{"database":"MassIVE","file_versions":[{"headers":{"Content-Type":["application/json"]},"body":{"files":{"Other":["ftp://massive.ucsd.edu/v06/MSV000092363/"]},"type":"primary"},"statusCode":"OK","statusCodeValue":200}],"scores":{"citationCount":0,"reanalysisCount":0,"viewCount":0,"searchCount":0},"additional":{"omics_type":["Proteomics"],"submitter":["Zoran Rankovic"],"instrument_platform":["solariX"],"species":["Influenza Virus A/california/04/2009"],"full_dataset_link":["https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=37bae135ec6044478358b9ffef4a8d9f"],"submitter_email":["Zoran.Rankovic@STJUDE.ORG"],"submitter_affiliation":["St. Jude Children's Research Hospital"],"sample_protocol":[""],"repository":["MassIVE"],"file_size":["50"],"ptm_modification":["MS:1002864 - No post-translational-modifications are included in the identified peptides of this dataset"],"data_protocol":[""],"pubmed_abstract":["Influenza is one of the leading causes of disease-related mortalities worldwide. Several strategies have been implemented during the past decades to hinder the replication cycle of influenza viruses, all of which have resulted in the emergence of resistant virus strains. The most recent example is baloxavir marboxil, where a single mutation in the active site of the target endonuclease domain of the RNA-dependent-RNA polymerase renders the recent FDA approved compound ∼1000-fold less effective. Raltegravir is a first-in-class HIV inhibitor that shows modest activity to the endonuclease. Here, we have used structure-guided approaches to create rationally designed derivative molecules that efficiently engage the endonuclease active site. The design strategy was driven by our previously published structures of endonuclease-substrate complexes, which allowed us to target functionally conserved residues and reduce the likelihood of resistance mutations. We succeeded in developing low nanomolar equipotent inhibitors of both wild-type and baloxavir-resistant endonuclease. We also developed macrocyclic versions of these inhibitors that engage the active site in the same manner as their 'open' counterparts but with reduced affinity. Structural analyses provide clear avenues for how to increase the affinity of these cyclic compounds.","<h4>Objective</h4>Acute pancreatitis (AP) stands as a frequent cause for clinical emergency hospital admissions. The X-box binding protein 1 (XBP1) was found to be implicated in pancreatic acinar cell apoptosis. The objective is to unveil the potential mechanisms governed by XBP1 and SIRT6 in the context of AP.<h4>Methods</h4>Caerulein-treated human pancreatic duct epithelial (HPDE) cells to establish an in vitro research model. The levels and regulatory role of SIRT6 in the treated cells were evaluated, including its effects on inflammatory responses, oxidative stress, apoptosis, and endoplasmic reticulum stress. The relationship between XBP1 and SIRT6 was explored by luciferase and ChIP experiments. Furthermore, the effect of XBP1 overexpression on the regulatory function of SIRT6 on cells was evaluated.<h4>Results</h4>Caerulein promoted the decrease of SIRT6 and the increase of XBP1 in HPDE cells. Overexpression of SIRT6 slowed down the secretion of inflammatory factors, oxidative stress, apoptosis level, and endoplasmic reticulum stress in HPDE cells. However, XBP1 negatively regulated SIRT6, and XBP1 overexpression partially reversed the regulation of SIRT6 on the above aspects.