Mass Spectrometry-Based Metabolic Profiling of Urinary Metabolites of N'-Nitrosonornicotine (NNN) in the Rat.

The tobacco-specific nitrosamine N'-nitrosonornicotine (NNN) and its close analogue 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) are classified as "carcinogenic to humans" (Group 1) by the International Agency for Research on Cancer. The currently used biomarker to monitor NNN exposure is urinary total NNN (free NNN plus its N-glucuronide). However, total NNN does not provide information about the extent of metabolic activation of NNN as related to its carcinogenicity. Targeted analysis of the major metabolites of NNN in laboratory animals recently led to the identification of N'-nitrosonornicotine-1N-oxide (NNN-N-oxide), a unique metabolite detected in human urine that is specifically formed from NNN. To further investigate NNN urinary metabolites that hold promise as new biomarkers for monitoring NNN exposure, uptake, and/or metabolic activation, we conducted a comprehensive profiling of NNN metabolites in the urine of F344 rats treated with NNN or [pyridine-d4]NNN. Using our optimized high-resolution mass spectrometry (HRMS)-based isotope-labeling method, 46 putative metabolites were identified with robust MS evidence. Out of the 46 candidates, all known major NNN metabolites were identified and structurally confirmed by comparing them to their isotopically labeled standards. More importantly, putative metabolites considered to be exclusively formed from NNN were also identified. The two new representative metabolites─4-(methylthio)-4-(pyridin-3-yl)butanoic acid (23, MPBA) and N-acetyl-S-(5-(pyridin-3-yl)-1H-pyrrol-2-yl)-l-cysteine (24, Py-Pyrrole-Cys-NHAc) ─were identified by comparing them to synthetic standards that were fully characterized by nuclear magnetic resonance and HRMS. They are hypothesized to be formed by NNN α-hydroxylation pathways and thus represent the first potential biomarkers to specifically monitor the uptake plus metabolic activation of NNN in tobacco users.

Identification of Formaldehyde-Induced DNA-RNA Cross-Links in the A/J Mouse Lung Tumorigenesis Model.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent lung carcinogen present in tobacco products, and exposure to it is likely one of the factors contributing to the development of lung cancer in cigarette smokers. To exert its carcinogenic effects, NNK must be metabolically activated into highly reactive species generating a wide spectrum of DNA damage. We have identified a new class of DNA adducts, DNA-RNA cross-links found for the first time in NNK-treated mice lung DNA using our improved high-resolution accurate mass segmented full scan data-dependent neutral loss MS3 screening strategy. The levels of these DNA-RNA cross-links were found to be significantly higher in NNK-treated mice compared to the corresponding controls, which is consistent with higher levels of formaldehyde due to NNK metabolism as compared to endogenous levels. We hypothesize that this DNA-RNA cross-linking occurs through reaction with NNK-generated formaldehyde and speculate that this phenomenon has broad implications for NNK-induced carcinogenesis. The structures of these cross-links were characterized using high-resolution LC-MS2 and LC-MS3 accurate mass spectral analysis and comparison to a newly synthesized standard. Taken together, our data demonstrate a previously unknown link between DNA-RNA cross-link adducts and NNK and provide a unique opportunity to further investigate how these novel NNK-derived DNA-RNA cross-links contribute to carcinogenesis in the future.

Targeted High Resolution LC/MS3 Adductomics Method for the Characterization of Endogenous DNA Damage.

