Pseudouridine detection improvement by derivatization with methyl vinyl sulfone and capillary HPLC–mass spectrometry

Emmerechts, Gert; Herdewijn, Piet; Rozenski, Jef

doi:10.1016/j.jchromb.2005.06.041

Cited by 29 publications

(27 citation statements)

References 11 publications

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“…In related work, alternative chemical derivatizations of pseudouridine have been used prior to mass spectrometric detection [12,13]. As with the CMC-based approach, these derivatization strategies place a mass tag on pseudouridine that facilitates its detection by mass spectrometry.…”

Section: Introductionmentioning

confidence: 99%

Improving CMC-derivatization of pseudouridine in RNA for mass spectrometric detection

Durairaj

Limbach

2008

Analytica Chimica Acta

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A protocol that utilizes matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and N-cyclohexyl-N′-β-(4-methylmorpholinium)ethylcarbodiimide (CMC) derivatization to detect the post-transcriptionally modified nucleoside, pseudouridine, in RNA has been optimized for RNase digests. Because pseudouridine is mass-silent (i.e. the mass of pseudouridine is the same as the mass of uridine), after CMC derivatization and alkaline treatment, all pseudouridine residues exhibit a mass shift of 252 Da that allows its presence to be easily detected by mass spectrometry. This protocol is illustrated by the direct MALDI-MS identification of pseudouridines within Escherichia coli tRNA TyrII starting from microgram amounts of sample. During this optimization study, it was discovered that the post-transcriptionally modified nucleoside 2-methylthio-N 6 -isopentenyladenosine, which is present in bacterial tRNAs, also retains a CMC unit after derivatization and incubation with base. Thus, care must be exercised when applying this MALDIbased CMC-derivatization approach for pseudouridine detection to samples containing transfer RNAs to minimize the misidentification of pseudouridine.

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Section: Introductionmentioning

confidence: 99%

Improving CMC-derivatization of pseudouridine in RNA for mass spectrometric detection

Durairaj

Limbach

2008

Analytica Chimica Acta

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“…64,67 The derivitization method typically involves treating the sample to chemically label pseudouridines and not uridines, with methylvinylsulfone, acrylonitrile or 1-cyclohexyl-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate (CMCT), which was originally used for biochemical pseudouridine detection by strong stops during primer extension. 66,69,70 The added mass after any of these chemical treatments can easily be detected and the sites of pseudouridylation identified. However, great care must be taken to ensure that pseudouridines are labeled selectively.…”

Section: Rna Modification Mapping Of Trnas and Rrnasmentioning

confidence: 99%

The identification and characterization of non-coding and coding RNAs and their modified nucleosides by mass spectrometry

Gaston

Limbach

2014

RNA Biology

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The analysis of ribonucleic acids (RNA) by mass spectrometry has been a valuable analytical approach for more than 25 years. In fact, mass spectrometry has become a method of choice for the analysis of modified nucleosides from RNA isolated out of biological samples. This review summarizes recent progress that has been made in both nucleoside and oligonucleotide mass spectral analysis. Applications of mass spectrometry in the identification, characterization and quantification of modified nucleosides are discussed. At the oligonucleotide level, advances in modern mass spectrometry approaches combined with the standard RNA modification mapping protocol enable the characterization of RNAs of varying lengths ranging from low molecular weight short interfering RNAs (siRNAs) to the extremely large 23 S rRNAs. New variations and improvements to this protocol are reviewed, including top-down strategies, as these developments now enable qualitative and quantitative measurements of RNA modification patterns in a variety of biological systems.

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“…38,39 Yet another recently evolved method is nucleoside-specific derivatization (e.g., of pseudouridine with acrylonitrile, with N-cyclohexyl-N'-β-(4-methylmorpholinium) ethylcarbodiimide p-tosylate (CMCT), or with methylvinylsulfone, see also Table 1) followed by MS analysis. 36 A particularly sophisticated instrumental setup was implemented by Emmerechts et al 40 using LC-Q-TOF-MS/MS. One of the most interesting latest developments was published by Hossain and Limbach 2009.…”

Section: Identification By Physicochemical Propertiesmentioning

confidence: 99%

“…5). In addition cyanoacrylate and methylvinylsulfone, which have been mentioned in context MS-based detection of Ψ, 36,40 4-methylbromo-7-methoxy-coumarine has also been reported to specifically react with Ψ residues. 73 Recent method development makes use of the chemical tagging for detection by mass spectrometry in RNA fragments obtained after digestion e.g., by RNase T1.…”

Section: Differential Chemical Reactivitymentioning

confidence: 99%

“…73 Recent method development makes use of the chemical tagging for detection by mass spectrometry in RNA fragments obtained after digestion e.g., by RNase T1. 36,40,74,75 Affinity purification and analysis. While tRNAs are highly modified and present up to 10% of the cellular RNA, most other RNA species are much more rare and presumably less modified.…”

Section: Differential Chemical Reactivitymentioning

confidence: 99%

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Detection of RNA modifications

Kellner

Burhenne²,

Helm³

2010

RNA Biology

120

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Pseudouridine detection improvement by derivatization with methyl vinyl sulfone and capillary HPLC–mass spectrometry

Cited by 29 publications

References 11 publications

Improving CMC-derivatization of pseudouridine in RNA for mass spectrometric detection

Improving CMC-derivatization of pseudouridine in RNA for mass spectrometric detection

The identification and characterization of non-coding and coding RNAs and their modified nucleosides by mass spectrometry

Detection of RNA modifications

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