<h4>Conclusion</h4>Our study illuminates the role of XBP1 in downregulating SIRT6 in HPDE cells, thereby promoting cellular injury. Inhibiting XBP1 or augmenting SIRT6 levels holds promise in preserving cell function and represents a potential therapeutic avenue in the management of AP."],"pubmed_title":["Chemical scaffold recycling: Structure-guided conversion of an HIV integrase inhibitor into a potent influenza virus RNA-dependent RNA polymerase inhibitor designed to minimize resistance potential.","X-Box binding protein 1 downregulates SIRT6 to promote injury in pancreatic ductal epithelial cells."],"pubmed_authors":["Slavish Peter J PJ, Cuypers Maxime G MG, Rimmer Mary Ashley MA, Abdolvahabi Alireza A, Jeevan Trushar T, Kumar Gyanendra G, Jarusiewicz Jamie A JA, Vaithiyalingam Sivaraja S, Jones Jeremy C JC, Bowling John J JJ, Price Jeanine E JE, DuBois Rebecca M RM, Min Jaeki J, Webby Richard J RJ, Rankovic Zoran Z, White Stephen W SW","Yang Zhuo Z, Li Shaojun S, Zhao Chuan C, Zhao Zongzheng Z, Tan Juan J, Zhang Lu L, Huang Yuanqing Y"],"name_synonyms":["phage f2 replicase, Data Set., Q-beta replicase activity, RNA synthetase activity, ribonucleic synthetase activity, Laser Desorption-Ionization Mass Spectrometry, MALDI-MS, ribonucleic replicase activity, Matrix-Assisted, PB1 proteins, ribonucleic acid-dependent ribonucleate nucleotidyltransferase activity, polymerase L, Matrix Assisted Laser Desorption Ionization Mass Spectrometry, Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry, MS-MALD, RNA replicase activity, PB2 proteins, RDRP, Mass Spectrometry, MALDI MS, Matrix Assisted, Matrix-Assisted Laser Desorption-Ionization, Laser Desorption Ionization Mass Spectrometry, ribonucleic acid-dependent ribonucleic acid polymerase activity, SELDI-TOF-MS, Matrix Assisted Laser Desorption Ionization, Spectroscopy, MALDI, ribonucleic acid replicase activity, nucleoside-triphosphate:RNA nucleotidyltransferase (RNA-directed), RNA nucleotidyltransferase (RNA-directed) activity, transcriptase, Mass, RNA-dependent RNA polymerase activity, RNA-dependent ribonucleate nucleotidyltransferase activity, RNA-dependent RNA replicase activity, 3D polymerase activity, Mass Spectroscopy, Surface Enhanced Laser Desorption Ionization Mass Spectrometry, MALD-MS"],"pubmed_abstract_synonyms":["Influenza in Human, Viridae, MGC130048, cyclic compounds., Ribonucleic, Human T-Cell Lymphotropic Virus Type III, insensitive, Activity, Ass-1, fond, AIDS Virus, A4, 4-C)pyrido(2, neutral molecular compounds, sci, CycEI, Human Immunodeficiency, 12, Mutations, Lymphadenopathy Associated Virus, 4)triazine-6, Ccne, Influenza Virus, diseases, 4)oxazino(3, AA408052, Virus, fold, HOW, How, 2, diseases and disorders, 4, 6, PAST, 8, Non Polyadenylated, Human immunodeficiency virus, molecule, l(3)j5D5, RNA Gene Products, molecula, 24B, gamma sarcoglycan, human disease, me75, molecules, Flu, hypoplasia, stru, Molekuel, l(3)S053606, CG10293, D17Mit170, T1, ASS, 8-difluoro-6, l(3)j5B5, Vira, flu, Human Flu, Influenza in Humans, gamma-sarcoglycan, Homo sapiens disease, HPAST1, Strains, Influenza with other manifestations (disorder), close to, Influenzas, 