Analysis of Nucleosides and Nucleotides in Milk and Infant Formula

Nucleotides have been routinely supplemented to infant formulas due to the important roles they play in metabolism and to replicate the higher concentrations typically found in human milk. A method utilising anion exchange solid-phase extraction clean-up and liquid chromatography was developed for the rapid, routine determination of supplemented cytidine 5′ monophosphate, uridine 5′ monophosphate, inosine 5′ monophosphate, guanosine 5′ monophosphate, and adenosine 5′ monophosphate in bovine milk-based infant formula. Chromatographic analyses were performed using a C18 stationary phase with gradient elution, UV detection, and quantitation by an internal standard technique. A single-laboratory validation was performed, with recoveries of 92–101% and repeatability of 1.0–2.3%. An extension study demonstrated the expansion in scope to a wider range of different infant formula products including milk protein and hydrolysate-based products, low and high fat products, soy protein-based and elemental products, adult nutritional and infant formulations, in both ready-to-feed and powder forms. The development of a method to measure the total potentially available nucleosides (TPAN) in human milk has made an important contribution to further understanding the distribution of nucleosides and nucleotides. This method was applied in a lactation study of bovine milk with colostrum and milk samples collected from two herds over the course of the first month post-partum, pooled within each herd by stage of lactation and the TPAN concentrations were determined. Sample analysis consisted of parallel enzymatic treatments, phenylboronate affinity gel extraction, and liquid chromatography to quantify contributions of nucleosides, monomeric nucleotides, nucleotide adducts, and polymeric nucleotides to the nutritionally available nucleoside pool. Bovine colostrum contained high levels of nucleosides and monomeric nucleotides, which rapidly decreased as lactation progressed into transitional milk. Mature milk was relatively consistent in nucleoside and monomeric nucleotide concentrations from approximately the tenth day post-partum. Differences in concentrations between summer-milk and winter-milk herds were largely attributable to variability in uridine and monomeric nucleotide concentrations. The TPAN method was subsequently applied to the analysis of mature bovine, caprine, and ovine milk. The contributions to TPAN from polymeric nucleotides, monomeric nucleotides, and nucleotide adducts were then calculated. Ovine milk contained the highest concentration of TPAN (374.1 µmol dL-1), with lower concentrations in caprine milk (97.4 µmol dL-1) and bovine milk (7.9 µmol dL-1). Ovine milk contained the highest concentrations of each of the different nucleoside and nucleotide forms, and bovine milk contained the lowest. A method for the simultaneous analysis of nucleosides and nucleotides in infant formula using reversed-phase liquid chromatography-tandem mass spectrometry was developed. Following sample dissolution, protein was removed by centrifugal ultrafiltration. Chromatographic analyses were performed using a C18 stationary phase and gradient elution, with mass spectrometric detection, and quantitation by stable isotope labelled internal standard technique. A single laboratory validation study was performed with recoveries of 80.1–112.9% and repeatability relative standard deviations of 1.9–7.2%. The method was validated for the analysis of bovine milk-based, soy-based, caprine milk-based and hydrolysate-based infant formula

Metabolic signature of breast cancer cell line MCF-7: profiling of modified nucleosides via LC-IT MS coupling

<p>Abstract</p> <p>Background</p> <p>Cancer, like other diseases accompanied by strong metabolic disorders, shows characteristic effects on cell turnover rate, activity of modifying enzymes and DNA/RNA modifications, resulting also in elevated amounts of excreted modified nucleosides. For a better understanding of the impaired RNA metabolism in breast cancer cells, we screened these metabolites in the cell culture supernatants of the breast cancer cell line MCF-7 and compared it to the human mammary epithelial cells MCF-10A. The nucleosides were isolated and analyzed via 2D-chromatographic techniques: In the first dimension by cis-diol specific boronate affinity extraction and subsequently by reversed phase chromatography coupled to an ion trap mass spectrometer.</p> <p>Results</p> <p>Besides the determination of ribonucleosides, additional compounds with cis-diol structure, deriving from cross-linked biochemical pathways, like purine-, histidine- and polyamine metabolism were detected. In total, 36 metabolites were identified by comparison of fragmentation patterns and retention time. Relation to the internal standard isoguanosine yielded normalized area ratios for each identified compound and enabled a semi-quantitative metabolic signature of both analyzed cell lines.</p> <p>13 of the identified 26 modified ribonucleosides were elevated in the cell culture supernatants of MCF-7 cells, with 5-methyluridine, <it>N</it>²,<it>N</it>²,7-trimethylguanosine, <it>N</it>⁶-methyl-<it>N</it>⁶-threonylcarbamoyladenosine and 3-(3-aminocarboxypropyl)-uridine showing the most significant differences. 1-ribosylimidazole-4-acetic acid, a histamine metabolite, was solely found in the supernatants of MCF-10A cells, whereas 1-ribosyl-4-carboxamido-5-aminoimidazole and S-adenosylmethionine occurred only in supernatants of MCF-7 cells.</p> <p>Conclusion</p> <p>The obtained results are discussed against the background of pathological changes in cell metabolism, resulting in new perspectives for modified nucleosides and related metabolites as possible biomedical markers for breast carcinoma <it>in vivo</it>.</p

