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.

Metabolomics Profiles of Smokers from Two Ethnic Groups with Differing Lung Cancer Risk.

African American (AA) smokers are at a higher risk of developing lung cancer compared to whites. The variations in the metabolism of nicotine and tobacco-derived carcinogens in these groups were reported previously with the levels of nicotine metabolites and carcinogen-derived metabolites measured using targeted approaches. While useful, these targeted strategies are not able to detect global metabolic changes for use in predicting the detrimental effects of tobacco use and ultimately lung cancer susceptibility among smokers. To address this limitation, we have performed global untargeted metabolomics profiling in urine of AA and white smokers to characterize the pattern of metabolites, identify differentially regulated pathways, and correlate these profiles with the observed variations in lung cancer risk between these two populations. Urine samples from AA (n = 30) and white (n = 30) smokers were used for metabolomics analysis acquired in both positive and negative electrospray ionization modes. LC-MS data were uploaded onto the cloud-based XCMS online (http://xcmsonline.scripps.edu) platform for retention time correction, alignment, feature detection, annotation, statistical analysis, data visualization, and automated systems biology pathway analysis. The latter identified global differences in the metabolic pathways in the two groups including the metabolism of carbohydrates, amino acids, nucleotides, fatty acids, and nicotine. Significant differences in the nicotine degradation pathway (cotinine glucuronidation) in the two groups were observed and confirmed using a targeted LC-MS/MS approach. These results are consistent with previous studies demonstrating AA smokers with lower glucuronidation capacity compared to whites. Furthermore, the d-glucuronate degradation pathway was found to be significantly different between the two populations, with lower amounts of the putative metabolites detected in AA compared to whites. We hypothesize that the differential regulation of the d-glucuronate degradation pathway is a consequence of the variations in the glucuronidation capacity observed in the two groups. Other pathways including the metabolism of amino acids, nucleic acids, and fatty acids were also identified, however, the biological relevance and implications of these differences across ethnic groups need further investigation. Overall, the applied metabolomics approach revealed global differences in the metabolic networks and endogenous metabolites in AA and whites, which could be used and validated as a new potential panel of biomarkers that could be used to predict lung cancer susceptibility among smokers in population-based studies.

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.

In Vivo Stable-Isotope Labeling and Mass-Spectrometry-Based Metabolic Profiling of a Potent Tobacco-Specific Carcinogen in Rats.

The tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is a potent lung carcinogen that exerts its carcinogenic effects upon metabolic activation. The identification and quantitation of NNK metabolites could identify potential biomarkers of bioactivation and detoxification of this potent carcinogen and may be used to predict lung cancer susceptibility among smokers. Here, we used in vivo isotope-labeling and high-resolution-mass-spectrometry-based methods for the comprehensive profiling of all known and unknown NNK metabolites. The sample-enrichment, LC-MS, and data-analysis workflow, including a custom script for automated d0- d4- m/ z-pair-peak detection, enabled unbiased identification of numerous NNK metabolites. The structures of the metabolites were confirmed using targeted LC-MS2 with retention-time ( tR) and MS2-fragmentation comparisons to those of standards when possible. Eleven known metabolites and unchanged NNK were identified simultaneously. More importantly, our workflow revealed novel NNK metabolites, including 1,3-Diol (13), α-OH-methyl-NNAL-Gluc (14), nitro-NK- N-oxide (15), nitro-NAL- N-oxide (16), γ-OH NNAL (17), and three N-acetylcysteine (NAC) metabolites (18a-c). We measured the differences in the relative distributions of a panel of nitroso-containing NNK-specific metabolites in rats before and after phenobarbital (PB) treatment, and this served as a demonstration of a general strategy for the detection of metabolic differences in animal and cell systems. Lastly, we generated a d4-labeled NNK-metabolite mixture to be used as internal standards ( d4-rat urine) for the relative quantitation of NNK metabolites in humans, and this new strategy will be used to assess carcinogen exposure and ultimately to evaluate lung-cancer risk and susceptibility in smokers.

