Quantification of DNA lesions constitutes one of the main tasks in toxicology and in assessing health risks accompanied by exposure to carcinogens. Tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N′-nitrosonornicotine (NNN) can undergo metabolic transformation to give a reactive intermediate that pyridyloxobutylates nucleobases and phosphate backbone of DNA. Here, we reported a highly sensitive method, relying on the use of nanoflow liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry (nLC-nESI-MS/MS), for the simultaneous quantifications of O6-[4-(3-pyridyl)-4-oxobut-1-yl]-2′-deoxyguanosine (O6-POBdG) as well as O2- and O4-[4-(3-pyridyl)-4-oxobut-1-yl]-thymidine (O2-POBdT and O4-POBdT). By using this method, we measured the levels of the three DNA adducts with the use of 10 μg of DNA isolated from cultured mammalian cells exposed to a model pyridyloxobutylating agent, 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc). Our results demonstrated, for the first time, the formation of O4-POBdT in naked DNA and in genomic DNA of cultured mammalian cells exposed with NNKOAc. We also revealed that the levels of the three lesions increased with the dose of NNKOAc and that O2-POBdT and O4-POBdT could be subjected to repair by the nucleotide excision repair (NER) pathway. The method reported here will be useful for investigations about the involvement of other DNA repair pathways in the removal of these lesions and for human toxicological studies in the future.

Arsenic is a ubiquitous environmental contaminant with widespread public health concern. Epidemiological studies have revealed that chronic human exposure to arsenic in drinking water is associated with the prevalence of skin, lung, and bladder cancers. Aberrant histone modifications (e.g., methylation, acetylation, and ubiquitination) were previously found to be accompanied by arsenic exposure; thus, perturbation of epigenetic pathways is thought to contribute to arsenic carcinogenesis. Arsenite is known to interact with zinc finger motifs of proteins, and zinc finger motif is present in and indispensable for the enzymatic activities of crucial histone-modifying enzymes especially the MYST family of histone acetyltransferases (e.g., TIP60). Hence, we reasoned that trivalent arsenic may target the zinc finger motif of these enzymes, disturb their enzymatic activities, and alter histone acetylation. Herein, we found that As3+ could bind directly to the zinc-finger motif of TIP60 in vitro and in cells. In addition, exposure to As3+ could lead to a dose-dependent decrease in TIP60 protein level via the ubiquitin-proteasome pathway. Thus, the results from the present study revealed, for the first time, that arsenite may target cysteine residues in the zinc-finger motif of the TIP60 histone acetyltransferase, thereby altering the H4K16Ac histone epigenetic mark. Our results also shed some new light on the mechanisms underlying the arsenic-induced epigenotoxicity and carcinogenesis in humans.

The guanine quadruplex (G4) structure in DNA is a secondary structure motif that plays important roles in DNA replication, transcriptional regulation, and maintenance of genomic stability. Here, we employed a quantitative mass spectrometry-based approach to profile the interaction proteomes of three well-defined G4 structures derived from the human telomere and the promoters of cMYC and cKIT genes. We identified SLIRP as a novel G4-interacting protein. We also demonstrated that the protein could bind directly with G4 DNA with Kd values in the low nanomolar range and revealed that the robust binding of the protein toward G4 DNA requires its RRM domain. We further assessed, by using CRISPR-Cas9-introduced affinity tag and ChIP-Seq analysis, the genome-wide occupancy of SLIRP, and showed that the protein binds preferentially to G-rich DNA sequences that can fold into G4 structures. Together, our results uncovered a novel cellular protein that can interact directly with G4 DNA, which underscored the complex regulatory networks involved in G4 biology.

