Absolute Quantitative Analysis Of Modified Ribonucleosides In . - Skyline

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Absolute quantitative analysis ofmodified ribonucleosides in tRNAand mRNA using SkylineSelene SwansonProteomics Center2019-06-02

AcknowledgementBrian PrattLaurence Florens2

Absolute quantitative analysis of modifiedribonucleosides in tRNA and mRNA using Skyline Introduction Why study modified ribonucleosides? Instrumentation and Methods Nucleoside standards Calibration curves Positional isomers Application Targeted analysis of global modification levels in eukaryotic mRNA Summary3

Why?Epitranscriptomics is a link between epigenomics and omicstranscriptomicsproteomics4

Why?Crick’s Wobble anosineuridinecytidine5Journal of Molecular Biology, Volume 366, Issue 1, 2007, 1–13

Why?Epigenetic marks with “writer”, “eraser”, and ineuridinecytidinemethylationm1A m1A and m6A are positional isomers that share the samemolecular formula, but different arrangement of the atoms in space.6The Scientist. 01.2016

Why?Nuclear RNA hylationm7Gm2Gm1GGm7Nucleic Acids Research, Vol. 44, 2016; Abcam

Pathways of RNA Modification8http://modomics.genesilico.pl/

Conservation and types of RNA modifications9Abcam; Genome Biology 2012 13:175

Approaches to Ribonucleosides Analysis Traditional methods: 2D thin-layer chromatography HPLC with UV-VIS spectrophotometrydetection Caveats: Labor- and time-intensiveRequire large RNA quantitiesAre at best semi-quantitativeAre not sensitive enough for lowabundance modifications Are modification-specific and cannotquantitate all RNA modifications at once May require radioactive labeling Sequencing-based techniques: Immuno-capturing and massively parallelsequencing Biochemical identification of A-to-I RNAediting sites by the inosine chemical erasingmethod RNA cytosine methylation analysis bybisulfite sequencing. Profiling of ribose methylations in RNA byhigh-throughput sequencing Transcriptome-wide mapping ofpseudouridines Caveats: Relatively poor quantitative capacity Application restricted to specific PTMs10

LC-MS/MS Approaches to Ribonucleosides AnalysisA Platform for Discovery and Quantification of Modified Ribonucleosides in RNA (Dedon et al. 2015) RNA Purification Data AnalysisxRNA RNA Hydrolysis MS QuantitationMononucleoside Mixture HPLC Resolution High AccuracyMass Spectrometry11Methods in Enzymology, Volume 560, 2015

Absolute quantitative analysis of modifiedribonucleosides in tRNA and mRNA using Skyline Introduction Why study modified ribonucleosides? Instrumentation and Methods Nucleoside standards Calibration curves Positional isomers Application Targeted analysis of global modification levels in eukaryotic mRNA Summary12

Stowers Proteomics CenterHPLC–MSn approachUltiMate 3000 RSLCnano Hypersil Gold aQ HPLC column3 μm particle size; 175Å20 cm x 75µ IDFlow rate: 0.3ul/minBuffer A: 0.1% formic acid v/vBuffer B: 80% acetonitrile with0.1% formic acid v/vLumos Tribrid Mass Spectrometer Positive ion modeScan range 50-400 m/zMS OT @ 60,000ddMS2 OT @ 30,000; 40% HCDddMS3 IT @ 50% HCD13

Analysis of ribonucleoside standardsNTMs2CCmm3Cm5CPrecursor m/zProduct m/z ug/ml260.0627 112.0509310258.1012 112.0509620258.1012 126.06642 100258.1012 126.06642 .0504225252525255014

Analysis of ribonucleoside standardsm3Cm6Am5C CmGm1AAmm2 2 GCYm5Um5Cm7GCYUUCmGmIGIUmm7GNucleotide Test MixRN standards15

Analysis of ribonucleoside standardsFrom raw data to Skylinem6A NCE30 #1 RT: 0.00 AV: 1 NL: 1.66E9T: FTMS p ESI Full ms2 275.00@hcd30.00 [50.00-500.00]150.08100m6A #1 RT: 0.00 AV: 1 NL: 2.03E9T: FTMS p ESI Full ms 060Relative 985050100160.1015020098.9888.085200.02 219.02304.10 332.12250300m/zPrecursor m/z350445.23 469.24400450170.12195.09 2.96400450500m/zProduct m/z16

Analysis of ribonucleoside standardsGenerating calibration curves17

Analysis of ribonucleoside standardsMore calibration curves18

Analysis of ribonucleoside standardsPositional isomers19

Analysis of ribonucleoside standardsPositional isomers – distinct MS3 ion pair20

Analysis of ribonucleoside standardsPositional isomers – RawConverter and in-house R scriptm1Am6AMS1MS2MS321

Analysis of ribonucleoside standardsPositional isomers – positive identification requires MS3 dataMS3 ion intensity22

