Mass Spectral Library for DNA Adductomics.

Endogenous electrophiles, ionizing and non-ionizing radiation, and hazardous chemicals present in the environment and diet can damage DNA by forming covalent adducts. DNA adducts can form in critical cancer driver genes and, if not repaired, may induce mutations during cell division, potentially leading to the onset of cancer. The detection and quantification of specific DNA adducts are some of the first steps in studying their role in carcinogenesis, the physiological conditions that lead to their production, and the risk assessment of exposure to specific genotoxic chemicals. Hundreds of different DNA adducts have been reported in the literature, and there is a critical need to establish a DNA adduct mass spectral database to facilitate the detection of previously observed DNA adducts and characterize newly discovered DNA adducts. We have collected synthetic DNA adduct standards from the research community, acquired MSn (n = 2, 3) fragmentation spectra using Orbitrap and Quadrupole-Time-of-Flight (Q-TOF) MS instrumentation, processed the spectral data and incorporated it into the MassBank of North America (MoNA) database, and created a DNA adduct portal Web site (https://sites.google.com/umn.edu/dnaadductportal) to serve as a central location for the DNA adduct mass spectra and metadata, including the spectral database downloadable in different formats. This spectral library should prove to be a valuable resource for the DNA adductomics community, accelerating research and improving our understanding of the role of DNA adducts in disease.

Screening DNA Damage in the Rat Kidney and Liver by Untargeted DNA Adductomics.

Air pollution, tobacco smoke, and red meat are associated with renal cell cancer (RCC) risk in the United States and Western Europe; however, the chemicals that form DNA adducts and initiate RCC are mainly unknown. Aristolochia herbaceous plants are used for medicinal purposes in Asia and worldwide. They are a significant risk factor for upper tract urothelial carcinoma (UTUC) and RCC to a lesser extent. The aristolochic acid (AA) 8-methoxy-6-nitrophenanthro-[3,4-d]-1,3-dioxolo-5-carboxylic acid (AA-I), a component of Aristolochia herbs, contributes to UTUC in Asian cohorts and in Croatia, where AA-I exposure occurs from ingesting contaminated wheat flour. The DNA adduct of AA-I, 7-(2'-deoxyadenosin-N6-yl)-aristolactam I, is often detected in patients with UTUC, and its characteristic A:T-to-T:A mutational signature occurs in oncogenes and tumor suppressor genes in AA-associated UTUC. Identifying DNA adducts in the renal parenchyma and pelvis caused by other chemicals is crucial to gaining insights into unknown RCC and UTUC etiologies. We employed untargeted screening with wide-selected ion monitoring tandem mass spectrometry (wide-SIM/MS2) with nanoflow liquid chromatography/Orbitrap mass spectrometry to detect DNA adducts formed in rat kidneys and liver from a mixture of 13 environmental, tobacco, and dietary carcinogens that may contribute to RCC. Twenty DNA adducts were detected. DNA adducts of 3-nitrobenzanthrone (3-NBA), an atmospheric pollutant, and AA-I were the most abundant. The nitrophenanthrene moieties of 3-NBA and AA-I undergo reduction to their N-hydroxy intermediates to form 2'-deoxyguanosine (dG) and 2'-deoxyadenosine (dA) adducts. We also discovered a 2'-deoxycytidine AA-I adduct and dA and dG adducts of 10-methoxy-6-nitro-phenanthro-[3,4-d]-1,3-dioxolo-5-carboxylic acid (AA-III), an AA-I isomer and minor component of the herbal extract assayed, signifying AA-III is a potent kidney DNA-damaging agent. The roles of AA-III, other nitrophenanthrenes, and nitroarenes in renal DNA damage and human RCC warrant further study. Wide-SIM/MS2 is a powerful scanning technology in DNA adduct discovery and cancer etiology characterization.

The Cooked Meat Carcinogen 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine Hair Dosimeter, DNA Adductomics Discovery, and Associations with Prostate Cancer Pathology Biomarkers.

