3-5, Polypeptide, Zinc DTPA, Rch1, d230, Rubisco, Circulatory, Dalpha2, FBN, Gene, photoreactivating enzyme activity, EfW1, Ribulose Bisphosphate Carboxylase Small Subunit, l(2)br37, Buffer, formic acid, Dark/Hac-1/dApaf1, Karbamid, potassium salt, Dark/Hac-1/dApaf-1, Octoxynols, reduced, Taf230, DPend, E421, Harnstoff, Ribulose Bisphosphate Carboxylase Large Subunit, Tina, stratum pigmentosa retinae, cloruro de hidrogeno, Portosystemic Shunt, Ribulose-1, Taf200, deoxyribocyclobutadipyrimidine pyrimidine-lyase activity, iones, Humus, chlorure de sodium, Taf1p, Mannitol, dark, eau, 5-Biphosphate, Flower Heads, OCTD, Hypovolemic, Imp-alpha2, DARK, Soils, Quecksilber, N-2-Hydroxyethylpiperazine-N'-2'-ethanesulfonic Acid, cromium (+3) salt, dArk, Chromatography-Mass Spectrometries, Dapaf-1, F23A5_3, CaNa-DTPA, sodium formate, Pentaind, Dimethylketon, 6-dimethyl-4, nickel formate, 3H-labeled, DmelCG1849, strontium formate, apaf1, Peptide, MODIFIER OF SNC1, capillary vessel, natural protein, Protein, deoxyribonucleic photolyase activity, Hydrochloride, TFIID, table salt, Vacuums, CyeE, Dark/Apaf-I, cyanomethane, WMS2, DDA, SAS, mercury, POC12, photolyase activity, uree, l(2)35Dd, N 2 Hydroxyethylpiperazine N' 2' ethanesulfonic Acid, TAF[II]250, P235, Trypure, Ion, Chromatography-Mass Spectrometry, Peptid, round, High Performance Liquid, assay, CG3938projections, nitric oxide homeostasis, Subfecundity, World, lamellae, protein complex, World Fertility Surveys, Proteins, lamina, Quality Controls., flanges, Gene, Fertility, protein, protein-containing complex, process of organ, World Fertility Survey, lamella, Fertility Preferences, protein polypeptide chains, Determinant, native protein, natural protein, polypeptide chain, Below Replacement, Protein, shelf, Gene Products, Natural Fertility, Survey, Preference, World Fertility, protein aggregate, Determinants, Fertility Surveys, flange, Fecundity, organ process, Fertility Incentives, Natural, shelves, Control, proteins, Quality, ridges, Controls, projection, Preferences, ridge, NO homeostasis, Fertility Determinants, Protein Gene Products, processes, Gene Proteins, Differential, Marital, papilla, Below Replacement Fertility, Fertility Determinant, Surveys, Fertility Preference, Fertility Incentive, Fecundability, laminae, Differential Fertility, Fertility Survey, Marital FertilityfalseDisrupted Nitric Oxide Homeostasis Impacts Fertility Through Multiple Processes Including Protein Quality ControlThe Organomercury-resin-assisted capture (MRC) of S-nitrosoproteins was done with modifications as described in (Doulias and Gould, 2018). Entire inflorescences pre-anthesis (corresponding to FD stages up to 12/13) of soil grown WT and hot5-2 plants were harvested, shock frozen in lN2 and ground to a fine powder. 5 mL of plant material was used to extract proteins using a 1:1 ratio of extraction buffer (250 mM Hepes pH 7.7, 1 mM DTPA, 0.1 mM neocuproine, 40mM MMTS, 1% TritonX-100). After 5 min of vortexing, samples were centrifuged at 16.000 x g at 4 C for 15 min to separate cell debris from soluble protein. Centrifugation was repeated, protein concentration measured using the BCA assay (Thermo Scientific, USA) and 10 mg of total protein per sample used for the subsequent S-nitrosoprotein enrichment. For photolysis controls 0.1 M mannitol and 5 mM MMTS were added to 10 mg of protein homogenate, vortexed briefly and placed into borosilicate glass vials. The vial was placed on top of an ice bucket and proteins were illuminated under a UV lamp for 7 