Solid‐Phase Synthesis of Branched Oligonucleotides

Branched nucleic acids (bNAs) have been of particular interest since the discovery of RNA forks and lariats as intermediates of nuclear mRNA splicing, as well as multicopy, single‐stranded DNA (msDNA). Such molecules contain the inherent trait of vicinal 2′,5′‐ and 3′,5′‐phosphodiester linkages. bNAs have many potential applications in nucleic acid biochemistry, particularly as tools for studying the substrate specificity of lariat debranching enzymes, and as biological probes for the investigation of branch recognition during pre‐mRNA splicing. The protocols described herein allow for the facile solid‐phase synthesis of branched DNA and/or RNA oligonucleotides of varying chain length, containing symmetrical or asymmetrical sequences immediate to an RNA branch point. The synthetic methodology utilizes widely adopted phosphoramidite chemistry. Methods for efficient purification of bNAs via anion‐exchange HPLC and PAGE are also illustrated.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/143634/1/cpnc0414.pd

Absolute quantification of exogenous stimuli-induced nucleic acid modification dynamics with LC-MS

Modifications of nucleic acids comply different functions and are involved in genome or-ganization, cell differentiation, silencing, structure stability and enzyme recognition. Modi-fication abundances can be regulated intrinsically, like the incorporation of cap modifica-tions on viral RNA to evade the host immune response, but also extrinsically as a cause of damage, which can result in mutations or translational defects. Either way, modifications are highly dynamic. It is of great importance to trace and quantify these changes in order to understand the underlying mechanisms, which may offer a more divers applica-bility of RNA therapeutics and even facilitate the establishment of personalized medicine. Mass Spectrometry is a common technique to examine nucleic acids. However, mass spectrometry per se offers solely a static insight into the versatile dynamics of nucleic acid modifications. In order to circumvent this obstacle, Nucleic Acid Isotope Labeling coupled Mass Spectrometry (NAIL-MS) was developed. This powerful technique allows for absolute quantification on the one hand and on the other hand for examination of modification dynamics originating from endogenous or exogenous actuators. In 2010, the stress-induced reprogramming of tRNA modification in S. cerevisiae was reported. However, the underlying mechanisms remained to be elucidated. Few years later, the dynamics of RNA modifications and mechanisms like dilution, degradation and (de-)modification could be identified by the application of NAIL-MS. The first part of my dissertation deals with the examination of the extent of damage-induced alterations on nucleic acids. Therefore, a novel biosynthetically produced stable isotope labeled internal standard (SILIS) was established, to avoid the interference of signals with isotopologues generated in the stable isotope labeled pulse-chase experiments. Furthermore, the L-methionine-[2H3]-methyl labeling in S. cerevisiae was optimized to achieve full efficient labeling and thus again avoiding signal interferences with isotopologues due to inefficient labeling. Additionally, the tandem size exclusion chromatography was developed, allowing the time efficient purification of 28S/25S, 18S rRNA and tRNA in a single step. The appli-cation of improved stable isotope labeling and the facilitated purification of RNA popula-tions allowed for the examination of the stress-induced alterations in the RNA modifica-tion profile of S. cerevisiae. Thereby, the knowledge on stress-induced reprogramming of tRNA modifications in yeast could be expanded. Original and new transcripts could be discerned and in addition endogenous methylation could be differentiated from damage induced methylation. It was shown, that stress-induced alterations occur on original tRNA transcripts, whereas new transcripts were not affected. Moreover, the fast decrease of damage-induced methylations on 25S, 18S rRNA and tRNA in S. cerevisiae was demon-strated. Additionally, the formation of base damage on 2’-O-methylated nucleosides in rRNA upon methyl methanesulfonate (MMS) exposure were detected and thereby novel damage products of MMS could be identified. Furthermore, the application of NAIL-MS was expanded to study the endogenous and damage-induced methylome on the genomic levels in S. cerevisiae and E. coli. In parallel to the aforementioned findings, the fast dis-appearance of damage-induced methylations in the genome and transcriptome of S. cerevisiae and E. coli was shown. Apart from that, m7dG and m7G could be identified as the main damage products in the genome and transcriptome of both organisms. In parallel to prokaryotes and eukaryotes, the modifications in viral RNA are highly dy-namic. RNA viruses have high mutation rates and their modification abundances can vary during infection. So, several mutants and variants of the RNA virus SARS-CoV-2 emerged since 2019. It is necessary to understand the characteristics of the viral genome and the differences in mutants and variants in order to identify novel drug targets and optimize the application of available therapeutics and vaccines. Our previous work on absolute quantification of nucleic acid modifications in various organisms showed the strength of our LC-MS based approach. In the course of this study it was aimed at inves-tigating