Well-done cooked red meat consumption is linked to aggressive prostate cancer (PC) risk. Identifying mutation-inducing DNA adducts in the prostate genome can advance our understanding of chemicals in meat that may contribute to PC. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a heterocyclic aromatic amine (HAA) formed in cooked meat, is a potential human prostate carcinogen. PhIP was measured in the hair of PC patients undergoing prostatectomy, bladder cancer patients under treatment for cystoprostatectomy, and patients treated for benign prostatic hyperplasia (BPH). PhIP hair levels were above the quantification limit in 123 of 205 subjects. When dichotomizing prostate pathology biomarkers, the geometric mean PhIP hair levels were higher in patients with intermediate and elevated-risk prostate-specific antigen values than lower-risk values <4 ng/mL (p = 0.03). PhIP hair levels were also higher in patients with intermediate and high-risk Gleason scores ≥7 compared to lower-risk Gleason score 6 and BPH patients (p = 0.02). PC patients undergoing prostatectomy had higher PhIP hair levels than cystoprostatectomy or BPH patients (p = 0.02). PhIP-DNA adducts were detected in 9.4% of the patients assayed; however, DNA adducts of other carcinogenic HAAs, and benzo[a]pyrene formed in cooked meat, were not detected. Prostate specimens were also screened for 10 oxidative stress-associated lipid peroxidation (LPO) DNA adducts. Acrolein 1,N2-propano-2'-deoxyguanosine adducts were detected in 54.5% of the patients; other LPO adducts were infrequently detected. Acrolein adducts were not associated with prostate pathology biomarkers, although DNA adductomic profiles differed between PC patients with low and high-grade Gleason scores. Many DNA adducts are of unknown origin; however, dG adducts of formaldehyde and a series of purported 4-hydroxy-2-alkenals were detected at higher abundance in a subset of patients with elevated Gleason scores. The PhIP hair biomarker and DNA adductomics data support the paradigm of well-done cooked meat and oxidative stress in aggressive PC risk.

DNA Damage and Oxidative Stress of Tobacco Smoke Condensate in Human Bladder Epithelial Cells.

Smoking is a major risk factor for bladder cancer (BC), with up to 50% of BC cases being attributed to smoking. There are 70 known carcinogens in tobacco smoke; however, the principal chemicals responsible for BC remain uncertain. The aromatic amines 4-aminobiphenyl (4-ABP) and 2-naphthylamine (2-NA) are implicated in BC pathogenesis of smokers on the basis of the elevated BC risk in factory workers exposed to these chemicals. However, 4-ABP and 2-NA only occur at several nanograms per cigarette and may be insufficient to induce BC. In contrast, other genotoxicants, including acrolein, occur at 1000-fold or higher levels in tobacco smoke. There is limited data on the toxicological effects of tobacco smoke in human bladder cells. We have assessed the cytotoxicity, oxidative stress, and DNA damage of tobacco smoke condensate (TSC) in human RT4 bladder cells. TSC was fractionated by liquid-liquid extraction into an acid-neutral fraction (NF), containing polycyclic aromatic hydrocarbons (PAHs), nitro-PAHs, phenols, and aldehydes, and a basic fraction (BF) containing aromatic amines, heterocyclic aromatic amines, and N-nitroso compounds. The TSC and NF induced a time- and concentration-dependent cytotoxicity associated with oxidative stress, lipid peroxide formation, glutathione (GSH) depletion, and apurinic/apyrimidinic (AP) site formation, while the BF showed weak effects. LC/MS-based metabolomic approaches showed that TSC and NF altered GSH biosynthesis pathways and induced more than 40 GSH conjugates. GSH conjugates of several hydroquinones were among the most abundant conjugates. RT4 cell treatment with synthetic hydroquinones and cresol mixtures at levels present in tobacco smoke accounted for most of the TSC-induced cytotoxicity and the AP sites formed. GSH conjugates of acrolein, methyl vinyl ketone, and crotonaldehyde levels also increased owing to TSC-induced oxidative stress. Thus, TSC is a potent toxicant and DNA-damaging agent, inducing deleterious effects in human bladder cells at concentrations of <1% of a cigarette in cell culture media.

