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A healthy lifestyle has been associated with decreased risk of developing breast cancer. Using untargeted metabolomics profiling, which provides unbiased information regarding lifestyle choices such as diet and exercise, we aim to identify the molecular mechanisms connecting lifestyle and breast cancer through network analysis. A total of 100 postmenopausal women, 50 with breast cancer and 50 cancer-free controls, were selected from the Long Island Breast Cancer Study Project (LIBCSP). We measured untargeted plasma metabolomics using liquid chromatography-high-resolution mass spectrometry (LC-HRMS). Using the "enet" package, we retained highly correlated metabolites representing active molecular network (AMN) clusters for analysis. LASSO was used to examine associations between cancer status and AMN metabolites and covariates such as BMI, age, and reproductive factors. LASSO was then repeated to examine associations between AMN metabolites and 10 lifestyle-related variables including smoking, physical activity, alcohol consumption, meat consumption, fruit and vegetable consumption, and supplemental vitamin use. Results were displayed as a network to uncover biological pathways linking lifestyle factors to breast cancer status. After filtering, 851 "active" metabolites out of 1797 metabolomics were retained in 197 correlation AMN clusters. Using LASSO, breast cancer status was associated with 71 "active" metabolites. Several of these metabolites were associated with lifestyle variables including meat consumption, alcohol consumption, and supplemental ß-carotene, B12, and folate use. Those metabolites could potentially serve as molecular-level biological intermediaries connecting healthy lifestyle factors to breast cancer, even though direct associations between breast cancer and the investigated lifestyles at the phenotype level are not evident. In particular, DiHODE, a metabolite linked with inflammation, was associated with breast cancer status and connected to ß-carotene supplement usage through an AMN. We found several plasma metabolites associated with lifestyle factors and breast cancer status. Future studies investigating the mechanistic role of inflammation in linking supplement usage to breast cancer status are warranted.
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The practical advantages of capillary whole blood collection over venipuncture plasma collection for human exposome research are well known. However, before epidemiologists, clinicians, and public health researchers employ these microvolume sample collections, a rigorous evaluation of pre-analytical storage conditions is needed to develop protocols that maximize sample stability and reliability over time. Therefore, we performed a controlled experiment of dried whole blood collected on 10 µL Mitra microsamplers (DBM), 5-mm punches of whole blood from a dried blood spot (DBS), and 10 µL of plasma, and evaluated the effects of storage conditions at 4 °C, -20 °C, or -80 °C for up to 6 months on the resulting metabolite profiles measured with untargeted liquid chromatography-high resolution mass spectrometry (LC-HRMS). At -80 °C storage conditions, metabolite profiles from DBS, DBM, and plasma showed similar stability. While DBS and DBM metabolite profiles remained similarly stable at -20 °C storage, plasma profiles showed decreased stability at -20 °C compared to -80 °C storage. At refrigerated temperatures (4 °C), metabolite profiles collected on DBM were more stable than plasma or DBS, particularly for lipid classes. These results inform robust capillary blood sample storage protocols for DBM and DBS at potentially warmer temperatures than -80 °C, which may facilitate blood collections for populations outside of a clinical setting.
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Plasma , Manejo de Especímenes , Humanos , Temperatura , Reproducibilidad de los ResultadosRESUMEN
De novo sequencing of oligonucleotides remains challenging, especially for oligonucleotides with post-transcriptional or synthetic modifications. Mass spectrometry (MS) sequencing can reliably detect and locate all of the modification sites in oligonucleotides via m/z variance. However, current MS-based sequencing methods exhibit complex spectra and low ion abundance and usually require coupled instrumentation. Herein, we demonstrate a method of oligonucleotide sequencing using TiO2/ZnAl-layered double oxide (LDO)-assisted laser desorption/ionization (LDI)-MS based on radical-induced dissociation (RID). ·CH2OH radicals can be produced on the surface of a TiO2/ZnAl-LDO matrix via ultraviolet light, inducing an attack on the active site of the oligonucleotide phosphate skeleton to create typical "a-, a-B-, c·-, d-, w-, and y"-type fragments. Compared with the spectra obtained via collision-based methods, such as collision-induced dissociation and higher-energy collisional dissociation, the LDI-MS spectra based on RID exhibit single-charged signals, fewer types of fragments, and a lower proportion of unknown noise peaks. We demonstrate full sequence coverage for a 6-mer 2'-O-methyl-modified oligonucleotide and a 21-mer small interfering RNA and show that RID can sequence oligonucleotides with modifications. Importantly, the mechanism responsible for the RID of the oligonucleotide phosphate skeleton was investigated through offline experiments, demonstrating consistent results with density functional theory calculations.
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Oligonucleótidos , Óxidos , Oligonucleótidos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , FosfatosRESUMEN
The exposome reflects multiple exposures across the life-course that can affect health. Metabolomics can reveal the underlying molecular basis linking exposures to health conditions. Here, we explore the concept and general data analysis framework of "molecular gatekeepers"âkey metabolites that link single or multiple exposure biomarkers with correlated clusters of endogenous metabolitesâto inform health-relevant biological targets. We performed untargeted metabolomics on plasma from 152 adolescent girls participating in the Growing Up Healthy Study in New York City. We then performed network analysis to link metabolites to exposure biomarkers including five trace elements (Cd, Mn, Pb, Se, and Hg) and five perfluorinated chemicals (PFCs; n-PFOS, Sm-PFOS, n-PFOA, PFHxS, and PFNA). We found 144 molecular gatekeepers and annotated 22 of them. Lysophosphatidylcholine (16:0) and taurodeoxycholate were correlated with both n-PFOA and n-PFOS, suggesting a shared dysregulation from multiple xenobiotic exposures. Sphingomyelin (d18:2/14:0) was significantly associated with age at menarche; yet, no direct association was detected between any exposure biomarkers and age at menarche. Thus, molecular gatekeepers can also discover molecular linkages between exposure biomarkers and health outcomes that may otherwise be obscured by complex interactions in direct measurements.