0904/17, ribose nucleic acid, ribonucleic acids, SG-gamma, influenza with non-respiratory manifestation, Cyc E, br37, Tl3, Tl2, BG:DS07108.3, Influenza Viruses, AIDS, SZ1, Ribonukleinsaeure, 1-f)(1, Diseases, 4)triazin-7-yl)oxy)methyl methyl carbonate, pentosenucleic acids, 12ahexahydro-1H-(1, sarcoglycan, Ribonucleic acids, l(2)05206, region, influenza, Orthomyxoviruses, Acquired Immune Deficiency Syndrome Virus, Acid, Lymphadenopathy-Associated, resistant, Human Influenza, influenza virus infection, PAST1, gamma (35kDa dystrophin-associated glycoprotein), disease, DMDA, l35Dd, hyaline, antagonists and inhibitors, 35kD dystrophin-associated glycoprotein, Acquired Immunodeficiency Syndrome Virus, baloxavir marboxil, Animal Viruses, (((12aR)-12-((11S)-7, anon-EST:Liang-2.39, Strains and Sprains, human immunodeficiency virus infectious disease, influenza infection, other disease, Orthomyxovirus, Influenza with other manifestations, cycline, SGCG_HUMAN, conformation, H-PAST, Influenza with other manifestations NOS (disorder), P62, DmcyclinE, Myxovirus, HTLV-III, cycE, l(2)br37, TYPE, CYCLE, Human, cdi7, DAGA4, cyclinE, reduced, resistance, 8-dioxo-3, 35DAG, Cdi7, CDI7, 1-F)(1, Gene Products, disease or disorder, 8-dione, Low, Animal, tiny, Human T-Cell Leukemia Virus Type III, MAM, gamma-SG, Sprains, SCG3, Xofluza, Animal Virus, Human T-Lymphotropic Virus Type III, CYCE, Myxoviruses, 12a-tetrahydro-1h-(1, l(3)s2612, Viruses, DmelCG3938, CyclE, 4-c)pyrido(2, 3938, inhibiteur, Immunodeficiency Viruses, DmcycE, Human T Cell Leukemia Virus Type III, non-neoplastic, Non-Polyadenylated RNA, l(2)k05007, dm-cycE, inhibidor, DmelCG10293, Lymphadenopathy-Associated Viruses, disorder, site, HIV, Influenza, Influenza with non-respiratory manifestation (disorder), Grippe, Human Immunodeficiency Viruses, relational structural quality, orthomyxoviridae disease or disorder, Lymphadenopathy-Associated Virus, small, inhibitors, RNA, Human Immunodeficiency Virus, Human T Lymphotropic Virus Type III, cou, 35 kDa dystrophin-associated glycoprotein, clone 2.39, Sprain, disorders, inhibitor, RNS, medical condition, qkr, l(3)S090417, SGCG, LGMD2C, near to, viruses, 11-dihydrodibenzo(b, Lr, l(2)k02514, DmCycE, E)thiepin-11-yl)-6, Immunodeficiency Virus, yeast nucleic acid, orthomyxoviridae caused disease or disorder, KH93F, Strain, condition, CyeE, Zoophaginae, AIDS virus, nm, who, antagonists, ribonucleic acid, l(2)k02602, e)thiepin-11-yl)-7-hydroxy-3, AIDS Viruses, DMDA1, underdeveloped, Non Polyadenylated RNA, l(2)35Dd, Non-Polyadenylated, Ribonucleic Acid, Who/How, (12aR)-12-((11S)-7, LAV-HTLV-III, orthomyxoviridae infectious disease, D-CycE, Human Influenzas, clear, approaches, vicinity of, SCARMD2, Bra, qkr[93F], CG3938, General activity, Endonuclease, Human T Cell Lymphotropic Virus Type III"],"pubmed_title_synonyms":["influenza, inhibitors, Influenza in Human, Influenzas, Viridae, influenza infection, Human Immunodeficiency Virus, Animal Virus, Influenza with other manifestations, resistance., Viruses, conformation, Influenza with other manifestations NOS (disorder), Human Influenza, Integration Protein, Flu, inhibiteur, inhibitor, influenza virus infection, influenza with non-respiratory manifestation, p31 Integrase Protein, HIV Integration Protein, Integrase, orthomyxoviridae