Imatinib mesylate, currently marketed by Novartis as Gleevec in the U.S., has emerged as the leading compound to treat the chronic phase of chronic myeloid leukemia (CML), through its inhibition of Bcr-Abl tyrosine kinases, and other cancers. However, resistance to imatinib develops frequently, particularly in late-stage disease. To identify new cellular pathways affected by imatinib treatment, we applied mass spectrometry together with stable isotope labeling by amino acids in cell culture (SILAC) for the comparative study of protein expression in K562 cells that were untreated or treated with a clinically relevant concentration of imatinib. Our results revealed that, among the 1344 quantified proteins, 73 had significantly altered levels of expression induced by imatinib and could be quantified in both forward and reverse SILAC labeling experiments. These included the down-regulation of thymidylate synthase, S-adenosylmethionine synthetase, and glycerol-3-phosphate dehydrogenase as well as the up-regulation of poly(ADP-ribose) polymerase 1, hemoglobins, and enzymes involved in heme biosynthesis. We also found, by assessing alteration in the acetylation level in histone H4 upon imatinib treatment, that the imatinib-induced hemoglobinization and erythroid differentiation in K562 cells are associated with global histone H4 hyperacetylation. Overall, these results provided potential biomarkers for monitoring the therapeutic intervention of CML using imatinib and offered important new knowledge for gaining insight into the molecular mechanisms of action of imatinib.Keywords: chronic myeloid leukemia; erythroid differentiation; histone acetylation; imatinib; SILAC;

Cadmium (Cd2+) is a toxic heavy metal and a well-known human carcinogen. The toxic effects of Cd2+ on biological systems are diverse and thought to be exerted through a complex array of mechanisms. Despite the large number of studies aimed to elucidate the toxic mechanisms of action of Cd2+, few have been targeted toward investigating the ability of Cd2+ to disrupt multiple cellular pathways simultaneously and the overall cellular responses toward Cd2+ exposure. In this study, we employed a quantitative proteomic method, relying on stable isotope labeling by amino acids in cell culture (SILAC) and LC–MS/MS, to assess the Cd2+-induced simultaneous alterations of multiple cellular pathways in cultured human skin fibroblast cells. By using this approach, we were able to quantify 2931 proteins, and 400 of them displayed significantly changed expression following Cd2+ exposure. Our results unveiled that Cd2+ treatment led to the marked upregulation of several antioxidant enzymes (e.g., metallothionein-1G, superoxide dismutase, pyridoxal kinase, etc.), enzymes associated with glutathione biosynthesis and homeostasis (e.g., glutathione S-transferases, glutathione synthetase, glutathione peroxidase, etc.), and proteins involved in cellular energy metabolism (e.g., glycolysis, pentose phosphate pathway, and the citric acid cycle). Additionally, we found that Cd2+ treatment resulted in the elevated expression of two isoforms of dimethylarginine dimethylaminohydrolase (DDAH I and II), enzymes known to play a key role in regulating nitric oxide biosynthesis. Consistent with these findings, we observed elevated formation of nitric oxide in human skin (GM00637) and lung (IMR-90) fibroblast cells following Cd2+ exposure. The upregulation of DDAH I and II suggests a role of nitric oxide synthesis in Cd2+-induced toxicity in human cells.Keywords: Cd2+; mass spectrometry; nitric oxide synthesis; protein quantitation; reactive oxygen species; stable isotope labeling by amino acids in cell culture;

Reactive oxygen species (ROS), resulting from endogenous metabolism and/or environmental exposure, can induce damage to the 2-deoxyribose moiety in DNA. Specifically, a hydrogen atom from each of the five carbon atoms in 2-deoxyribose can be abstracted by hydroxyl radical, and improper chemical repair of the ensuing radicals formed at the C1′, C3′, and C4′ positions can lead to the stereochemical inversion at these sites to yield epimeric 2-deoxyribose lesions. Although ROS-induced single-nucleobase lesions have been well studied, the biological consequences of the C3′-epimeric lesions of 2′-deoxynucleosides, i.e., 2′-deoxyxylonucleosides (dxN), have not been comprehensively investigated. Herein, we assessed the impact of dxN lesions on the efficiency and fidelity of DNA replication in Escherichia coli cells by conducting a competitive replication and adduct bypass assay with single-stranded M13 phage containing a site-specifically incorporated dxN. Our results revealed that, of the four dxN lesions, only dxG constituted a strong impediment to DNA replication, and intriguingly, dxT and dxC conferred replication bypass efficiencies higher than those of the unmodified counterparts. In addition, the three SOS-induced DNA polymerases (Pol II, Pol IV, and Pol V) did not play any appreciable role in bypassing these lesions. Among the four dxNs, only dxA directed a moderate frequency of dCMP misincorporation. These results provided important insights into the impact of the C3′-epimeric lesions on DNA replication in E. coli cells.