Comprehensive Analysis of DNA Adducts Using Data-Independent wSIM/MS2 Acquisition and wSIM-City.

A novel software has been created to comprehensively characterize covalent modifications of DNA through mass spectral analysis of enzymatically hydrolyzed DNA using the neutral loss of 2'-deoxyribose, a nearly universal MS2 fragmentation process of protonated 2'-deoxyribonucleosides. These covalent modifications termed DNA adducts form through xenobiotic exposures or by reaction with endogenous electrophiles and can induce mutations during cell division and initiate carcinogenesis. DNA adducts are typically present at trace levels in the human genome, requiring a very sensitive and comprehensive data acquisition and analysis method. Our software, wSIM-City, was created to process mass spectral data acquired by a wide selected ion monitoring (wSIM) with gas-phase fractionation and coupled to wide MS2 fragmentation. This untargeted approach can detect DNA adducts at trace levels as low as 1.5 adducts per 109 nucleotides. This level of sensitivity is sufficient for comprehensive analysis and characterization of DNA modifications in human specimens.

Effects of GSTT1 Genotype on the Detoxification of 1,3-Butadiene Derived Diepoxide and Formation of Promutagenic DNA-DNA Cross-Links in Human Hapmap Cell Lines.

Smoking is a leading cause of lung cancer, accounting for 81% of lung cancer cases. Tobacco smoke contains over 5000 compounds, of which more than 70 have been classified as human carcinogens. Of the many tobacco smoke constituents, 1,3-butadiene (BD) has a high cancer risk index due to its tumorigenic potency and its abundance in cigarette smoke. The carcinogenicity of BD has been attributed to the formation of several epoxide metabolites, of which 1,2,3,4-diepoxybutane (DEB) is the most toxic and mutagenic. DEB is formed by two oxidation reactions carried out by cytochrome P450 monooxygenases, mainly CYP2E1. Glutathione-S-transferase theta 1 (GSTT1) facilitates the conjugation of DEB to glutathione as the first step of its detoxification and subsequent elimination via the mercapturic acid pathway. Human biomonitoring studies have revealed a strong association between GSTT1 copy number and urinary concentrations of BD-mercapturic acids, suggesting that it plays an important role in the metabolism of BD. To determine the extent that GSTT1 genotype affects the susceptibility of individuals to the toxic and genotoxic properties of DEB, GSTT1 negative and GSTT1 positive HapMap lymphoblastoid cell lines were treated with DEB, and the extent of apoptosis and micronuclei (MN) formation was assessed. These toxicological end points were compared to the formation of DEB-GSH conjugates and 1,4-bis-(guan-7-yl)-2,3-butanediol (bis-N7G-BD) DNA-DNA cross-links. GSTT1 negative cell lines were more sensitive to DEB-induced apoptosis as compared to GSTT1 positive cell lines. Consistent with the protective effect of GSH conjugation against DEB-derived apoptosis, GSTT1 positive cell lines formed significantly more DEB-GSH conjugate than GSTT1 negative cell lines. However, GSTT1 genotype did not affect formation of MN or bis-N7G-BD cross-links. These results indicate that GSTT1 genotype significantly influences BD metabolism and acute toxicity.

Kinetics of DNA Adducts and Abasic Site Formation in Tissues of Mice Treated with a Nitrogen Mustard.