infectious disease, Human, viruses, Human Influenzas, inhibidor, Vira, orthomyxoviridae caused disease or disorder, p31 pol Gene Product, flu, Human Flu, Influenza in Humans, Virus, Animal Viruses, HIV, Influenza, Influenza with non-respiratory manifestation (disorder), Animal, Grippe, Zoophaginae, Influenza with other manifestations (disorder), relational structural quality, orthomyxoviridae disease or disorder"],"description_synonyms":["sodium salt, Viridae, dec, l(4)13, Ribonucleic, ammonium formate, 13C-labeled, Laser Desorption-Ionization Mass Spectrometry, cadmium salt, MeCN, PhrB photolyase activity, ion, PNT-P1, INSULYSIN, Lasers, EY3-1, E-Cadherin, magnesium formate, mg/ml, insulin-degrading neutral proteinase activity, zinc salt, LEF-1, Long Term, Integrase, Dmel_CG32005, Personal, manganese dichloride, cobalt(II) formate dihydrate, insulin-degrading enzyme activity, Method, CH3-C#N, responsivity, p31 pol Gene Product, LEF1/TCF, 3, RNA Directed RNA Polymerase, TEP1, NUP96, DmelCG14228, Analysis, D-ets-2, 3520, insulinase activity, epithelium, Non Polyadenylated, WMS, dec1, thymus nucleic acid, fs(1)14-963, Analyses, rya-r44F, DCAD2, Flu, DCad2, Nonmigrant, insulin proteinase activity, MS-MALD, HardwareType, Replicase, cTCF, DECad, Transient, Shots, CG3722, SUPPRESSOR OF AUXIN RESISTANCE 3, Rya-R44F, AI047805, Social, Matrix Assisted Laser Desorption Ionization, DE Cad, epiphyseal dysplasia, CG10844, Vira, scientific observation, manganese chloride (MnCl2), flu, Human Flu, fs(1)M1021, Social Power, RNA-Directed, DEcad, fs(1)12-3907, Pan, PAN, nickel salt, SGS, accumulated, Power, DE, EIEE13, cobalt (+2) salt, G protein-coupled estrogen receptor 1, G-protein coupled receptor 41, Flow-induced endothelial G-protein coupled receptor 1, Longterm Effect, Shg, dy, influenza with non-respiratory manifestation, Procedure, fs(1)12-1873, ACMICD, sodium (4:1:1) salt, visualization., GPR30, Dm1, magnesium salt, nmf12, PTEN1, ECadh, dipyrimidine photolyase (photosensitive), Ribonucleic acids, 5830440B04, desoxyribose nucleic acid, Migrants and Transients, mKIAA4087, influenza, Acid, Q-Switched Laser, pntP2, D E-cad, Pointed-P1, instrument, E-cadherin, F24J5.12, Classical Polyarteritis Nodosa, Panarteritis Nodosa, Ets94F, formate, DTCF, DTcf, hexaammoniate, cromium (+3), Pulsed Laser, Methodological, study protocol, surveillance, gp150, Fc, F24J5_12, RNA Dependent RNA Polymerase, multiple, EMR2, LERGU, antagonists and inhibitors, Emr2, ds DNA, ammonium (4:1) salt, G-protein coupled receptor 30, Animal Viruses, Migrants, DNA, methyl cyanide, FEG-1, CT12481, lead (+2) salt, DCad, ECad2, dTCF, dTcf, CG34403, Psychological Power, 0998/12, 2-ME, Influenza with other manifestations, DNS, nickel formate dihydrate, DE Cadh, conformation, (Deoxyribonucleotide)n, lead formate, GLM2, Effects, Biocatalysts, monoammoniate, Periarteritis Nodosa, fs(1)14A-114, aluminum salt, number, thomson, p31 Integrase Protein, CG7826, DOI, Human, DmelCG2175, l(2)k03401, mass-to-charge ratio, DMPOINT1A, Deoxyribonucleic Acid, LEMD5, Gene Products, CG7835, CG42273, Classic Polyarteritis Nodosa, Technique, doi, CadE, Ecad, Animal Virus, medea, Pulsed Lasers, pointed-RC, thallium (+1) salt, occurrence, Longterm, cadh, rubidium salt, prevalence, F23A5.3, Double Stranded, Fcp7C, Deoxyribonucleic acid, Matrix