An alpha-synuclein MRM assay with diagnostic potential for Parkinson's disease and monitoring disease progression.

AIM: The alpha-synuclein (α-syn) level in human cerebrospinal fluid (CSF), as measured by immunoassays, is promising as a Parkinson's disease (PD) biomarker. However, the levels of total α-syn are inconsistent among studies with large cohorts and different measurement platforms. Total α-syn level also does not correlate with disease severity or progression. Here, the authors developed a highly sensitive MRM method to measure absolute CSF α-syn peptide concentrations without prior enrichment or fractionation, aiming to discover new candidate biomarkers. RESULTS: Six peptides covering 73% of protein sequence were reliably identified, and two were consistently quantified in cross-sectional and longitudinal cohorts. Absolute concentration of α-syn in human CSF was determined to be 2.1 ng/mL. A unique α-syn peptide, TVEGAGSIAAATGFVK (81-96), displayed excellent correlation with previous immunoassay results in two independent PD cohorts (p < 0.001), correlated with disease severity, and its changes significantly tracked the disease progression longitudinally. CONCLUSIONS: An MRM assay to quantify human CSF α-syn was developed and optimized. Sixty clinical samples from cross-sectional and longitudinal PD cohorts were analyzed with this approach. Although further larger scale validation is needed, the results suggest that α-syn peptide could serve as a promising biomarker in PD diagnosis and progression.

A High Resolution/Accurate Mass (HRAM) Data-Dependent MS3 Neutral Loss Screening, Classification, and Relative Quantitation Methodology for Carbonyl Compounds in Saliva.

Reactive carbonyl compounds (RCCs) are ubiquitous in the environment and are generated endogenously as a result of various physiological and pathological processes. These compounds can react with biological molecules inducing deleterious processes believed to be at the basis of their toxic effects. Several of these compounds are implicated in neurotoxic processes, aging disorders, and cancer. Therefore, a method characterizing exposures to these chemicals will provide insights into how they may influence overall health and contribute to disease pathogenesis. Here, we have developed a high resolution accurate mass (HRAM) screening strategy allowing simultaneous identification and relative quantitation of DNPH-derivatized carbonyls in human biological fluids. The screening strategy involves the diagnostic neutral loss of hydroxyl radical triggering MS3 fragmentation, which is only observed in positive ionization mode of DNPH-derivatized carbonyls. Unique fragmentation pathways were used to develop a classification scheme for characterizing known and unanticipated/unknown carbonyl compounds present in saliva. Furthermore, a relative quantitation strategy was implemented to assess variations in the levels of carbonyl compounds before and after exposure using deuterated d 3 -DNPH. This relative quantitation method was tested on human samples before and after exposure to specific amounts of alcohol. The nano-electrospray ionization (nano-ESI) in positive mode afforded excellent sensitivity with detection limits on-column in the high-attomole levels. To the best of our knowledge, this is the first report of a method using HRAM neutral loss screening of carbonyl compounds. In addition, the method allows simultaneous characterization and relative quantitation of DNPH-derivatized compounds using nano-ESI in positive mode. Graphical Abstract ᅟ.

The Aryl Hydrocarbon Receptor is a Repressor of Inflammation-associated Colorectal Tumorigenesis in Mouse.

