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

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

Diversity of Archaeosine Synthesis in Crenarchaeota

Archaeosine (G⁺) is found at position 15 of many archaeal tRNAs. In Euryarchaeota, the G⁺ precursor, 7-cyano-7-deazaguanine (preQ₀), is inserted into tRNA by tRNA-guanine transglycosylase (arcTGT) before conversion into G⁺ by ARChaeosine Synthase (ArcS). However, many Crenarchaeota known to harbor G⁺ lack ArcS homologues. Using comparative genomics approaches, two families that could functionally replace ArcS in these organisms were identified: (1) GAT-QueC, a two-domain family with an N-terminal glutamine amidotransferase class-II domain fused to a domain homologous to QueC, the enzyme that produces preQ₀ and (2) QueF-like, a family homologous to the bacterial enzyme catalyzing the reduction of preQ₀ to 7-aminomethyl-7-deazaguanine. Here we show that these two protein families are able to catalyze the formation of G⁺ in a heterologous system. Structure and sequence comparisons of crenarchaeal and euryarchaeal arcTGTs suggest the crenarchaeal enzymes have broader substrate specificity. These results led to a new model for the synthesis and salvage of G⁺ in Crenarchaeota