Nitrogen mustards (NM) are an important class of chemotherapeutic drugs used in the treatment of malignant tumors. The accepted mechanism of action of NM is through the alkylation of DNA bases. NM-adducts block DNA replication in cancer cells by forming cytotoxic DNA interstrand cross-links. We previously characterized several adducts formed by reaction of bis(2-chloroethyl)ethylamine (NM) with calf thymus (CT) DNA and the MDA-MB-231 mammary tumor cell line. The monoalkylated N7-guanine (NM-G) adduct and its cross-link (G-NM-G) were major lesions. The cationic NM-G undergoes a secondary reaction through depurination to form an apurinic (AP) site or reacts with hydroxide to yield the stable ring-opened N5-substituted formamidopyrimidine (NM-Fapy-G) adduct. Both of these lesions are mutagenic and may contribute to secondary tumor development, a major clinical limitation of NM chemotherapy. We established a kinetic model with NM-treated female mice and measured the rates of formation and removal of NM-DNA adducts and AP sites. We employed liquid chromatography-mass spectrometry (LC-MS) to measure NM-G, G-NM-G, and NM-Fapy-G adducts in liver, lung, and spleen over 168 h. NM-G reached a maximum level within 6 h in all organs and then rapidly declined. The G-NM-G cross-link and NM-FapyG were more persistent with half-lives over three-times longer than NM-G. We quantified AP site lesions in the liver and showed that NM treatment increased AP site levels by 3.7-fold over the basal levels at 6 h. The kinetics of AP site repair closely followed the rate of removal of NM-G; however, AP sites remained 1.3-fold above basal levels 168 h post-treatment with NM. Our data provide new insights into NM-induced DNA damage and biological processing in vivo. The quantitative measurement of the spectrum of NM adducts and AP sites can serve as biomarkers in the design and assessment of the efficacy of novel chemotherapeutic regimens.

Development of a DNA Adductome Mass Spectral Database.

Mass spectrometry-based DNA adductomics is an emerging approach for the human biomonitoring of hazardous chemicals. A mass spectral database of DNA adducts will be created for the scientific community to investigate the associations between chemical exposures, DNA damage, and disease risk.

Truncated rank correlation (TRC) as a robust measure of test-retest reliability in mass spectrometry data.

In mass spectrometry (MS) experiments, more than thousands of peaks are detected in the space of mass-to-charge ratio and chromatographic retention time, each associated with an abundance measurement. However, a large proportion of the peaks consists of experimental noise and low abundance compounds are typically masked by noise peaks, compromising the quality of the data. In this paper, we propose a new measure of similarity between a pair of MS experiments, called truncated rank correlation (TRC). To provide a robust metric of similarity in noisy high-dimensional data, TRC uses truncated top ranks (or top m-ranks) for calculating correlation. A comprehensive numerical study suggests that TRC outperforms traditional sample correlation and Kendall's τ. We apply TRC to measuring test-retest reliability of two MS experiments, including biological replicate analysis of the metabolome in HEK293 cells and metabolomic profiling of benign prostate hyperplasia (BPH) patients. An R package trc of the proposed TRC and related functions is available at https://sites.google.com/site/dhyeonyu/software.

Methods and Challenges for Computational Data Analysis for DNA Adductomics.

Frequent exposure to chemicals in the environment, diet, and endogenous electrophiles leads to chemical modification of DNA and the formation of DNA adducts. Some DNA adducts can induce mutations during cell division and, when occurring in critical regions of the genome, can lead to the onset of disease, including cancer. The targeted analysis of DNA adducts over the past 30 years has revealed that the human genome contains many types of DNA damages. However, a long-standing limitation in conducting DNA adduct measurements has been the inability to screen for the total complement of DNA adducts derived from a wide range of chemicals in a single assay. With the advancement of high-resolution mass spectrometry (MS) instrumentation and new scanning technologies, nontargeted "omics" approaches employing data-dependent acquisition and data-independent acquisition methods have been established to simultaneously screen for multiple DNA adducts, a technique known as DNA adductomics. However, notable challenges in data processing must be overcome for DNA adductomics to become a mature technology. DNA adducts occur at low abundance in humans, and current softwares do not reliably detect them when using common MS data acquisition methods. In this perspective, we discuss contemporary computational tools developed for feature finding of MS data widely utilized in the disciplines of proteomics and metabolomics and highlight their limitations for conducting nontargeted DNA-adduct biomarker discovery. Improvements to existing MS data processing software and new algorithms for adduct detection are needed to develop DNA adductomics into a powerful tool for the nontargeted identification of potential cancer-causing agents.

Customized Consensus Spectral Library Building for Untargeted Quantitative Metabolomics Analysis with Data Independent Acquisition Mass Spectrometry and MetaboDIA Workflow.

