Mass spectrometric techniques in nucleic acid research

Crain, Pamela F.

doi:10.1002/mas.1280090504

Cited by 118 publications

(67 citation statements)

References 177 publications

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“…It has been previously reported that the nucleosidederived protonated nucleobase (i.e., the [B ϩ H] ϩ ion), gives rise to indistinguishable tandem mass spectrum from the [M ϩ H] ϩ ion formed from the corresponding nucleobase [18,33]. Therefore, the fragmentation results discussed here, which were obtained for the protonated nucleobase ions from the fragmentation of the corresponding 2=-deoxynucleosides, can generally be extended to nucleobases from either DNA or RNA.…”

Section: Discussionmentioning

confidence: 57%

Collisionally activated dissociation of protonated 2′-deoxycytidine, 2′-deoxyuridine, and their oxidatively damaged derivatives

Cao

Wang

2006

J. Am. Soc. Mass Spectrom.

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We examined the collisionally activated dissociation (CAD) pathways of protonated 2=-deoxycytidine (dC), 5-formyl-2=-deoxycytidine (5-FmdC), 5-hydroxy-2=-deoxycytidine (5-OHdC), 5-hydroxymethyl-2=-deoxycytidine (5-HmdC), and their corresponding stable isotope-labeled compounds to gain insights into the effects of modifications on the fragmentation pathways of the pyrimidine bases. Multi-stage MS (MS n ) results showed that protonated cytosine, its 5-hydroxyland 5-hydroxymethyl-substituted derivatives, but not its 5-formyl-substituted analog, could undergo Dimroth-like rearrangement in the gas-phase. The elimination of HNCO was one of the major fragmentation pathways observed for the protonated ions of all dC derivatives except for 5-hydroxymethylcytosine, which underwent this loss only after a H 2 O molecule had been eliminated. In addition, the protonated cytosine and 5-hydroxycytosine can undergo a facile elimination of NH 3 molecule. This loss, however, was not observed for protonated 5-hydroxymethylcytosine, 5-formylcytosine, and their uracil analogs. Taken together, our study demonstrated that modifications could alter markedly the CAD patterns of the protonated pyrimidine bases. The results from this study provided a basis for the identifications of other modified pyrimidine bases/nucleosides by tandem mass spectrometry. (J Am Soc Mass Spectrom 2006, 17, 1335-1341

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Section: Discussionmentioning

confidence: 57%

Collisionally activated dissociation of protonated 2′-deoxycytidine, 2′-deoxyuridine, and their oxidatively damaged derivatives

Cao

Wang

2006

J. Am. Soc. Mass Spectrom.

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“…However, regardless of the accuracy afforded by the available instrumentation, positive identification cannot be based solely on matching mass values, but must receive further corroboration by gas-phase fragmentation data consistent with the putative analyte structure. The facile cleavage of the N-glycosidic bond represents a characteristic dissociation channel that is diagnostic of nucleotide structures (Biemann and McCloskey 1962;Crain 1990a) and can be frequently used to discriminate between isomeric forms present simultaneously in a sample (Quinn et al 2013). Cleavage products can immediately reveal whether the modifying group may be situated on the phosphoribose or nucleobase moiety of the PTM structure.…”

Section: Assignment Confirmationmentioning

confidence: 99%

“…Mass spectrometry (MS)-based approaches have historically played a determinant role in the discovery and characterization of RNA modifications (McCloskey 1979(McCloskey , 1985Crain 1990a;Nordhoff et al 1996). This platform affords the ability to recognize the characteristic mass signatures associated with the different variants, as well as the unique fragmentation patterns necessary to confirm their structures (Banoub and Limbach 2010 and reference therein).…”

Section: Introductionmentioning

confidence: 99%

Profiling ribonucleotide modifications at full-transcriptome level: a step toward MS-based epitranscriptomics