OBJECTIVE: To determine the role of the aryl hydrocarbon receptor (AHR) in colitis-associated colorectal tumorigenesis. BACKGROUND: Colorectal cancer (CRC) is the third most commonly diagnosed cancer in United States. Chronic intestinal inflammation increases the risk for the development of CRC. We investigated the involvement of AHR, a ligand-activated transcriptional regulator, in colitis-associated colorectal tumorigenesis. METHODS: We used a mouse model of colitis-associated colorectal tumorigenesis that employs treatment with azoxymethane and dextran sodium sulfate. We examined the role of AHR using both an Ahr-deletion mouse model (Ahr) and treatment with the AHR pro-agonist indole-3-carbinol (I3C). Incidence, multiplicity, and location of tumors were visually counted. Tumors were defined as neoplasms. Intestinal inflammation was assessed by quantitative PCR for proinflammatory markers and colon length. Data were evaluated and compared using GraphPad Prism software (version 6, La Jolla, CA). RESULTS: Tumor incidence was increased 32% in Ahr null mice and tumor multiplicity was approximately increased 3-fold compared with wild-type mice (2.4 vs 7; P < 0.05). Furthermore, tumor multiplicity was reduced 92% by treatment of I3C in wild-type mice, whereas the suppressor effect of I3C was not observed in Ahr null mice (P < 0.05). CONCLUSIONS: We found that AHR plays a protective role in colitis-associated colorectal tumorigenesis. This conclusion is based on the observations that Ahr null mice showed increased number of colorectal tumors, and mice treated with I3C exhibited fewer tumors. This study supports the use of AHR agonists such as I3C as a chemopreventive therapy for IBD-associated CRC in human patients.

Cerebrospinal fluid peptides as potential Parkinson disease biomarkers: a staged pipeline for discovery and validation.

Finding robust biomarkers for Parkinson disease (PD) is currently hampered by inherent technical limitations associated with imaging or antibody-based protein assays. To circumvent the challenges, we adapted a staged pipeline, starting from our previous proteomic profiling followed by high-throughput targeted mass spectrometry (MS), to identify peptides in human cerebrospinal fluid (CSF) for PD diagnosis and disease severity correlation. In this multicenter study consisting of training and validation sets, a total of 178 subjects were randomly selected from a retrospective cohort, matching age and sex between PD patients, healthy controls, and neurological controls with Alzheimer disease (AD). From ∼14,000 unique peptides displaying differences between PD and healthy control in proteomic investigations, 126 peptides were selected based on relevance and observability in CSF using bioinformatic analysis and MS screening, and then quantified by highly accurate and sensitive selected reaction monitoring (SRM) in the CSF of 30 PD patients versus 30 healthy controls (training set), followed by diagnostic (receiver operating characteristics) and disease severity correlation analyses. The most promising candidates were further tested in an independent cohort of 40 PD patients, 38 AD patients, and 40 healthy controls (validation set). A panel of five peptides (derived from SPP1, LRP1, CSF1R, EPHA4, and TIMP1) was identified to provide an area under curve (AUC) of 0.873 (sensitivity = 76.7%, specificity = 80.0%) for PD versus healthy controls in the training set. The performance was essentially confirmed in the validation set (AUC = 0.853, sensitivity = 82.5%, specificity = 82.5%). Additionally, this panel could also differentiate the PD and AD groups (AUC = 0.990, sensitivity = 95.0%, specificity = 97.4%). Furthermore, a combination of two peptides belonging to proteins TIMP1 and APLP1 significantly correlated with disease severity as determined by the Unified Parkinson's Disease Rating Scale motor scores in both the training (r = 0.381, p = 0.038)j and the validation (r = 0.339, p = 0.032) sets. The novel panel of CSF peptides, if validated in independent cohorts, could be used to assist in clinical diagnosis of PD and has the potential to help monitoring or predicting disease progression.

Proteomic profiling in MPTP monkey model for early Parkinson disease biomarker discovery.