Data independent acquisition-mass spectrometry (DIA-MS) coupled with liquid chromatography is a promising approach for rapid, automatic sampling of MS/MS data in untargeted metabolomics. However, wide isolation windows in DIA-MS generate MS/MS spectra containing a mixed population of fragment ions together with their precursor ions. This precursor-fragment ion map in a comprehensive MS/MS spectral library is crucial for relative quantification of fragment ions uniquely representative of each precursor ion. However, existing reference libraries are not sufficient for this purpose since the fragmentation patterns of small molecules can vary in different instrument setups. Here we developed a bioinformatics workflow called MetaboDIA to build customized MS/MS spectral libraries using a user's own data dependent acquisition (DDA) data and to perform MS/MS-based quantification with DIA data, thus complementing conventional MS1-based quantification. MetaboDIA also allows users to build a spectral library directly from DIA data in studies of a large sample size. Using a marine algae data set, we show that quantification of fragment ions extracted with a customized MS/MS library can provide as reliable quantitative data as the direct quantification of precursor ions based on MS1 data. To test its applicability in complex samples, we applied MetaboDIA to a clinical serum metabolomics data set, where we built a DDA-based spectral library containing consensus spectra for 1829 compounds. We performed fragment ion quantification using DIA data using this library, yielding sensitive differential expression analysis.

Performance of a High-Pressure Liquid Chromatography-Ion Mobility-Mass Spectrometry System for Metabolic Profiling.

A commercial liquid chromatography/drift tube ion mobility-mass spectrometer (LC/IM-MS) was evaluated for its utility in global metabolomics analysis. Performance was assessed using 12 targeted metabolite standards where the limit of detection (LOD), linear dynamic range, resolving power, and collision cross section (Ω) are reported for each standard. Data were collected in three different instrument operation modes: flow injection analysis with IM-MS (FIA/IM-MS), LC/MS, and LC/IM-MS. Metabolomics analyses of human plasma and HaCaT cells were used to compare the above three operation modes. LC/MS provides linearity in response, data processing automation, improved limits of detection, and ease of use. Advantages of LC/IM-MS and FIA/IM-MS include the ability to develop mobility-mass trend lines for structurally similar biomolecules, increased peak capacity, reduction of chemical/matrix noise, improvement in signal-to-noise, and separations of isobar/isomer compounds that are not resolved by LC. We further tested the feasibility of incorporating IM-MS into conventional LC/MS metabolomics workflows. In general, the addition of ion mobility dimension has increased the separation of compounds in complex biological matrixes and has the potential to largely improve the throughput of metabolomics analysis.

LuciPHOr: algorithm for phosphorylation site localization with false localization rate estimation using modified target-decoy approach.

The localization of phosphorylation sites in peptide sequences is a challenging problem in large-scale phosphoproteomics analysis. The intense neutral loss peaks and the coexistence of multiple serine/threonine and/or tyrosine residues are limiting factors for objectively scoring site patterns across thousands of peptides. Various computational approaches for phosphorylation site localization have been proposed, including Ascore, Mascot Delta score, and ProteinProspector, yet few address direct estimation of the false localization rate (FLR) in each experiment. Here we propose LuciPHOr, a modified target-decoy-based approach that uses mass accuracy and peak intensities for site localization scoring and FLR estimation. Accurate estimation of the FLR is a difficult task at the individual-site level because the degree of uncertainty in localization varies significantly across different peptides. LuciPHOr carries out simultaneous localization on all candidate sites in each peptide and estimates the FLR based on the target-decoy framework, where decoy phosphopeptides generated by placing artificial phosphorylation(s) on non-candidate residues compete with the non-decoy phosphopeptides. LuciPHOr also reports approximate site-level confidence scores for all candidate sites as a means to localize additional sites from multiphosphorylated peptides in which localization can be partially achieved. Unlike the existing tools, LuciPHOr is compatible with any search engine output processed through the Trans-Proteomic Pipeline. We evaluated the performance of LuciPHOr in terms of the sensitivity and accuracy of FLR estimates using two synthetic phosphopeptide libraries and a phosphoproteomic dataset generated from complex mouse brain samples.