Assisted, Long-Term, methanoic acid, Worker, manganous chloride, ions, E(zen)3, 3-hydroxy-2-pyridinecarboxylic acid, EK3-2, ECAD, ECad, Study, l(3)07825, Continuous Wave Laser, Enzyme, Q-Switched Lasers, D-cad, hexahydrate, CG17077, DmelCG10844, (Deoxyribonucleotide)m, species, Influenza, CG5517, CADH, MALD-MS, Drl, inhibitors, measuring, Human Immunodeficiency Virus, RNA Dependent RNA Replicase, RNA, Ets, DRR, dec[[1]], 1-(14)C-labeled, DRY, DNAn+1, BG01543, Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry, CG2175, RNS, potassium formate, copper (+2) salt, acetonitrile, Cadh, Mdu, fs(1)C1, Nucleoside-Triphosphate:RNA Nucleotidyltransferase (RNA-directed), fs(1)12-403, Acoustic, N-cad, Temperatures, dry, mda, A-7, orthomyxoviridae caused disease or disorder, MED, CWS1, cupric formate, chemical analysis, Long Term Effects, MFS1, MIXL, aluminum formate, Tcf/LEF, pnt-P1, pnt-P2, outbreaks, Mer, ami, periarteritis, AU020952, RPE, lithium formate, l(2)k00424, LERGU2, DmelCG5517, med, Ribonucleic Acid, ethanenitrile, Childhood, Matrix-Assisted Laser Desorption-Ionization, mer, mER, E-cad, CG32005, Longterm Effects, p. pigmentosa retinae, ME-IV, plan specification, l(2)k04913, NaCh6, Lef, Nomads, Power (Psychology), 2-mercapto-, Desoxyribonukleinsaeure, fs(1)13-453, epidemics, deoxyribonucleate pyrimidine dimer lyase (photosensitive), PN4, l(3)XIIm137, General activity, CEPR, Endonuclease, POINT, Data Analyses, E-CAD, E-Cad, l(2)k10220, CG8705, Sta, STA, Influenza in Human, fs(1)A384, Polyarteritis Nodosa, Activity, NCMe, determination, DmelCG17077, instrument configuration, Matrix Assisted Laser Desorption Ionization Mass Spectrometry, zinc formate, lead salt, DmelCG34403, Eyk, Pnt, RNA Replicase, pigmented epithelium, IA5, CG11121, fs(1)A257, Ethanol, Techniques, 52Mn-labeled, old, ammonium tetraformate, png, Virus, Min, fs(1)5, Prei1, merosin, Chemoattractant receptor-like 2, Effect, Surface Enhanced Laser Desorption Ionization Mass Spectrometry, MIX, RNA Gene Products, DmelCG42273, fs(1)M102, Pnt-P1, Psychological, EDM1, EDM2, TCF/LEF, EDM3, Maser, Migrant Worker, fs(1)11-549, pigmented retina, 2 Mercaptoethanol, min, mAPC, abeta-degrading protease, insulysin, Mass Spectrometry, MALDI MS, EDMD, copper, SO, DNA cyclobutane dipyrimidine photolyase activity, HIV Integration Protein, Continuous Wave, reaction, insulinase, lithium salt, anon-EST:Posey121, DE-CAD2, dEcad, Methodological Studies, dya, Influenza in Humans, Squatters, Double-Stranded DNA, DECadh, deoxyribonucleic acids, DNAn, EPD1, Influenza with other manifestations (disorder), ICR, GPHYSD2, polyarteritis, ammonium (2:1) salt, Long-Term Effects, So, Influenzas, l(3)12m-137, PRE, DJ39G22.4, IDD, ribose nucleic acid, IDE, deoxyribonucleic cyclobutane dipyrimidine photolyase activity, frequency, metalloinsulinase activity, ribonucleic acids, Dmel_CG7826, Q Switched Lasers, D-Ets-2, Th, ets94F, 0123/09, DE-Cad2, Double-Stranded, Psychological Powers, CG17820, Laser Desorption Ionization Mass Spectrometry, LEF/TCF, Dm Pan, DE-cadh, Spectroscopy, (Deoxyribonucleotide)n+m, fs(1)1501, Immunization, fs(1)12-365, retinal pigment, Ionen, DE-Cadherin, PERIANTHIA, Ribonukleinsaeure, Migrant, RNA-Dependent RNA, DE-cadherin, pentosenucleic acids, Workers, nickel (+2) salt, dSmad4, retinal pigment