Nucleoside Analysis by Hydrophilic Interaction Liquid Chromatography Coupled with Mass Spectrometry

Sakaguchi, Yuriko; Miyauchi, Katsumi; Kang, Byeong-il; Suzuki, Tsutomu

doi:10.1016/bs.mie.2015.03.015

Cited by 64 publications

(58 citation statements)

References 30 publications

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“…LC/MS analyses of total nucleosides were performed essentially as described previously (9,33–34), using an LCQ Advantage ion-trap (IT) mass spectrometer (Thermo Fisher Scientific) equipped with an ESI source and an HP1100 liquid chromatography system (Agilent Technologies) or a Q Exactive hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific) equipped with an ESI source and an Ultimate 3000 liquid chromatography system (Dionex).…”

Section: Methodsmentioning

confidence: 99%

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Identification of 2-methylthio cyclic N6-threonylcarbamoyladenosine (ms2ct6A) as a novel RNA modification at position 37 of tRNAs

Kang

Miyauchi

Matuszewski

et al. 2016

Nucleic Acids Research

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Transfer RNA modifications play pivotal roles in protein synthesis. N6-threonylcarbamoyladenosine (t6A) and its derivatives are modifications found at position 37, 3΄-adjacent to the anticodon, in tRNAs responsible for ANN codons. These modifications are universally conserved in all domains of life. t6A and its derivatives have pleiotropic functions in protein synthesis including aminoacylation, decoding and translocation. We previously discovered a cyclic form of t6A (ct6A) as a chemically labile derivative of t6A in tRNAs from bacteria, fungi, plants and protists. Here, we report 2-methylthio cyclic t6A (ms2ct6A), a novel derivative of ct6A found in tRNAs from Bacillus subtilis, plants and Trypanosoma brucei. In B. subtilis and T. brucei, ms2ct6A disappeared and remained to be ms2t6A and ct6A by depletion of tcdA and mtaB homologs, respectively, demonstrating that TcdA and MtaB are responsible for biogenesis of ms2ct6A.

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Section: Methodsmentioning

confidence: 99%

“…For HILIC/ESI-MS, a ZIC-cHILIC column (3 μm particle size, 2.1 × 150 mm, Merck-Millipore) was used on a Q Exactive instrument (33). The mobile phase consisted of 5 mM ammonium acetate (pH 5.3) (solvent A) and ACN (solvent B).…”

Section: Methodsmentioning

confidence: 99%

Identification of 2-methylthio cyclic N6-threonylcarbamoyladenosine (ms2ct6A) as a novel RNA modification at position 37 of tRNAs

Kang

Miyauchi

Matuszewski

et al. 2016

Nucleic Acids Research

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“…While addition of 10 mM sodium or ammonium acetate into the aqueous buffer at a pH ~4.5 enhances the separation and resolution of individual nucleosides, the chromatography parameters must be tailored to the individual needs of each study, focusing on complete resolution of the analytes of interest. This concern has spurred the application of hydrophilic interaction liquid chromatography (HILIC), in order to increase retention and resolution of nucleosides carrying polar modifications [91,141,142]. Though LC with UV-Visible detection has been an invaluable tool for analysis of tRNA modifications in years past, it is limited by sensitivity and the requirement of commercially available standards (Table 3) to validate retention times.…”

Section: Methods For Investigation and Quantification Of Trna Modimentioning

confidence: 99%

Diverse Mechanisms of Sulfur Decoration in Bacterial tRNA and Their Cellular Functions

2017

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Sulfur-containing transfer ribonucleic acids (tRNAs) are ubiquitous biomolecules found in all organisms that possess a variety of functions. For decades, their roles in processes such as translation, structural stability, and cellular protection have been elucidated and appreciated. These thionucleosides are found in all types of bacteria; however, their biosynthetic pathways are distinct among different groups of bacteria. Considering that many of the thio-tRNA biosynthetic enzymes are absent in Gram-positive bacteria, recent studies have addressed how sulfur trafficking is regulated in these prokaryotic species. Interestingly, a novel proposal has been given for interplay among thionucleosides and the biosynthesis of other thiocofactors, through participation of shared-enzyme intermediates, the functions of which are impacted by the availability of substrate as well as metabolic demand of thiocofactors. This review describes the occurrence of thio-modifications in bacterial tRNA and current methods for detection of these modifications that have enabled studies on the biosynthesis and functions of S-containing tRNA across bacteria. It provides insight into potential modes of regulation and potential evolutionary events responsible for divergence in sulfur metabolism among prokaryotes.

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“…Total nucleosides were prepared by digesting E. coli total RNA with nuclease P1 (Wako Pure Chemical Industries) and bacterial alkaline phosphatase (BAP) ( E. coli strain C75, TAKARA BIO INC.) under acidic conditions (31). For these reactions, a solution (typically 40 μl) containing 1 μg/μl total RNA, 20 mM trimethylamine-acetate (pH 5.3), nuclease P1 (0.1 units for 40 μg of RNA), and BAP (0.16 units for 40 μg of RNA) was incubated at 37°C for 1 h.…”

Section: Methodsmentioning

confidence: 99%

A hydantoin isoform of cyclic N6-threonylcarbamoyladenosine (ct6A) is present in tRNAs

Matuszewski

Wojciechowski

Miyauchi

et al. 2016

Nucleic Acids Research

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N6-Threonylcarbamoyladenosine (t6A) and its derivatives are universally conserved modified nucleosides found at position 37, 3΄ adjacent to the anticodon in tRNAs responsible for ANN codons. These modifications have pleiotropic functions of tRNAs in decoding and protein synthesis. In certain species of bacteria, fungi, plants and protists, t6A is further modified to the cyclic t6A (ct6A) via dehydration catalyzed by TcdA. This additional modification is involved in efficient decoding of tRNALys. Previous work indicated that the chemical structure of ct6A is a cyclic active ester with an oxazolone ring. In this study, we solved the crystal structure of chemically synthesized ct6A nucleoside. Unexpectedly, we found that the ct6A adopted a hydantoin isoform rather than an oxazolone isoform, and further showed that the hydantoin isoform of ct6A was actually present in Escherichia coli tRNAs. In addition, we observed that hydantoin ct6A is susceptible to epimerization under mild alkaline conditions, warning us to avoid conventional deacylation of tRNAs. A hallmark structural feature of this isoform is the twisted arrangement of the hydantoin and adenine rings. Functional roles of ct6A37 in tRNAs should be reconsidered.

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Nucleoside Analysis by Hydrophilic Interaction Liquid Chromatography Coupled with Mass Spectrometry

Cited by 64 publications

References 30 publications

Identification of 2-methylthio cyclic N6-threonylcarbamoyladenosine (ms2ct6A) as a novel RNA modification at position 37 of tRNAs

Identification of 2-methylthio cyclic N6-threonylcarbamoyladenosine (ms2ct6A) as a novel RNA modification at position 37 of tRNAs

Diverse Mechanisms of Sulfur Decoration in Bacterial tRNA and Their Cellular Functions

A hydantoin isoform of cyclic N6-threonylcarbamoyladenosine (ct6A) is present in tRNAs

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