Absolute quantitative analysis of modifiedribonucleosides in tRNA and mRNA using Skyline Introduction Why study modified ribonucleosides? Instrumentation and Methods Nucleoside standards Calibration curves Positional isomers Application Targeted analysis of global modification levels in eukaryotic mRNA Summary23

Targeted analysis of global modification levels in eukaryotic mRNAE. coli tRNAWT mRNAKD mRNA(Positive Control)Benzonase, Phosphodiesterase I,Alkaline phosphatase, 37 C for 3 h24QIAGEN

Targeted analysis of global modification levels in eukaryotic mRNART:RT: 0.000.00 -- 000tRNAmRNAtRNA - PinkmRNA - Purple32.96850038000NL:NL:4.47E49.88E3UV VIS 1UV VIS 1UVUV tRNA 001000400056.6420.3353.8921.4814.31 0.942020252530303535TimeTime (min)(min)54.9243.9126.6326.96 29.219.69 11.49101058.3753.0844.2340404558.34 60.2750556060656565.87707025

Targeted analysis of global modification levels in eukaryotic mRNAReplicate(ng)AGCtRNA 1154.2176 185.6838tRNA 2168.3678 186.8875tRNA 3163.1332 186.371tRNA 4192.8613 188.5319WT 131.2655 26.4209WT 234.626229.057WT 333.9048 29.1903WT 436.0858 31.3199KD 138.467336.336KD 243.7527 39.9296KD 343.1213 40.3504KD 444.8967 37.9349m1A/m6UAmGmCmm7GIAm2Gm5Cm5U38.96018.568 -0.22622.07480.47164.4535 -0.04820.50353.28490.02690.643340.89439.981 -0.22411.87570.52864.5976 -0.06760.49193.11790.02950.719938.84099.2623 -0.22242.02920.5264.6448 -0.00720.49643.24660.03260.764237.96969.6352 04214.30246.0406 -0.0991 -2.3721-0.1230.89470.12340.0818 -0.44950.0208 -0.821915.32555.4026 -0.0926 -2.3575 -0.10980.89890.17150.0738 -0.43720.0082 -0.812914.94355.4159 -0.1011 -2.3433 #N/A0.89650.08780.0502 -0.42520.0077 -0.821615.41975.4381 -0.0918 -2.3397 -0.14730.89780.11250.0354 -0.42220.034-0.82216.92195.5723 -0.0368 -2.3185 -0.14540.890.4385 -0.0042 -0.40440.0083 -0.822414.73695.1281-0.05 -2.3073 -0.14890.89320.4696-0.001-0.3950.0079 -0.822116.63054.7533 -0.0363 -2.3057 -0.15430.89290.3667 -0.0164 -0.39370.01 -0.822217.2665.1784 -0.0426 -2.3088 #N/A0.89370.2985 -0.0397 -0.39630.0085 -0.822126

Targeted analysis of global modification levels in eukaryotic mRNACanonical RNsWT mRNAE. coli tRNAKD mRNA20040ngngng401002020000AGCUAGCUAGCUAGCU27

Targeted analysis of global modification levels in eukaryotic mRNAModified RNsWT mRNAKD A/m6Am2Gm5Cm5U5U45m55C5mE. coli tRNA28

Targeted analysis of global modification levels in eukaryotic mRNARelative abundance level of RNs inKnock-down vs. Wide-type cellsNormalized Abundance FactorNAFX X/(X Y)*100 Decrease in level of m7G and m5C Increase in the level of inosine m1A/m6A was not detected in the KD cells43*3KD/WT22*11**05CIm1A 0WNormalized Abundance FactorX Absolute quantity of modified RNXY Absolute quantity of canonical RNs29

Summary Using two degrees of separation (retention time of [MH ] and m/z of MS/MS product ions), naturaland modified ribonucleosides were detected and quantified at the sub-nanogram level. Natural and modified ribonucleosides were observed from 500ng of E. coli tRNA and mammalianmRNA. Predicted fragmentation parameters from nucleosides standards were imported into Skyline 4.2.0 forinterpretation and quantification in quadruplicates. Positional isomers with same product ion mass and similar retention time could be distinguished usingRawConverter (Anal. Chem., 2015, 87) and an in-house R-script. Further method development would be required to expand on the number of detectable andquantifiable modified ribonucleosides in hope of providing a comprehensive epitranscriptomic profileof RNA modifications in different cellular states. A G U Cs are not entirely boring, especially in their modified forms!30

Ideas and questions welcome!

Traditional methods: 2D thin-layer chromatography HPLC with UV-VIS spectrophotometry detection . Biochemical identification of A-to-I RNA editing sites by the inosine chemical erasing method . A Platform for Discovery and Quantification of Modified Ribonucleosides in RNA (Dedon et al. 2015) Methods in Enzymology, Volume 560, 2015 11.

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