layer, Dmel_CG7835, CERIII, Mnb, MNB, MILD1, CMKRL2, shg/DE-Cad, ammonium salt, RyR, Human Influenza, RNA-Directed RNA Polymerase, MMAC1, cobaltous formate, influenza virus infection, MASS, CG17964, AW124434, Methodological Study, morbidity, ptd, PntP2, data analysis, phr A photolyase activity, Q-Switched, DNA-photoreactivating enzyme, DmelCG3722, RYR, Ryr, PntP1, 2-Mercaptoethanol, E(E2F)3D, copper salt, Biocatalyst, RNA-Dependent, PNTP2, Fc1, Laser, Powers, BZS, com5, PNTP1, influenza infection, fs(1)12-2514, CIAT, DJ885L7.4.1, DRY12, DmelCG1775, MALDI-MS, Procedures, ryr, Professional Power, Influenza with other manifestations NOS (disorder), Integration Protein, PSACH, ETS2, Ets2, fs(1)384, LEF/TCF-1, cesium salt, FBN, antibiotic A 7, MnCl2, photoreactivating enzyme activity, presence, RNA Polymerase, Migrant Workers, Deoxyribonucleic acids, hardware, l(2)10469, formic acid, MHAM, method, pntegfr, CG14228, IL8-related receptor DRY12, potassium salt, 0608/07, DmelCG11121, ECTOL1, 14C-labeled, Pulsed, method used in an experiment, resistance, l(3)SG36, RyRs, Mass, Studies, stratum pigmentosa retinae, Animal, Masers, manganese chloride, Mass Spectroscopy, reactivity, somda, MOS3, deoxyribocyclobutadipyrimidine pyrimidine-lyase activity, Viruses, PRECOCIOUS, iones, Transients, inhibiteur, Nonmigrants, Tcf-1, Dmerlin, d-TCF, calcium formate, Membrane estrogen receptor, SELDI-TOF-MS, Non-Polyadenylated RNA, OCTD, fs(1)12-3512, dihydrate, Squatter, SMAD4, D-Mer, Table, inhibidor, data processing, TCF/LEF1, Nav1.6, GPCR-Br, chimpanzees, Smad4, HIV, cromium (+3) salt, Long-Term Effect, Influenza with non-respiratory manifestation (disorder), Lymphocyte-derived G-protein coupled receptor, GPER, Grippe, relational structural quality, l(3)j1B7, F23A5_3, sodium formate, incidence, orthomyxoviridae disease or disorder, DE[cyto], insulin-specific protease activity, tetrahydrate, DYRK1, LYGPR, dIDE, dIde, fs(1)13C-73, nickel formate, 3H-labeled, Continuous Wave Lasers, Dmel_CG17964, DE-cad, strontium formate, inhibitor, l(3)11m-254, strontium salt, light amplification by the stimulated emission of radiation, viruses, count in organism, MODIFIER OF SNC1, DmelCG17820, Cad, pan.dTCF, insulin-glucagon protease activity, fs(1)12-4860, STL5, yeast nucleic acid, deoxyribonucleic photolyase activity, Nomad, DE-CAD, DE-Cad, fs(1)14E19, Professional, Dyrk1, Tcf, TCF, ds-DNA, Zoophaginae, ACETONITRILE, CG1775, l(3)s118306, cyanomethane, WMS2, Lef1, antagonists, ribonucleic acid, AW550900, cad, LEF1, 10q23del, dRyR, photolyase activity, insulin protease, Matrix-Assisted, RNA-Dependent RNA Replicase, Non Polyadenylated RNA, CC1, THBS5, Non-Polyadenylated, tcf, l(3)SG70, LyGPR, l(4)102ABb, chromic formate, endemics, orthomyxoviridae infectious disease, Nyk, DEC, Personal Power, Human Influenzas, Ets58AB, MALDI, Ion, concentration, Data, SSKS, calcium salt, insulin protease activity, fs(1)13C-57, assay, lef1, response, diammoniate, e-cad, Rya-r76CD"],"citation_count":["0"],"additional_accession":[]},"is_claimable":false,"name":"RdRp MALDI Data Set - 2022, Slavish/Rimmer","description":"Chemical scaffold recycling: Structure-guided conversion of an HIV integrase inhibitor into a potent influenza virus RNA-dependent RNA polymerase inhibitor designed to minimize resistance potential\n\nEur J Med Chem. 2023 Feb 5;247:115035. doi: 10.1016/j.ejmech.2022.115035. Epub 2022 Dec 22.