min in time mode using a UV-crosslinker with a 254 nm light source (Stratalinker Model 1800, Stratagene, USA). Each sample had a corresponding UV-treated negative control analyzed under identical conditions. Proteins were obtained after acetone precipitation and centrifugation at 4500 x g for 15 min at 4 C by resuspending in blocking buffer (250 mM Hepes pH 7.7, 1 mM DTPA, 0.1 mM neocuproine, 50 mM MMTS, 2.5 % SDS). All samples were alkylated for 35 min in the dark in a 50 C water bath with frequent vortexing, followed by another round of acetone precipitation to remove any non-reacted MMTS. Proteins were resuspended to 0.5 mg/ ml using loading buffer (250 mM MES pH 6.0, 1 mM DTPA, 1 % SDS). Organo-mercury resin was synthesized as mention in (Doulias and Gould, 2018). After alkylation, proteins were loaded onto 15 ml of activated organo-mercury resin slurry (50 %) and incubated for 60 min at RT in the dark to allow binding of S-nitrosated proteins to the resin material. Following the incubation, columns were washed with 50 CV of 50 mM Tris-HCl pH 7.5, 0.3 M NaCl, 0.5 % SDS, 50 CV of 50 mM Tris-HCl pH 7.5, 0.3 M NaCl, 0.05 % SDS, 50 CV of 50 mM Tris-HCl pH 7.5, 0.3 M NaCl, 1 % Triton X-100, 50 CV of 50 mM Tris-HCl pH 7.5, 0.3 M NaCl, 0.1 % Triton X-100, 0.1 M Urea and 200 CV of H2O. For on-column trypsin digest, columns were pre-equilibrated with 5 CV of 0.1 M NH4CO3 and then loaded with 1 CV of 0.1 M NH4CO3 containing 1 ug/ml trypsin gold (Promega V5280, Promega, USA). The flow-through containing the digested peptides of S-nitrosated proteins (elution 1) was collected in glass tubes after incubation overnight at RT in the dark. The remaining S-nitrosated peptides that were still bound to the organo-mercury resin were washed with 40 CV of 1 M NH4CO3, 0.3 M NaCl, 40 CV of 1 M NH4CO3, 40 CV of 0.1 M NH4CO3 and 200 CV of H2O. Bound peptides (elution 2) were then oxidatively released by incubation with 1 CV of 1 % performic acid for 45 min at RT and then collected in glass tubes. Following lyophilization of the samples and resuspension in 300 ul of 0.1 % formic acid, sample volume was reduced to 30 ul using a vacuum concentrator (Thermo Savant SPD111V, Thermo Scientific, USA) and then centrifuged for 20 min at 16,000 x g at 4C to remove any precipitate. Peptides were desalted using C18 tips as recommended by the manufacturer (Pierce C18 Tips, Thermo Scientific, USA) and resuspended into 15 ul of 1 % formic acid. For the LC-MS\MS run, a 5 uL injection was loaded by a Thermo Easy nLC 1000 UPLC on to a 2 cm trapping column and desalted with 12 uL mobile phase A (0.1% formic acid in water). Peptides were eluted at 300 nL/min on to a 75 um x 15 cm RSLC column (Thermo) using a linear gradient of 5-35% mobile phase B (0.1% formic acid in acetonitrile) over 90 minutes. Ions were introduced by positive ESI using a stainless steel capillary at 2.1 kV into a Thermo Orbitrap Fusion tribrid mass spectrometer. Mass spectra were acquired over m/z 350-2000 at 120,000 resolution (m/z 200) and 1 second cycle time with an AGC target of 1e6, and data-dependent acquisition selected the top speed most abundant precursor ions for tandem mass spectrometry by HCD fragmentation using an isolation width of 2 Da, and automatic gain settings. Peptides were fragmented with a normalized collision energy of 27 %, and fragment spectra acquired in the linear ion trap. Targeted mass exclusion was activated to avoid MS/MS of ions previously identified as Rubisco or keratin peptides Wed Feb 21 14:19:00 GMT 2024MSV000094148