Multiple Enzymatic Digestions and Ion Mobility Separation Improve Quantification of Bacterial Ribosomal Proteins by Data Independent Acquisition Liquid Chromatography−Mass Spectrometry

Mass spectrometry-based quantification of ribosomal proteins (r-proteins) associated with mature ribosomes and ribosome assembly complexes is typically accomplished by relative quantification strategies. These strategies provide information on the relative stoichiometry of proteins within the complex compared to a wild-type strain. Here we have evaluated the applicability of a label-free approach, enhanced liquid chromatography–mass spectrometry (LC–MS^E), for absolute “ribosome-centric” quantification of r-proteins in Escherichia coli mature ribosomes. Because the information obtained in this experiment is related to the number of peptides identified per protein, experimental conditions that allow accurate and reproducible quantification of r-proteins were found. Using an additional dimension of gas-phase separation through ion mobility and the use of multiple endoproteinase digestion significantly improved quantification of proteins associated with mature ribosomes. The actively translating ribosomes (polysomes) contain amounts of proteins consistent with their known stoichiometry within the complex. These measurements exhibited technical and biological reproducibilities at %CV less than 15% and 35%, respectively. The improved LC–MS^E approach described here can be used to characterize in vivo ribosome assembly complexes captured during ribosome biogenesis and assembly under different perturbations (e.g., antibiotics, deletion mutants of assembly factors, oxidative stress, nutrient deprivation). Quantitative analysis of these captured complexes will provide information relating to the interplay and dynamics of how these perturbations interfere with the assembly process

RNAModMapper: RNA Modification Mapping Software for Analysis of Liquid Chromatography Tandem Mass Spectrometry Data

Liquid chromatography tandem mass spectrometry (LC-MS/MS) has proven to be a powerful analytical tool for the characterization of modified ribonucleic acids (RNAs). The typical approach for analyzing modified nucleosides within RNA sequences by mass spectrometry involves ribonuclease digestion followed by LC-MS/MS analysis and data interpretation. Here we describe a new software tool, RNAModMapper (RAMM), to assist in the interpretation of LC-MS/MS data. RAMM is a stand-alone package that requires user-submitted DNA or RNA sequences to create a local database against which collision-induced dissociation (CID) data of modified oligonucleotides can be compared. RAMM can interpret MS/MS data containing modified nucleosides in two modes: fixed and variable. In addition, RAMM can also utilize interpreted MS/MS data for RNA modification mapping back against the input sequence(s). The applicability of RAMM was first tested using total tRNA isolated from Escherichia coli. It was then applied to map modifications found in 16S and 23S rRNA from Streptomyces griseus

Oxidized Residues Identified on the Stromally Exposed Regions of the D1 and D2 Proteins in the Vicinity of Q_A and Pheo_D1.

<p>The <i>T. vulcanus</i> residues corresponding to the oxidatively modified spinach residues (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058042#pone-0058042-t001" target="_blank">Table 1</a>) are highlighted. These oxidized residues are shown as spheres superimposed on monomer I of the <i>T. vulcanus</i> structure. For clarity, only the D1 and D2 proteins and their associated cofactors are shown. A. the view from outside Monomer I, looking towards the dimeric complex from within the plane of the membrane. B. the view from Monomer II looking towards its interface with Monomer I within the plane of the membrane. The D1 protein is shown in pale green and the D2 protein is shown in pale yellow. The oxidatively modified residues of D1 are shown in dark green while those of D2 are shown in orange. Various cofactors of both D1 and D2 are labeled and colored pale green or yellow, respectively. Pheo_D1 is shown in bright green. The non-heme iron is shown in bright red. The Mn₄O₅Ca cluster and its associated chloride ions are labeled as the OEC. Figs. 2–4 were produced using PYMOL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058042#pone.0058042-Software1" target="_blank">[53]</a>.</p

Oxidized Amino Acid Residues in the Vicinity of Q_A and Pheo_D1 of the Photosystem II Reaction Center: Putative Generation Sites of Reducing-Side Reactive Oxygen Species