the viral RNA modification profile in the different mutants and variants of SARS-CoV-2. The absolute quantification of RNA modifications and the comparison to pub-lished reports lead to the assumption that observed modification densities are highly de-pendent on the cultivation and infection conditions as well as the purification method and verification of sample integrity is crucial for valid analysis. As outlined above, less is known about the genome of SARS-CoV-2 in terms of internal modifications. While the cap modification of the 5’ end of the SARS-CoV-2 genome is confirmed from many sides and is ascribed to regulate the host innate immune response and the viral replication. Hence, a better understanding of the viral capping mechanism is required in order to limit its contagiousness. Besides the interest in biological capping processes, the investigations on cap modifications become more relevant nowadays be-cause of mRNA therapeutics. The cap modification on engineered mRNA is necessary to prevent immunogenicity, improve intercellular stability and translation efficiency. Thus, therapeutic mRNA is engineered to resemble mature and processed eukaryotic mRNA, including the 5’ cap and the 3’ poly A tail. Currently, there are only a few published LC-MS methods for detection of cap modifications. Nevertheless, these methods include labor intensive sample preparation, long analyses times and have moderate sensitivity. In the course of my dissertation, the development and optimization of a time efficient and highly sensitive LC-MS method for absolute quantification of cap modifications is pre-sented. It includes an extensive method development, optimizing chromatographic and mass spectrometric parameters under consideration of short analysis time, low detection and quantification limits. For absolute quantification of cap modifications, an in vitro tran-scribed cap-SILIS was generated. Furthermore, limits of detection and quantification as well as the dynamic range for size and amount of macromolecules to be analyzed were determined. The high sensitivity allows for the analysis of RNA from synthetic but also from biological sources. The time efficiency is aspirational for ecologic and economic rea-sons, thus making this method suitable for high throughput analyses and industry. The identification and quantification of RNA modifications is getting more important with the significance of RNA therapeutics. In this work, efficient LC-MS based tools to study the extent of nucleic acid modifications are described. Insight into the stress-dependent regulation of the genome and transcriptome of common model organisms is given and a powerful method to quantify cap modifications is presented. These techniques can be used to study nucleic acid dynamics in clinical studies but also for quality control of RNA therapeutics

Mass Spectrometry for the Sensitive Analysis of Intracellular Nucleotides and Analogues

Nowadays, mass spectrometry is very important and widely applied tool in nucleotide analysis. As a result of technological advances in sample preparation, separation and mass spectrometry detection, the developed methods allow sensitive and selective measurement of polar compounds occurring in low levels in various biological matrices. This enables more potential uses in clinical field. Direct methods require no special sample pre‐treatment before analysis in contrast to indirect methods, where fractionation, dephosphorylation and purification are needed. The use of ion‐pairing agent, ion exchange chromatography with pH gradient, porous graphitic carbon columns and HILIC in liquid chromatography represents the most common methods of nucleotide analysis. High separation efficiency is also achieved with the use of CE with MS detection. Analysis of nucleotides was also described by the means of MALDI‐TOF, but poor reproducibility and lack of applications make a limitation for this approach. The chapter summarizes different techniques and approaches for determination of endogenous nucleotides and its analogues in various clinical applications

Production and application of stable isotope-labeled internal standards for RNA modification analysis

Post-transcriptional RNA modifications have been found to be present in a wide variety of organisms and in different types of RNA. Nucleoside modifications are interesting due to their already known roles in translation fidelity, enzyme recognition, disease progression, and RNA stability. In addition, the abundance of modified nucleosides fluctuates based on growth phase, external stress, or possibly other factors not yet explored. With modifications ever changing, a method to determine absolute quantities for multiple nucleoside modifications is required. Here, we report metabolic isotope labeling to produce isotopically labeled internal standards in bacteria and yeast. These can be used for the quantification of 26 different modified nucleosides. We explain in detail how these internal standards are produced and show their mass spectrometric characterization. We apply our internal standards and quantify the modification content of transfer RNA (tRNA) from bacteria and various eukaryotes. We can show that the origin of the internal standard has no impact on the quantification result. Furthermore, we use our internal standard for the quantification of modified nucleosides in mouse tissue messenger RNA (mRNA), where we find different modification profiles in liver and brain tissue