Human exposure to arsenic in drinking water is known to be associated with the development of bladder, lung, kidney, and skin cancers. The molecular mechanisms underlying the carcinogenic effects of arsenic species remain incompletely understood. DNA interstrand cross-links (ICLs) are among the most cytotoxic type of DNA lesions that block DNA replication and transcription, and these lesions can be induced by endogenous metabolism and by exposure to exogenous agents. Fanconi anemia (FA) is a congenital disorder manifested with elevated sensitivity toward DNA interstrand cross-linking agents, and monoubiquitination of FANCD2 by FANCL is a crucial step in FA-mediated DNA repair. Here, we demonstrated that As3+ could bind to the PHD/RING finger domain of FANCL in vitro and in cells. This binding led to compromised ubiquitination of FANCD2 in cells and diminished recruitment of FANCD2 to chromatin and DNA damage sites induced by 4,5′,8-trimethylpsoralen plus UVA irradiation. Furthermore, clonogenic survival assay results showed that arsenite coexposure rendered cells more sensitive toward DNA interstrand cross-linking agents. Together, our study suggested that arsenite may compromise genomic stability via perturbation of the Fanconi anemia pathway, thereby conferring its carcinogenic effect.

A variety of endogenous and exogenous agents can induce DNA damage and lead to genomic instability. Reactive oxygen species (ROS), an important class of DNA damaging agents, are constantly generated in cells as a consequence of endogenous metabolism, infection/inflammation, and/or exposure to environmental toxicants. A wide array of DNA lesions can be induced by ROS directly, including single-nucleobase lesions, tandem lesions, and hypochlorous acid (HOCl)/hypobromous acid (HOBr)-derived DNA adducts. ROS can also lead to lipid peroxidation, whose byproducts can also react with DNA to produce exocyclic DNA lesions. A combination of bioanalytical chemistry, synthetic organic chemistry, and molecular biology approaches have provided significant insights into the occurrence, repair, and biological consequences of oxidatively induced DNA lesions. The involvement of these lesions in the etiology of human diseases and aging was also investigated in the past several decades, suggesting that the oxidatively induced DNA adducts, especially bulky DNA lesions, may serve as biomarkers for exploring the role of oxidative stress in human diseases. The continuing development and improvement of LC-MS/MS coupled with the stable isotope-dilution method for DNA adduct quantification will further promote research about the clinical implications and diagnostic applications of oxidatively induced DNA adducts.

The genetic integrity of living organisms is constantly threatened by environmental and endogenous sources of DNA damaging agents that can induce a plethora of chemically modified DNA lesions. Unrepaired DNA lesions may elicit cytotoxic and mutagenic effects and contribute to the development of human diseases including cancer and neurodegeneration. Understanding the deleterious outcomes of DNA damage necessitates the investigation about the effects of DNA adducts on the efficiency and fidelity of DNA replication and transcription.Conventional methods for measuring lesion-induced replicative or transcriptional alterations often require time-consuming colony screening and DNA sequencing procedures. Recently, a series of mass spectrometry (MS)-based strategies have been developed in our laboratory as an efficient platform for qualitative and quantitative analyses of the changes in genetic information induced by DNA adducts during DNA replication and transcription. During the past few years, we have successfully used these MS-based methods for assessing the replicative or transcriptional blocking and miscoding properties of more than 30 distinct DNA adducts. When combined with genetic manipulation, these methods have also been successfully employed for revealing the roles of various DNA repair proteins or translesion synthesis DNA polymerases (Pols) in modulating the adverse effects of DNA lesions on transcription or replication in mammalian and bacterial cells. For instance, we found that Escherichia coli Pol IV and its mammalian ortholog (i.e., Pol κ) are required for error-free bypass of N2-(1-carboxyethyl)-2′-deoxyguanosine (N2-CEdG) in cells. We also found that the N2-CEdG lesions strongly inhibit DNA transcription and they are repaired by transcription-coupled nucleotide excision repair in mammalian cells.In this Account, we focus on the development of MS-based approaches for determining the effects of DNA adducts on DNA replication and transcription, where liquid chromatography–tandem mass spectrometry is employed for the identification, and sometimes quantification, of the progeny products arising from the replication or transcription of lesion-bearing DNA substrates in vitro and in mammalian cells. We also highlight their applications to lesion bypass, mutagenesis, and repair studies of three representative types of DNA lesions, that is, the methylglyoxal-induced N2-CEdG, oxidatively induced 8,5′-cyclopurine-2′-deoxynucleosides, and regioisomeric alkylated thymidine lesions. Specially, we discuss the similar and distinct effects of the minor-groove DNA lesions including N2-CEdG and O2-alkylated thymidine lesions, as well as the major-groove O4-alkylated thymidine lesions on DNA replication and transcription machinery. For example, we found that the addition of an alkyl group to the O4 position of thymine may facilitate its preferential pairing with guanine and thus induce exclusively the misincorporation of guanine nucleotide opposite the lesion, whereas alkylation of thymine at the O2 position may render the nucleobase unfavorable in pairing with any of the canonical nucleobases and thus exhibit promiscuous miscoding properties during DNA replication and transcription. The MS-based strategies described herein should be generally applicable for quantitative measurement of the biological consequences and repair of other DNA lesions in vitro and in cells.