Comprehensive analysis of protein digestion using six trypsins reveals the origin of trypsin as a significant source of variability in proteomics.

Trypsin is an endoprotease commonly used for sample preparation in proteomics experiments. Importantly, protein digestion is dependent on multiple factors, including the trypsin origin and digestion conditions. In-depth characterization of trypsin activity could lead to improved reliability of peptide detection and quantitation in both targeted and discovery proteomics studies. To this end, we assembled a data analysis pipeline and suite of visualization tools for quality control and comprehensive characterization of preanalytical variability in proteomics experiments. Using these tools, we evaluated six available proteomics-grade trypsins and their digestion of a single purified protein, human serum albumin (HSA). HSA was aliquoted and then digested for 2 or 18 h for each trypsin, and the resulting digests were desalted and analyzed in triplicate by reversed-phase liquid chromatography-tandem mass spectrometry. Peptides were identified and quantified using the NIST MSQC pipeline and a comprehensive HSA mass spectral library. We performed a statistical analysis of peptide abundances from different digests and further visualized the data using the principal component analysis and quantitative protein "sequence maps". While the performance of individual trypsins across repeat digests was reproducible, significant differences were observed depending on the origin of the trypsin (i.e., bovine vs porcine). Bovine trypsins produced a higher number of peptides containing missed cleavages, whereas porcine trypsins produced more semitryptic peptides. In addition, many cleavage sites showed variable digestion kinetics patterns, evident from the comparison of peptide abundances in 2 h vs 18 h digests. Overall, this work illustrates effects of an often neglected source of variability in proteomics experiments: the origin of the trypsin.

Proteomic profiling of the effect of metabolic acidosis on the apical membrane of the proximal convoluted tubule.

The physiological response to the onset of metabolic acidosis requires pronounced changes in renal gene expression. Adaptations within the proximal convoluted tubule support the increased extraction of plasma glutamine and the increased synthesis and transport of glucose and of NH(4)(+) and HCO(3)(-) ions. Many of these adaptations involve proteins associated with the apical membrane. To quantify the temporal changes in these proteins, proteomic profiling was performed using brush-border membrane vesicles isolated from proximal convoluted tubules (BBMV(PCT)) that were purified from normal and acidotic rats. This preparation is essentially free of contaminating apical membranes from other renal cortical cells. The analysis identified 298 proteins, 26% of which contained one or more transmembrane domains. Spectral counts were used to assess changes in protein abundance. The onset of acidosis produced a twofold, but transient, increase in the Na(+)-dependent glucose transporter and a more gradual, but sustained, increase (3-fold) in the Na(+)-dependent lactate transporter. These changes were associated with the loss of glycolytic and gluconeogenic enzymes that are contained in the BBMV(PCT) isolated from normal rats. In addition, the levels of γ-glutamyltranspeptidase increased twofold, while transporters that participate in the uptake of neutral amino acids, including glutamine, were decreased. These changes could facilitate the deamidation of glutamine within the tubular lumen. Finally, pronounced increases were also observed in the levels of DAB2 (3-fold) and myosin 9 (7-fold), proteins that may participate in endocytosis of apical membrane proteins. Western blot analysis and accurate mass and time analyses were used to validate the spectral counting.

Metabolism and biomarkers of heterocyclic aromatic amines in humans.

