This study investigated the ion mobility (IM) and the collision cross section (CCS) of fatty acids (FAs) using electrospray IM MS. The IM analysis of 18 FA ions showed intriguing differences among the saturated FAs, monounsaturated FAs, multi-unsaturated FAs, and cis-isomer/trans-isomer with respect to the aliphatic tail chains. The length of aliphatic tail chain present in the ion structures had a strong influence on the differentiation of drift, while the number of double bond showed a weaker influence. The tiny drift differences between cis-isomer and trans-isomer were also observed. In the CCS measurements, two internal standards were involved in the mobility calibration and accuracy estimation. It insured our empirical CCS values were of high experimental precision (±0.35% or better) and accuracy (±0.25% or better). Moreover, the mass-to-charge ratio (m/z) - mobility plots obtained by ion mobility spectrometry with mass spectrometry analysis of FAs - was used to investigate the structural relationship between the molecules. Each series of FAs sharing a similar structure was aligned in the linear plot. Finally, the developed procedure was applied to the determination of FAs in rat adipose tissues, and it allowed the presence of 13 FAs to be confirmed with their exact masses and CCS values. These studies reveal the direct relationship between the behaviors in IM and the molecular structures and thus may provide further validations to the FA identification process.

To cope with unrepaired DNA lesions, cells are equipped with DNA damage tolerance mechanisms, including translesion synthesis (TLS). While TLS polymerases are well documented in facilitating replication across damaged DNA templates, it remains unknown whether TLS polymerases participate in transcriptional bypass of DNA lesions in cells. Herein, we employed the competitive transcription and adduct bypass assay to examine the efficiencies and fidelities of transcription across N 2-alkyl-2′-deoxyguanosine (N 2-alkyl-dG, alkyl = methyl, ethyl, n-propyl, or n-butyl) lesions in HEK293T cells. We found that N 2-alkyl-dG lesions strongly blocked transcription and elicited CC → AA tandem mutations in nascent transcripts, where adenosines were misincorporated opposite the lesions and their adjacent 5′ nucleoside. Additionally, genetic ablation of Pol η, but not Pol κ, Pol ι, or Pol ζ, conferred marked diminutions in the transcriptional bypass efficiencies of the N 2-alkyl-dG lesions, which is exacerbated by codepletion of Rev1 in Pol η-deficient background. We also observed that the repair of N 2-nBu-dG was not pronouncedly affected by genetic depletion of Pol η or Rev1. Hence, our results provided insights into transcriptional perturbations induced by N 2-alkyl-dG lesions and expanded the biological functions of TLS DNA polymerases.

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Quantitative measurement of DNA adducts in carcinogen-exposed cells provides the information about the frequency of formation and the rate of removal of DNA lesions in vivo, which yields insights into the initial events of mutagenesis. Metabolic activation of tobacco-specific nitrosamines, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its reduction product 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), leads to pyridyloxobutylation and pyridylhydroxybutylation of DNA. In this study, we employed a highly robust nanoflow liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry (nLC-nESI-MS/MS) coupled with the isotope-dilution method for simultaneous quantification of O 6-[4-(3-pyridyl)-4hydroxylbut-1-yl]-2′-deoxyguanosine (O 6-PHBdG) and O 2-and O 4-[4-(3-pyridyl)-4hydroxylbut-1-yl]-thymidine (O 2-PHBdT and O 4-PHBdT). Cultured mammalian cells were exposed to a model pyridylhydroxybutylating agent, 4-(acetoxymethylnitrosamino)-1-(3pyridyl)-1-butanol (NNALOAc), followed by DNA extraction, enzymatic digestion, and sample enrichment prior to nLC-nESI-MS/MS quantification. Our results demonstrate, for the first time, that O 4-PHBdT is quantifiable in cellular DNA and naked DNA upon NNALOAc exposure. We also show that nucleotide excision repair (NER) machinery may counteract the formation of O 2-PHBdT and O 4-PHBdT, and O 6-alkylguanine DNA alkyltransferase (AGT) may be responsible for the repair of O 6-PHBdG and O 4-PHBdT in mammalian cells. Together, our study provides new knowledge about the occurrence and repair of NNAL-induced DNA lesions in mammalian cells.

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