<div><p>Under a variety of stress conditions, Photosystem II produces reactive oxygen species on both the reducing and oxidizing sides of the photosystem. A number of different sites including the Mn₄O₅Ca cluster, P₆₈₀, Pheo_D1, Q_A, Q_B and cytochrome b₅₅₉ have been hypothesized to produce reactive oxygen species in the photosystem. In this communication using Fourier-transform ion cyclotron resonance mass spectrometry we have identified several residues on the D1 and D2 proteins from spinach which are oxidatively modified and in close proximity to Q_A (D1 residues ²³⁹F, ²⁴¹Q, ²⁴²E and the D2 residues ²³⁸P, ²³⁹T, ²⁴²E and ²⁴⁷M) and Pheo_D1 (D1 residues ¹³⁰E, ¹³³L and ¹³⁵F). These residues may be associated with reactive oxygen species exit pathways located on the reducing side of the photosystem, and their modification may indicate that both Q_A and Pheo_D1 are sources of reactive oxygen species on the reducing side of Photosystem II.</p> </div

Identification of Oxidized Amino Acid Residues in the Vicinity of the Mn₄CaO₅ Cluster of Photosystem II: Implications for the Identification of Oxygen Channels within the Photosystem

As a light-driven water–plastoquinone oxidoreductase, Photosystem II produces molecular oxygen as an enzymatic product. Additionally, under a variety of stress conditions, reactive oxygen species are produced at or near the active site for oxygen evolution. In this study, Fourier-transform ion cyclotron resonance mass spectrometry was used to identify oxidized amino acid residues located in several core Photosystem II proteins (D1, D2, CP43, and CP47) isolated from spinach Photosystem II membranes. While the majority of these oxidized residues (81%) are located on the oxygenated solvent-exposed surface of the complex, several residues on the CP43 protein (³⁵⁴E, ³⁵⁵T, ³⁵⁶M, and ³⁵⁷R) which are in close proximity (<15 Å) to the Mn₄CaO₅ active site are also modified. These residues appear to be associated with putative oxygen/reactive oxygen species exit channel(s) in the photosystem. These results are discussed within the context of a number of computational studies which have identified putative oxygen channels within the photosystem

Detail of the Oxidized Residues in the Vicinity of Q_A.

<p>A close-up of the Q_A – Non-Heme Iron – Q_B region is shown. The <i>T. vulcanus</i> residues corresponding to the oxidatively modified spinach residues (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058042#pone-0058042-t001" target="_blank">Table 1</a>) are highlighted and labeled. The D1 protein is shown in pale green and the D2 protein is shown in pale yellow. The oxidatively modified residues of D1 are shown in dark green while those of D2 are shown in orange, with the individual modified residues being labeled. Q_A is shown in yellow, Q_B in green and the non-heme iron is shown in bright red.</p

The Effects of Ultraviolet Radiation on Nucleoside Modifications in RNA

Ultraviolet radiation (UVR) is a known genotoxic agent. Although its effects on DNA have been well-documented, its impact on RNA and RNA modifications is less studied. By using <i>Escherichia coli</i> tRNA (tRNA) as a model system, we identify the UVA (370 nm) susceptible chemical groups and bonds in a large variety of modified nucleosides. We use liquid chromatography tandem mass spectrometry to identify specific nucleoside photoproducts under <i>in vitro</i> and <i>in vivo</i> conditions, which were then verified by employing stable-isotope labeled tRNAs. These studies suggest that the -amino or -oxy groups of modified nucleosides, in addition to sulfur, are labile in the oxidative environment generated by UVA exposure. Further, these studies document a range of RNA photoproducts and post-transcriptional modifications that arise because of UVR-induced cellular stress

Example Mass Spectrometry Data from the Unmodified Peptide.