Rose

Quinn

Sayre

et al. 2015

RNA

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The elucidation of the biological significance of RNA post-transcriptional modifications is hampered by the dearth of effective high-throughput sequencing approaches for detecting, locating, and tracking their levels as a function of predetermined experimental factors. With the goal of confronting this knowledge gap, we devised a strategy for completing global surveys of all ribonucleotide modifications in a cell, which is based on the analysis of whole cell extracts by direct infusion electrospray ionization mass spectrometry (ESI-MS). Our approach eschews chromatographic separation to promote instead the direct application of MS techniques capable of providing detection, differentiation, and quantification of post-transcriptional modifications (PTMs) in complex ribonucleotide mixtures. Accurate mass analysis was used to carry out database-aided identification of PTMs, whereas multistep tandem mass spectrometry (MS n ) and consecutive reaction monitoring (CRM) provided the necessary structural corroboration. We demonstrated that heat-map plots afforded by ion mobility spectrometry mass spectrometry (IMS-MS) can provide comprehensive modification profiles that are unique for different cell types and metabolic states. We showed that isolated tRNA samples can be used as controlled sources of PTMs in standard-additions quantification. Intrinsic internal standards enable direct comparisons of heat-maps obtained under different experimental conditions, thus offering the opportunity to evaluate the global effects of such conditions on the expression levels of all PTMs simultaneously. This type of comparative analysis will be expected to support the investigation of the system biology of RNA modifications, which will be aimed at exploring mutual correlations of their expression levels and providing new valuable insights into their biological significance.

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“…Release factors RF1 and RF2 were purified from wild-type strains (Tate & Caskey, 1990) or from release factor overexpressing strains (Adamski et al+, 1994)+ RF3 was purified as described (Grentzmann et al+, 1994) by HPLC on DEAE (Waters)+ Contamination by elongation factors was efficiently eliminated by a supplementary step on CM-Sephadex (Pharmacia) Grentzmann et al+, 1994)+ Release factor fractions were checked for GTP contamination using electrospray mass spectrometry (Crain, 1990;Straub & Voyksner, 1993)+ Concentrations of purified release factor fractions in terms of amount of protein per unit of volume was not constant compared to the number of molecules of active protein per unit of volume+ For example, overexpression of release factors has been shown to result in loss of specific activity (Adamski et al+, 1994) and a possible site for posttranslational modification for RF2 has been reported (Uno et al+, 1996)+ We therefore estimated the quantities of active release factors in units of activity as described previously (Caskey et al+, 1971;Grentzmann et al+, 1994)+ tRNAfmet (Subriden) was charged and purified according to Tate and Caskey (1990)+ Tight couple ribosomes were purified as described (Spedding, 1990)+ UUC AUG UAA, UUC AUG UAG, and UUC AUG UGA RNA oligonucleotides were synthesized on an ABI synthesizer, deprotected, and purified on Sephadex G-25 (Pharmacia)+ Sequences were verified by mass spectroscopy (Limbach et al+, 1995)+ RRF was purified from an overexpressing strain (Ichikawa & Kaji, 1989) as described (Hirashima & Kaji, 1972)+ EF-G was purified and its activity was tested as described (Kaziro et al+, 1972)+…”

Section: Purified Elements For In Vitro Reactionsmentioning

confidence: 99%

Release factor RF-3 GTPase activity acts in disassembly of the ribosome termination complex

Grentzmann

Kelly

Laalami

et al. 1998

RNA

188

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RF3 was initially characterized as a factor that stimulates translational termination in an in vitro assay. The factor has a GTP binding site and shows sequence similarity to elongation factors EF-Tu and EF-G. Paradoxically, addition of GTP abolishes RF3 stimulation in the classical termination assay, using stop triplets.We here show GTP hydrolysis, which is only dependent on the simultaneous presence of RF3 and ribosomes. Applying a new termination assay, which uses a minimessenger RNA instead of separate triplets, we show that GTP in the presence of RF3 stimulates termination at rate-limiting concentrations of RF1. We show that RF3 can substitute for EF-G in RRF-dependent ribosome recycling reactions in vitro. This activity is GTP-dependent. In addition, excess RF3 and RRF in the presence of GTP caused release of nonhydrolyzed fmet-tRNA. This supports previous genetic experiments, showing that RF3 might be involved in ribosomal drop off of peptidyl-tRNA. In contrast to GTP involvement of the above reactions, stimulation of termination with RF2 by RF3 was independent of the presence of GTP. This is consistent with previous studies, indicating that RF3 enhances the affinity of RF2 for the termination complex without GTP hydrolysis. Based on our results, we propose a model of how RF3 might function in translational termination and ribosome recycling.

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Mass spectrometric techniques in nucleic acid research

Cited by 118 publications

References 177 publications

Collisionally activated dissociation of protonated 2′-deoxycytidine, 2′-deoxyuridine, and their oxidatively damaged derivatives

Collisionally activated dissociation of protonated 2′-deoxycytidine, 2′-deoxyuridine, and their oxidatively damaged derivatives

Profiling ribonucleotide modifications at full-transcriptome level: a step toward MS-based epitranscriptomics

Release factor RF-3 GTPase activity acts in disassembly of the ribosome termination complex

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