Heterocyclic aromatic amines (HAAs) form during the high-temperature cooking of meats, poultry, and fish. Some HAAs also arise during the combustion of tobacco. HAAs are multisite carcinogens in rodents, inducing cancer of the liver, gastrointestinal tract, pancreas, mammary, and prostate glands. HAAs undergo metabolic activation by N-hydroxylation of the exocyclic amine groups to produce the proposed reactive intermediate, the heteroaryl nitrenium ion, which is the critical metabolite implicated in DNA damage and genotoxicity. Humans efficiently convert HAAs to these reactive intermediates, resulting in HAA protein and DNA adduct formation. Some epidemiologic studies have reported an association between frequent consumption of well-done cooked meats and elevated cancer risk of the colorectum, pancreas, and prostate. However, other studies have reported no associations between cooked meat and these cancer sites. A significant limitation in epidemiology studies assessing the role of HAAs and cooked meat in cancer risk is their reliance on food frequency questionnaires (FFQ) to gauge HAA exposure. FFQs are problematic because of limitations in self-reported dietary history accuracy, and estimating HAA intake formed in cooked meats at the parts-per-billion level is challenging. There is a critical need to establish long-lived biomarkers of HAAs for implementation in molecular epidemiology studies designed to assess the role of HAAs in health risk. This review article highlights the mechanisms of HAA formation, mutagenesis and carcinogenesis, the metabolism of several prominent HAAs, and the impact of critical xenobiotic-metabolizing enzymes on biological effects. The analytical approaches that have successfully biomonitored HAAs and their biomarkers for molecular epidemiology studies are presented.

Proteomic analysis of brush-border membrane vesicles isolated from purified proximal convoluted tubules.

The renal proximal convoluted tubule is the primary site of water, electrolyte and nutrient reabsorption and of active secretion of selected molecules. Proteins in the apical brush-border membrane facilitate these functions and initiate some of the cellular responses to altered renal physiology. The current study uses two-dimensional liquid chromatography/mass spectrometry to compare brush border membrane vesicles isolated from rat renal cortex (BBMV(CTX)) and from purified proximal convoluted tubules (BBMV(PCT)). Both proteomic data and Western blot analysis indicate that the BBMV(CTX) contain apical membrane proteins from cortical cells other than the proximal tubule. This heterogeneity was greatly reduced in the BBMV(PCT). Proteomic analysis identified 193 proteins common to both samples, 21 proteins unique to BBMV(CTX), and 57 proteins unique to BBMV(PCT). Spectral counts were used to quantify relative differences in protein abundance. This analysis identified 42 and 50 proteins that are significantly enriched (p values

A Perspective and Framework for Developing Sample Type Specific Databases for LC/MS-Based Clinical Metabolomics.

Metabolomics has the potential to greatly impact biomedical research in areas such as biomarker discovery and understanding molecular mechanisms of disease. However, compound identification (ID) remains a major challenge in liquid chromatography mass spectrometry-based metabolomics. This is partly due to a lack of specificity in metabolomics databases. Though impressive in depth and breadth, the sheer magnitude of currently available databases is in part what makes them ineffective for many metabolomics studies. While still in pilot phases, our experience suggests that custom-built databases, developed using empirical data from specific sample types, can significantly improve confidence in IDs. While the concept of sample type specific databases (STSDBs) and spectral libraries is not entirely new, inclusion of unique descriptors such as detection frequency and quality scores, can be used to increase confidence in results. These features can be used alone to judge the quality of a database entry, or together to provide filtering capabilities. STSDBs rely on and build upon several available tools for compound ID and are therefore compatible with current compound ID strategies. Overall, STSDBs can potentially result in a new paradigm for translational metabolomics, whereby investigators confidently know the identity of compounds following a simple, single STSDB search.

A prototypic small molecule database for bronchoalveolar lavage-based metabolomics.

The analysis of bronchoalveolar lavage fluid (BALF) using mass spectrometry-based metabolomics can provide insight into lung diseases, such as asthma. However, the important step of compound identification is hindered by the lack of a small molecule database that is specific for BALF. Here we describe prototypic, small molecule databases derived from human BALF samples (n=117). Human BALF was extracted into lipid and aqueous fractions and analyzed using liquid chromatography mass spectrometry. Following filtering to reduce contaminants and artifacts, the resulting BALF databases (BALF-DBs) contain 11,736 lipid and 658 aqueous compounds. Over 10% of these were found in 100% of samples. Testing the BALF-DBs using nested test sets produced a 99% match rate for lipids and 47% match rate for aqueous molecules. Searching an independent dataset resulted in 45% matching to the lipid BALF-DB compared to<25% when general databases are searched. The BALF-DBs are available for download from MetaboLights. Overall, the BALF-DBs can reduce false positives and improve confidence in compound identification compared to when general databases are used.