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Ácidos Alcanesulfónicos , Fluorocarburos , Oligoelementos , Adolescente , Biomarcadores , Caprilatos , Femenino , Humanos , Metabolómica , Ciudad de Nueva York , Flujo de TrabajoRESUMEN
BACKGROUND: Teeth have unique histology that make this biomatrix a time-capsule for retrospective exposure analysis of fetal and early life. However, most analytic methods require pulverizing the whole tooth, which eliminates exposure timing information. Further, the range of chemicals and endogenous exposures that can be measured in teeth has yet to be fully characterized. METHODS: We performed untargeted metabolomics on micro-dissected layers from naturally shed deciduous teeth. Using four liquid-chromatography high-resolution mass spectrometry analytical modes, we profiled small molecules (<1000 Da) from prenatal and postnatal tooth fractions. In addition, we employed linear regression on the tooth fraction pairs from 31 children to identify metabolites that discriminate between prenatal and postnatal exposures. RESULTS: Of over 10,000 features measured in teeth dentin, 390 unique compounds were annotated from 62 chemical classes. The class with the largest number of compounds was carboxylic acids and their derivatives (36%). Of the annotated exogenous metabolites (phthalates, parabens, perfluoroalkyl compounds, and cotinine) and endogenous metabolites (fatty acids, steroids, carnitines, amino acids, and others), 91 are linked to 256 health conditions through published literature. Differential analysis revealed 267 metabolites significantly different between the prenatal and the postnatal tooth fractions (adj. p-value < 0.05, Bonferroni correction), and 21 metabolites exclusive to the prenatal fraction. CONCLUSIONS: The prenatal and early postnatal exposome revealed from dental biomarkers represents a broad range of endogenous and exogenous metabolites for a comprehensive characterization in environmental health research. Most importantly, this technology provides a direct window into fetal exposures that is not possible by maternal biomarkers. Indeed, we identified several metabolites exclusively in the prenatal fraction, suggesting unique fetal exposures that are markedly different to postnatal exposures. Expansion of databases that include tooth matrix metabolites will strengthen biological interpretation and shed light on exposures during gestation and early life that may be causally linked with later health conditions.
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Exposoma , Biomarcadores , Niño , Cromatografía Liquida , Exposición a Riesgos Ambientales/análisis , Femenino , Humanos , Metabolómica , Embarazo , Estudios RetrospectivosRESUMEN
In this work, highly oxidized multifunctional molecules (HOMs) in fresh and aged secondary organic aerosol (SOA) derived from biogenic precursors are characterized with high-resolution mass spectrometry. Fresh SOA was generated by mixing ozone with a biogenic precursor (ß-pinene, limonene, α-pinene) in a flow tube reactor. Aging was performed by passing the fresh SOA through a photochemical reactor where it reacted with hydroxyl radicals. Although these aerosols were as a whole not highly oxidized, molecular analysis identified a significant number of HOMs embedded within it. HOMs in fresh SOA consisted mostly of monomers and dimers, which is consistent with condensation of extremely low-volatility organic compounds (ELVOCs) that have been detected in the gas phase in previous studies and linked to SOA particle formation. Aging caused an increase in the average number of carbon atoms per molecule of the HOMs, which is consistent with particle phase oxidation of (less oxidized) oligomers already existing in fresh SOA. HOMs having different combinations of oxygen-to-carbon ratio, hydrogen-to-carbon ratio and average carbon oxidation state are discussed and compared to low volatility oxygenated organic aerosol (LVOOA), which has been identified in ambient aerosol based on average elemental composition but not fully understood at a molecular level. For the biogenic precursors and experimental conditions studied, HOMs in fresh biogenic SOA have molecular formulas more closely resembling LVOOA than HOMs in aged SOA, suggesting that aging of biogenic SOA is not a good surrogate for ambient LVOOA.
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Compuestos Bicíclicos con Puentes/química , Ciclohexenos/química , Monoterpenos/química , Ozono/química , Terpenos/química , Aerosoles/química , Monoterpenos Bicíclicos , Limoneno , Espectrometría de Masas , Oxidación-ReducciónRESUMEN
Unexpected [M + 15](+) ions were formed during the analysis of aromatic aldehydes by use of methanol in positive-ion electrospray ionization mass spectrometry. Aromatic aldehydes with electron-withdrawing groups or electron-donating groups were all tested to make sure the universality. All the aromatic aldehydes studied with methanol as the solvent could generate [M + 15](+) ion, and for most of them, the [M + 15](+) ion was more intense than the [M + H](+) ion. Deuterium-labeling experiment, high-performance liquid chromatography-MS experiment, collision-induced dissociation experiment, and theoretical calculations were performed to identify the formation of [M + 15](+) ion. The proposed reaction mechanism is a gas-phase aldol reaction between protonated aromatic aldehydes and methanol occurring in electrospray source. Understanding and using this unique gas-phase ion/molecule reaction can indeed offer a novel and fast approach for the direct identification of aromatic aldehydes.