<p>²³⁵AFNPTQAEETYSMVTAN²⁵²R and the Oxidatively Modified Peptide ²³⁵AFNPTQAEETYS²⁴⁷M+16 VTAN²⁵²R of the D2 Protein A. Top, spectrum of the collision-induced dissociation (CID) of the unmodified peptide ²³⁵AFNPTQAEETYSMVTAN²⁵²R. Various identified ions are labeled. Bottom, table of all predicted masses for the y- and b- ions generated from this peptide sequence. Ions identified in the CID spectrum (above) are shown in red. The b'⁺⁺, b'⁺ y'⁺⁺ and y'⁺ ions are generated by the neutral loss of water while the b*⁺⁺, b*⁺ y*⁺⁺ and y*⁺ ions are generated from the loss of ammonia. B. Top, spectrum of the CID dissociation of the modified ²³⁵AFNPTQAEETYS²⁴⁷M+16 VTAN²⁵²R. Various identified ions are labeled. Bottom, table of all predicted masses for the y- and b- ions generated from this peptide sequence. Ions identified in the CID spectrum are shown in red. The b'⁺⁺, b'⁺ y'⁺⁺ and y'⁺ ions are generated by the neutral loss of water while the b*⁺⁺, b*⁺ y*⁺⁺ and y*⁺ ions are generated from the loss of ammonia. For comparison the b¹³⁺–b¹⁷⁺ ions of the unmodified peptide are highlighted in blue and those of the modified peptide are highlighted in cyan. All b ions longer than b¹²⁺ in the modified peptide are 16 Da larger than the corresponding ions observed from the unmodified peptide. This indicates that ²⁴⁷M contains an oxidative modification. Additionally, the y⁶⁺–y¹⁵⁺ions of the unmodified peptide are highlighted in green, while those of the modified peptide are highlighted in yellow. All y ions longer than y⁵⁺ in the modified peptide are 16 Da larger than the corresponding ions observed from the unmodified peptide. This verifies that ²⁴⁷M contains an oxidative modification. The p values for the unmodified and modified peptide were 10⁻¹³ and 10⁻¹¹, respectively.</p

Identification of a Novel Epoxyqueuosine Reductase Family by Comparative Genomics

The reduction of epoxyqueuosine (oQ) is the last step in the synthesis of the tRNA modification queuosine (Q). While the epoxyqueuosine reductase (EC 1.17.99.6) enzymatic activity was first described 30 years ago, the encoding gene <i>queG</i> was only identified in <i>Escherichia coli</i> in 2011. Interestingly, <i>queG</i> is absent from a large number of sequenced genomes that harbor Q synthesis or salvage genes, suggesting the existence of an alternative epoxyqueuosine reductase in these organisms. By analyzing phylogenetic distributions, physical gene clustering, and fusions, members of the Domain of Unknown Function 208 (DUF208) family were predicted to encode for an alternative epoxyqueuosine reductase. This prediction was validated with genetic methods. The Q modification is present in <i>Lactobacillus salivarius</i>, an organism missing <i>queG</i> but harboring the <i>duf208</i> gene. <i>Acinetobacter baylyi</i> ADP1 is one of the few organisms that harbor both QueG and DUF208, and deletion of both corresponding genes was required to observe the absence of Q and the accumulation of oQ in tRNA. Finally, the conversion oQ to Q was restored in an <i>E. coli queG</i> mutant by complementation with plasmids harboring <i>duf208</i> genes from different bacteria. Members of the DUF208 family are not homologous to QueG enzymes, and thus, <i>duf208</i> is a non-orthologous replacement of <i>queG</i>. We propose to name DUF208 encoding genes as <i>queH</i>. While QueH contains conserved cysteines that could be involved in the coordination of a Fe/S center in a similar fashion to what has been identified in QueG, no cobalamin was identified associated with recombinant QueH protein

Plant, Animal, and Fungal Micronutrient Queuosine Is Salvaged by Members of the DUF2419 Protein Family

Queuosine (Q) is a modification found at the wobble position of tRNAs with GUN anticodons. Although Q is present in most eukaryotes and bacteria, only bacteria can synthesize Q <i>de novo</i>. Eukaryotes acquire queuine (q), the free base of Q, from diet and/or microflora, making q an important but under-recognized micronutrient for plants, animals, and fungi. Eukaryotic type tRNA-guanine transglycosylases (eTGTs) are composed of a catalytic subunit (QTRT1) and a homologous accessory subunit (QTRTD1) forming a complex that catalyzes q insertion into target tRNAs. Phylogenetic analysis of eTGT subunits revealed a patchy distribution pattern in which gene losses occurred independently in different clades. Searches for genes co-distributing with eTGT family members identified DUF2419 as a potential Q salvage protein family. This prediction was experimentally validated in <i>Schizosaccharomyces pombe</i> by confirming that Q was present by analyzing tRNA^Asp with anticodon GUC purified from wild-type cells and by showing that Q was absent from strains carrying deletions in the QTRT1 or DUF2419 encoding genes. DUF2419 proteins occur in most Eukarya with a few possible cases of horizontal gene transfer to bacteria. The universality of the DUF2419 function was confirmed by complementing the <i>S. pombe</i> mutant with the <i>Zea mays</i> (maize), human, and <i>Sphaerobacter thermophilus</i> homologues. The enzymatic function of this family is yet to be determined, but structural similarity with DNA glycosidases suggests a ribonucleoside hydrolase activity