DNA can be damaged through covalent modifications of the nucleobases by endogenous processes. These modifications, commonly referred to as DNA adducts, can persist and may lead to mutations, and ultimately to the initiation of cancer. A screening methodology for the majority of known endogenous DNA adducts would be a powerful tool for investigating the etiology of cancer and for the identification of individuals at high-risk to the detrimental effects of DNA damage. This idea led to the development of a DNA adductomic approach using high resolution data-dependent scanning, an extensive MS2 fragmentation inclusion list of known endogenous adducts, and neutral loss MS3 triggering to profile all DNA modifications. In this method, the detection of endogenous DNA adducts is performed by observation of their corresponding MS3 neutral loss triggered events and their relative quantitation using the corresponding full scan extracted ion chromatograms. The method's inclusion list consists of the majority of known endogenous DNA adducts, compiled, and reported here, as well as adducts specific to tobacco exposure included to compare the performance of the method with previously developed targeted approaches. The sensitivity of the method was maximized by reduction of extraneous background signal through the purification and minimization of the amount of commercially obtained enzymes used for the DNA hydrolysis. In addition, post-hydrolysis sample purification was performed using off-line HPLC fraction collection to eliminate the highly abundant unmodified bases, and to avoid introduction of plasticizers found in solid-phase extraction cartridges. Also, several instrument parameters were evaluated to optimize the ion signal intensities and fragmentation spectra quality. The method was tested on an animal model of lung carcinogenesis where A/J mice were exposed to the tobacco specific lung carcinogen 4-methylnitrosamino-1-3-pyridyl-1-butanone (NNK) with its effects enhanced by co-exposure to the pro-inflammatory agent lipopolysaccharide (LPS). Lung DNA were screened for endogenous DNA adducts known to result from oxidative stress and LPS-induced lipid peroxidation, as well as for adducts due to NNK exposure. The relative quantitation of the detected DNA adducts was performed using parallel reaction monitoring MS2 analysis, demonstrating a general workflow for analysis of endogenous DNA adducts.

Bioanalytical and Mass Spectrometric Methods for Aldehyde Profiling in Biological Fluids.

Human exposure to aldehydes is implicated in multiple diseases including diabetes, cardiovascular diseases, neurodegenerative disorders (i.e., Alzheimer's and Parkinson's Diseases), and cancer. Because these compounds are strong electrophiles, they can react with nucleophilic sites in DNA and proteins to form reversible and irreversible modifications. These modifications, if not eliminated or repaired, can lead to alteration in cellular homeostasis, cell death and ultimately contribute to disease pathogenesis. This review provides an overview of the current knowledge of the methods and applications of aldehyde exposure measurements, with a particular focus on bioanalytical and mass spectrometric techniques, including recent advances in mass spectrometry (MS)-based profiling methods for identifying potential biomarkers of aldehyde exposure. We discuss the various derivatization reagents used to capture small polar aldehydes and methods to quantify these compounds in biological matrices. In addition, we present emerging mass spectrometry-based methods, which use high-resolution accurate mass (HR/AM) analysis for characterizing carbonyl compounds and their potential applications in molecular epidemiology studies. With the availability of diverse bioanalytical methods presented here including simple and rapid techniques allowing remote monitoring of aldehydes, real-time imaging of aldehydic load in cells, advances in MS instrumentation, high performance chromatographic separation, and improved bioinformatics tools, the data acquired enable increased sensitivity for identifying specific aldehydes and new biomarkers of aldehyde exposure. Finally, the combination of these techniques with exciting new methods for single cell analysis provides the potential for detection and profiling of aldehydes at a cellular level, opening up the opportunity to minutely dissect their roles and biological consequences in cellular metabolism and diseases pathogenesis.

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.

In vivo X-ray footprinting of pre-30S ribosomes reveals chaperone-dependent remodeling of late assembly intermediates.

Assembly of 30S ribosomal subunits from their protein and RNA components requires extensive refolding of the 16S rRNA and is assisted by 10-20 assembly factors in bacteria. We probed the structures of 30S assembly intermediates in E. coli cells, using a synchrotron X-ray beam to generate hydroxyl radical in the cytoplasm. Widespread differences between mature and pre-30S complexes in the absence of assembly factors RbfA and RimM revealed global reorganization of RNA-protein interactions prior to maturation of the 16S rRNA and showed how RimM reduces misfolding of the 16S 3' domain during transcription in vivo. Quantitative (14)N/(15)N mass spectrometry of affinity-purified pre-30S complexes confirmed the absence of tertiary assembly proteins and showed that N-terminal acetylation of proteins S18 and S5 correlates with correct folding of the platform and central pseudoknot. Our results indicate that cellular factors delay specific RNA folding steps to ensure the quality of assembly.