Humans are exposed to N-nitroso compounds through environmental exposure and endogenous metabolism. Some N-nitroso compounds can be metabolically activated to yield diazoacetate, which is known to induce DNA carboxymethylation. DNA lesion measurement remains one of the core tasks in toxicology and in evaluating human health risks associated with carcinogen exposure. In this study, we developed a highly sensitive nanoflow liquid chromatography-nanoelectrospray ionization-multistage tandem mass spectrometry (nLC-nESI-MS3) method for the simultaneous quantification of O6-carboxymethyl-2′-deoxyguanosine (O6-CMdG), O6-methyl-2′-deoxyguanosine (O6-MedG), and N6-carboxymethyl-2′-deoxyadenosine (N6-CMdA). We were able to measure the levels of these three lesions with the use of low-microgram quantities of DNA from cultured human skin fibroblasts and human colorectal carcinoma cells treated with azaserine, a DNA carboxymethylating agent. Our results revealed that the levels of O6-CMdG and O6-MedG increased when the dose of azaserine was increased from 0 to 450 μM. We, however, did not observe an apparent dose-dependent induction of N6-CMdA, suggesting the presence of repair mechanism(s) for the rapid clearance of this lesion in cells. This is the first report about the application of nLC-nESI-MS3 technique for the simultaneous quantification of O6-CMdG, O6-MedG, and N6-CMdA. The method reported here will be useful for future investigations about the repair of the carboxymethylated DNA lesions and about the implications of these lesions in carcinogenesis.

Kinases are one of the most important families of enzymes that are involved in numerous cell signaling processes. Existing methods for studying kinase expression and activation have limited kinome coverage. Herein we established a multiple-reaction monitoring (MRM)-based targeted proteomic method that provided an unprecedented coverage (∼80%) of the human kinome. We employed this method for profiling comprehensively the alterations of the global kinome of HEK293T human embryonic kidney cells upon treatment with methylglyoxal, a glycolysis byproduct that is present at elevated levels in blood and tissues of diabetic patients and is thought to contribute to diabetic complications. Our results led to the quantification of 328 unique kinases. In particular, we found that methylglyoxal treatment gave rise to altered expression of a number of kinases in the MAPK pathway and diminished expression of several receptor tyrosine kinases, including epidermal growth factor receptor (EGFR), insulin growth factor 2 receptor (IGF2R), fibroblast growth factor receptor (FGFR), etc. Furthermore, we demonstrated that the diminished expression of EGFR occurred through a mechanism that is distinct from the reduced expression of IGF2R and FGFR1. Together, our targeted kinome profiling method offers a powerful resource for exploring kinase-mediated signaling pathways that are altered by extracellular stimuli, and the results from the present study suggest new mechanisms underlying the development of diabetic complications.