Hydrophilic interaction chromatography – mass spectrometry for metabolomics and proteomics:state-of-the-art and current trends

Among all the –omics approaches, proteomics and metabolomics have received increased attention over the last decade. Both approaches have reached a certain level of maturity, showing their relevance in numerous clinical applications, including biomarkers discovery, improved diagnosis, staging, and prognosis of diseases, as well as a better knowledge on various (patho-)physiological processes. Analytically, reversed-phase liquid chromatography – mass spectrometry (RPLC-MS) is considered the golden standard in proteomics and metabolomics, due to its ease of use and reproducilibity. However, RPLC-MS alone is not sufficient to resolve the complexity of the proteome, while very polar metabolites are typically poorly retained. In this context, hydrophilic interaction chromatography (HILIC) represents an attractive complementary approach, due to its orthogonal separation mechanism. This review presents an overview of the literature reporting the application of HILIC-MS in metabolomics and proteomics. For metabolomics the focus is on the analysis of bioactive lipids, amino acids, organic acids, and nucleotides/nucleosides, whereas for proteomics the analysis of complex samples and protein post-translational modifications therein using bottom-up, middle up/down proteomics and intact protein analysis is discussed. The review handles the technological aspects related to the use of HILIC-MS in both proteomics and metabolomics, paying attention to stationary phases, mobile phase conditions, injection volume and column temperature. Recent trends and developments in the application of HILIC-MS in proteomics and metabolomics are also presented and discussed, highlighting the advantages the technique can provide in addition or complementary to RPLC-MS, as well as the current limitations and possible solutions

Mechanistic investigations of pseudouridine synthases : a surprising glycal intermediate lies on the reaction pathway.

Pseudouridine synthases (ΨSs) catalyze the isomerization of uridine (U) in RNA to pseudouridine (Ψ), which is the most common post-transcriptional modification in RNAs and is ubiquitous within all three domains of life. ΨSs are classified into six different families based on sequence alignments and have a universally conserved aspartic acid residue (‘conserved Asp’ for simplicity) that is absolutely essential for activity. RNA containing 5-fluorouridine ([F5U]RNA) has been used as a mechanistic probe. Upon incubation with [F5U]RNA, TruA and RluA get irreversibly inhibited and appear in a protein-RNA adduct band on denaturing PAGE gels, which is consistent with the ‘Michael mechanism’ that proposes a covalent adduct between the conserved Asp and the pyrimidine ring. E. coli TruB does not get inhibited upon incubation with [F5U]RNA, nor does it form an adduct and instead converts F5U into two rearranged, hydrated products. 18O labeling studies with the E. coli ΨSs TruA, TruB, RluA, and TruD show that the hydration of the products of F5U occurs directly from solution and not through the hydrolysis of a Michael adduct. These 18O labeling studies were extended here to Thermotoga maritima TruB (TmTruB) which showed a behavior intermediate between that of E. coli TruB (no observed adduct; multiple turnovers) and RluA and TruA (stoichiometric adduction; single turnover). 18O label is incorporated into RNA directly from solvent during the reaction of [F5U]RNA catalyzed by TmTruB. These results are consistent with the scheme proposed earlier for the handling of F5U by ΨSs. Among examined ΨSs, TmTruB uniquely shows the interesting ability to catalyze the dehydration and rehydration of the products of F5U. The minor product of F5U from the action of ΨSs is an arabino isomer and its generation requires epimerization at C2′, which suggested that the ΨS mechanism may proceed by deprotonation of C2′ to eliminate the uracil and form a glycal intermediate. To test this mechanistic possibility, substrate RNA stem-loops containing [2′-2H]uridine were prepared and used to measure the deuterium kinetic isotope effect on the conversion of U to Ψ. Deuteration at C2′ reduced both Vmax and Vmax/Km for the reaction as catalyzed by TruB (2.54-fold and 3.58-fold, respectively) and RluA (1.79-fold and 2.17-fold, respectively). These results conclusively indicate that the deprotonation of C2′ occurs in a partially rate-determining step in the conversion of U to Ψ, which is consistent with the ‘glycal mechanism’ but inconsistent with the proposed alternatives