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.
The challenges posed by the analysis of mono-nucleotide mixtures by direct infusion electrospray ionization (ESI) were examined in the context of recent advances of MS technologies. In particular, we evaluated the merits of high-resolution mass analysis, multistep gas-phase dissociation, and ion mobility determinations for the characterization of species with very similar or identical elemental composition. The high resolving power afforded by a linear trap quadrupole (LTQ)-orbitrap allowed the complete differentiation of overlapping isotopic distributions produced by nucleotides that differed by a single mass unit. Resolving 12C signals from nearly overlapped 13C contributions provided the exact masses necessary to calculate matching elemental compositions for unambiguous formulae assignment. However, it was the ability to perform sequential steps of gas-phase dissociation (i.e., MSn-type analysis) that proved more valuable for discriminating between truly isobaric nucleotides, such as the AMP/dGMP and UMP/ψMP couples, which were differentiated in the mixture from their unique fragmentation patterns. The identification of diagnostic fragments enabled the deconvolution of dissociation spectra containing the products of coexisting isobars that could not be individually isolated in the mass-selection step. Approaches based on ion mobility spectrometry (IMS)-MS provided another dimension upon which isobaric nucleotides could be differentiated according to their distinctive mobility behaviors. Subtle structural variations, such as the different positions of an oxygen atom in AMP/dGMP or the glycosidic bond in UMP/ψMP, produced detectable differences in the respective ion mobility profiles, which enabled the differentiation of the isobaric couples in the mixture. Parallel activation of all ions emerging from the ion mobility element provided an additional dimension for differentiating these analytes on the basis of both mobility and fragmentation properties.
Oxygen isotope ratios of quartz inclusions (QI) within garnet from granulite and amphibolite facies gneisses in the Adirondack Mountains, NY were analysed and used to determine metamorphic temperatures. Primary QI for eight of 12 samples have δ18O values significantly lower than matrix quartz (MQ). The primary QI retain δ18O values representative of thermal conditions during garnet crystallization, whereas the δ18O values of MQ were raised by diffusive exchange with other matrix minerals (e.g. mica and feldspar) during cooling. The δ18O differences between QI and MQ show that garnet (a mineral with slow diffusion of oxygen) can armour QI from isotopic exchange with surrounding matrix, even during slow cooling. These differences between δ18O in MQ and QI can further be used to test cooling rates by Fast Grain Boundary diffusion modelling. Criteria for identifying QI that preserve primary compositions and are suitable for thermometry were developed based on comparative tests. Relations between δ18O and inclusion size, distance of inclusion to host–garnet rim, core–rim zonation of individual inclusions, and presence or absence of petrological features (healed cracks in QI, inclusions in contact with garnet cracks lined by secondary minerals, and secondary minerals along the inclusion grain boundary) were investigated. In this study, 61% of QI preserve primary δ18O and 39% were associated with features that were linked to reset δ18O values. If δ18O in garnet is homogeneous and inclusions are removed, laser‐fluorination δ18O values of bulk garnet are more precise, more accurate, and best for thermometry. Intragrain δ18O(Grt) profiles measured in situ by ion microprobe show no δ18O zonation. Almandine–rich garnet (Alm60–75) from each sample was measured by laser‐fluorination mass‐spectrometry (LF‐MS) for δ18O and compared with ion microprobe measurements of δ18O in QI for thermometry. The Δ18O(Qz–Grt) values for Adirondack samples range from 2.66 to 3.24‰, corresponding to temperatures of 640–740 °C (A[Qz–Alm] = 2.71). Out of 12 samples that were used for thermometry, nine are consistent with previous estimates of peak temperature (625–800 °C) based on petrological and carbon–isotope thermometry for regional granulite and upper amphibolite facies metamorphism. The three samples that disagree with independent thermometry for peak metamorphism are from the anorthosite–mangerite–charnockite–granite suite in the central Adirondacks and yield temperatures of 640–665 °C, ~100 °C lower than previous estimates. These low temperatures could be interpreted as thermal conditions during late (post‐peak) crystallization of garnet on the retrograde path.
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