Identification of reliable and robust biomarkers is crucial to enable early diagnosis of Parkinson disease (PD) and monitoring disease progression. While imperfect, the slow, chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced non-human primate animal model system of parkinsonism is an abundant source of pre-motor or early stage PD biomarker discovery. Here, we present a study of a MPTP rhesus monkey model of PD that utilizes complementary quantitative iTRAQ-based proteomic, glycoproteomics and phosphoproteomics approaches. We compared the glycoprotein, non-glycoprotein, and phosphoprotein profiles in the putamen of asymptomatic and symptomatic MPTP-treated monkeys as well as saline injected controls. We identified 86 glycoproteins, 163 non-glycoproteins, and 71 phosphoproteins differentially expressed in the MPTP-treated groups. Functional analysis of the data sets inferred the biological processes and pathways that link to neurodegeneration in PD and related disorders. Several potential biomarkers identified in this study have already been translated for their usefulness in PD diagnosis in human subjects and further validation investigations are currently under way. In addition to providing potential early PD biomarkers, this comprehensive quantitative proteomic study may also shed insights regarding the mechanisms underlying early PD development. This article is part of a Special Issue entitled: Neuroproteomics: Applications in neuroscience and neurology.

Plasma exosomal α-synuclein is likely CNS-derived and increased in Parkinson's disease.

Extracellular α-synuclein is important in the pathogenesis of Parkinson's disease (PD) and also as a potential biomarker when tested in the cerebrospinal fluid (CSF). The performance of blood plasma or serum α-synuclein as a biomarker has been found to be inconsistent and generally ineffective, largely due to the contribution of peripherally derived α-synuclein. In this study, we discovered, via an intracerebroventricular injection of radiolabeled α-synuclein into mouse brain, that CSF α-synuclein was readily transported to blood, with a small portion being contained in exosomes that are relatively specific to the central nervous system (CNS). Consequently, we developed a technique to evaluate the levels of α-synuclein in these exosomes in individual plasma samples. When applied to a large cohort of clinical samples (267 PD, 215 controls), we found that in contrast to CSF α-synuclein concentrations, which are consistently reported to be lower in PD patients compared to controls, the levels of plasma exosomal α-synuclein were substantially higher in PD patients, suggesting an increased efflux of the protein to the peripheral blood of these patients. Furthermore, although no association was observed between plasma exosomal and CSF α-synuclein, a significant correlation between plasma exosomal α-synuclein and disease severity (r = 0.176, p = 0.004) was observed, and the diagnostic sensitivity and specificity achieved by plasma exosomal α-synuclein were comparable to those determined by CSF α-synuclein. Further studies are clearly needed to elucidate the mechanism involved in the transport of CNS α-synuclein to the periphery, which may lead to a more convenient and robust assessment of PD clinically.

Targeted discovery and validation of plasma biomarkers of Parkinson's disease.

Despite extensive research, an unmet need remains for protein biomarkers of Parkinson's disease (PD) in peripheral body fluids, especially blood, which is easily accessible clinically. The discovery of such biomarkers is challenging, however, due to the enormous complexity and huge dynamic range of human blood proteins, which are derived from nearly all organ systems, with those originating specifically from the central nervous system (CNS) being exceptionally low in abundance. In this investigation of a relatively large cohort (∼300 subjects), selected reaction monitoring (SRM) assays (a targeted approach) were used to probe plasma peptides derived from glycoproteins previously found to be altered in the CNS based on PD diagnosis or severity. Next, the detected peptides were interrogated for their diagnostic sensitivity and specificity as well as the correlation with PD severity, as determined by the Unified Parkinson's Disease Rating Scale (UPDRS). The results revealed that 12 of the 50 candidate glycopeptides were reliably and consistently identified in plasma samples, with three of them displaying significant differences among diagnostic groups. A combination of four peptides (derived from PRNP, HSPG2, MEGF8, and NCAM1) provided an overall area under curve (AUC) of 0.753 (sensitivity: 90.4%; specificity: 50.0%). Additionally, combining two peptides (derived from MEGF8 and ICAM1) yielded significant correlation with PD severity, that is, UPDRS (r = 0.293, p = 0.004). The significance of these results is at least two-fold: (1) it is possible to use a targeted approach to identify otherwise very difficult to detect CNS related biomarkers in peripheral blood and (2) the novel biomarkers, if validated in independent cohorts, can be employed to assist with clinical diagnosis of PD as well as monitoring disease progression.

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.