\nSlavish, Jake et al.\n\nThe enzymatic activity of PAN endonuclease in the presence of inhibitors was measured using a MALDI-FTICR-MS-based functional assay. An ECHO 650 \n acoustic dispenser plate reformat protocol (Labcyte) was used to make 16-point dose responses of each compound (67.5 nL, from 45 uM to 0.8 pM) in 384-well plate format (Greiner Bio-One). The compounds were pre-incubated for 1 h with 10 uL of the enzyme assay mix; 300 nM PAN endonuclease, 5 mM manganese chloride (Aldrich), 20 mM 2-mercaptoethanol (Calbiochem) in 10 mM Tris pH 8.0 (Fisher Scientific). The reaction was initiated by the addition of 10 uL of RNA (Integrated DNA Technologies) to a final concentration of 50 uM. The plate was incubated at room temperature for 90 min before being\nquenched with the addition of 10% formic acid (EMD Millipore Corp.). Two microliters of each reaction were mixed with 8 uL of 40 mg/mL 3-hydroxypicolinic acid (Sigma-Aldrich) in 70% acetonitrile (Fisher Scientific) containing 10 mg/ml ammonium citrate (Fisher Scientific), manually spotted on a 384 Anchorchip MALDI target (Bruker Daltonics), and let dry at room temperature. The experiments were performed on a 7-T SolariX XR FT-ICR Mass Spectrometer (Bruker Daltonics) in MALDI negative mode using ftmsControl v2.3. The spectrometer instrument method was developed specifically to improve its dynamic range for substrate/product RNA with minimal sample preparation. Thus, with a mass range of 150 to 5000 m/z, signal from a minimum 10 transients were accumulated and each transient analyzing ions produced from 300 MALDI laser shots at a 500 Hz frequency. Other FTMS settings were as follows: sweep excitation power of 44%, excitation waveform calculation version 2, Time of Flight of 0.002 s, Q1 mass of 300 m/z, ion accumulation time of 0.025 s, laser power 40 lp. The readout for this assay was the disappearance of a single-stranded, 17-mer RNA substrate (5'-UCUCUAGCAGUGGCGCC -3'; m/z = 2687.3 for doubly-charged species) and appearance of the 7-mer hydrolyzed product (5'-UCUCUAG-3' m/z = 2139.6 for singly-charged species). Five assay replicates\nacross three days were performed with two instrument replicates per assay. The highest 2500 local maxima with an absolute intensity threshold of 100,000 were extracted, and those within +/- 0.1 m/z of target m/z were used to calculate product and substrate intensity. Enzyme turnover (T) was calculated as a ratio of product intensity (I) over total intensity of substrate plus product (TRdRp = I2139.6/I2687.3 + I2139.6). Peak extraction and data analysis were performed in R using the RStudio IDE [59,60]. Z-prime was calculated for each individual FTMS target and targets with a resulting Z-prime below 0 were eliminated. FTMS replicates were then combined and Z-prime for each assay replicate was calculated (Table 6). Turnover values were normalized to negative controls and dose-response curves were fit to a four-parameter log-logistic model using non-linear least-squares optimization. Resulting fits were plotted using the ggplot2 visualization package. R code is available on request. ","dates":{"publication":"Thu Jul 06 15:23:00 BST 2023"},"accession":"MSV000092363","cross_references":{"pubmed":["36603507"]}}