Environmental and endogenous genotoxic agents can result in a variety of alkylated and carboxymethylated DNA lesions, including N3-ethylthymidine (N3-EtdT), O2-EtdT, and O4-EtdT as well as N3-carboxymethylthymidine (N3-CMdT) and O4-CMdT. By using nonreplicative double-stranded vectors harboring a site-specifically incorporated DNA lesion, we assessed the potential roles of alkyladenine DNA glycosylase (Aag); alkylation repair protein B homologue 2 (Alkbh2); or Alkbh3 in modulating the effects of N3-EtdT, O2-EtdT, O4-EtdT, N3-CMdT, or O4-CMdT on DNA transcription in mammalian cells. We found that the depletion of Aag did not significantly change the transcriptional inhibitory or mutagenic properties of all five examined lesions, suggesting a negligible role of Aag in the repair of these DNA adducts in mammalian cells. In addition, our results revealed that N3-EtdT, but not other lesions, could be repaired by Alkbh2 and Alkbh3 in mammalian cells. Furthermore, we demonstrated the direct reversal of N3-EtdT by purified human Alkbh2 protein in vitro. These findings provided important new insights into the repair of the carboxymethylated and alkylated thymidine lesions in mammalian cells.

Environmental exposure and endogenous metabolism can give rise to DNA alkylation. Among alkylated nucleosides, O4-alkylthymidine (O4-alkyldT) lesions are poorly repaired in mammalian systems and may compromise the efficiency and fidelity of cellular DNA replication. To cope with replication-stalling DNA lesions, cells are equipped with translesion synthesis DNA polymerases that are capable of bypassing various DNA lesions. In this study, we assessed human DNA polymerase η (Pol η)-mediated bypass of various O4-alkyldT lesions, with the alkyl group being Me, Et, nPr, iPr, nBu, iBu, (R)-sBu, or (S)-sBu, in template DNA by conducting primer extension and steady-state kinetic assays. Our primer extension assay results revealed that human Pol η, but not human polymerases κ and ι or yeast polymerase ζ, was capable of bypassing all O4-alkyldT lesions and extending the primer to generate full-length replication products. Data from steady-state kinetic measurements showed that Pol η preferentially misincorporated dGMP opposite O4-alkyldT lesions with a straight-chain alkyl group. The nucleotide misincorporation opposite most lesions with a branched-chain alkyl group was, however, not selective, where dCMP, dGMP, and dTMP were inserted at similar efficiencies opposite O4-iPrdT, O4-iBudT, and O4-(R)-sBudT. These results provide important knowledge about the effects of the length and structure of the alkyl group in O4-alkyldT lesions on the fidelity and efficiency of DNA replication mediated by human Pol η.

DNA alkylation represents a major type of DNA damage and is generally unavoidable due to ubiquitous exposure to various exogenous and endogenous sources of alkylating agents. Among the alkylated DNA lesions, O2-alkylthymidines (O2-alkyldT) are known to be persistent and poorly repaired in mammalian systems and have been shown to accumulate in the esophagus, lung, and liver tissue of rats treated with tobacco-specific N-nitrosamines, i.e., 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N′-nitrosonornicotine (NNN). In this study, we assessed the replicative bypass of a comprehensive set of O2-alkyldT lesions, with the alkyl group being a Me, Et, nPr, iPr, nBu, iBu, or sBu, in template DNA by conducting primer extension assays with the use of major translesion synthesis DNA polymerases. The results showed that human Pol η and, to a lesser degree, human Pol κ, but not human polymerase ι or yeast polymerase ζ, were capable of bypassing all O2-alkyldT lesions and extending the primer to generate full-length replication products. Data from steady-state kinetic measurements showed that human Pol η exhibited high frequencies of misincorporation of dCMP opposite those O2-alkyldT lesions bearing a longer straight-chain alkyl group. However, the nucleotide misincorporation opposite branched-chain lesions was not selective, with dCMP, dGMP, and dTMP being inserted at similar efficiencies, though the total frequencies of nucleotide misincorporation opposite the branched-chain lesions differed and followed the order of O2-iPrdT > O2-iBudT > O2-sBudT. Together, the results from the present study provided important knowledge about the effects of the length and structure of the alkyl group in the O2-alkyldT lesions on the fidelity and efficiency of DNA replication mediated by human Pol η.

Exogenous and endogenous sources of chemical species can react, directly or after metabolic activation, with DNA to yield DNA adducts. If not repaired, DNA adducts may compromise cellular functions by blocking DNA replication and/or inducing mutations. Unambiguous identification of the structures and accurate measurements of the levels of DNA adducts in cellular and tissue DNA constitute the first and important step towards understanding the biological consequences of these adducts. The advances in mass spectrometry (MS) instrumentation in the past 2–3 decades have rendered MS an important tool for structure elucidation, quantification, and revelation of the biological consequences of DNA adducts. In this review, we summarized the development of MS techniques on these fronts for DNA adduct analysis. We placed our emphasis of discussion on sample preparation, the combination of MS with gas chromatography- or liquid chromatography (LC)-based separation techniques for the quantitative measurement of DNA adducts, and the use of LC-MS along with molecular biology tools for understanding the human health consequences of DNA adducts. The applications of mass spectrometry-based DNA adduct analysis for predicting the therapeutic outcome of anti-cancer agents, for monitoring the human exposure to endogenous and environmental genotoxic agents, and for DNA repair studies were also discussed.

The rising interest in understanding the functions, regulation, and maintenance of the epitranscriptome calls for robust and accurate analytical methods for the identification and quantification of post-transcriptionally modified nucleosides in RNA. Monomethylations of cytidine and adenosine are common post-transcriptional modifications in RNA. Herein, we developed an LC–MS/MS/MS coupled with the stable isotope-dilution method for the sensitive and accurate quantifications of 5-methylcytidine (m5C), 2′-O-methylcytidine (Cm), N6-methyladenosine (m6A), and 2′-O-methyladenosine (Am) in RNA isolated from mammalian cells and tissues. Our results showed that the distributions of m5C, Cm and Am are tissue-specific. In addition, the 2′-O-methylated ribonucleosides (Cm and Am) are present at higher levels than the corresponding methylated nucleobase products (m5C and m6A) in total RNA isolated from mouse brain, pancreas, and spleen but not mouse heart. We also found that the levels of m5C, Cm, and Am are significantly lower (by 6.5–43-fold) in mRNA than in total RNA isolated from HEK293T cells, whereas the level of m6A was slightly higher (by 1.6-fold) in mRNA than in total RNA. The availability of this analytical method, in combination with genetic manipulation, may facilitate the future discovery of proteins involved in the maintenance and regulation of these RNA modifications.

Arsenic toxicity is a serious public health problem worldwide that brings more than 100 million people into the risk of arsenic exposure from groundwater and food contamination. Although there is accumulating evidence linking arsenic exposure with aberrant cytosine methylation in the global genome or at specific genomic loci, very few have investigated the impact of arsenic on the oxidation of 5-methylcytosine (5-mC) mediated by the Ten-eleven translocation (Tet) family of proteins. Owing to the high binding affinity of As(III) toward cysteine residues, we reasoned that the highly conserved C3H-type zinc fingers situated in Tet proteins may constitute potential targets for arsenic binding. Herein, we found that arsenite could bind directly to the zinc fingers of Tet proteins in vitro and in cells, and this interaction substantially impaired the catalytic efficiency of Tet proteins in oxidizing 5-mC to 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-foC), and 5-carboxylcytosine (5-caC). Treatments with arsenite also led to a dose-dependent decrease in the level of 5-hmC, but not 5-mC, in DNA isolated from HEK293T cells overexpressing the catalytic domain of any of the three Tet proteins and from mouse embryonic stem cells. Together, our study unveiled, for the first time, that arsenite could alter epigenetic signaling by targeting the zinc fingers of Tet proteins and perturbing the Tet-mediated oxidation of 5-mC in vitro and in cells. Our results offer important mechanistic understanding of arsenic epigenotoxicity and carcinogenesis in mammalian systems and may lead to novel approaches for the chemoprevention of arsenic toxicity.

Ionizing radiation is widely used in cancer therapy; however, cancer cells often develop radioresistance, which compromises the efficacy of cancer radiation therapy. Quantitative assessment of the alteration of the entire kinome in radioresistant cancer cells relative to their radiosensitive counterparts may provide important knowledge to define the mechanism(s) underlying tumor adaptive radioresistance and uncover novel target(s) for effective prevention and treatment of tumor radioresistance. By employing a scheduled multiple-reaction monitoring analysis in conjunction with isotope-coded ATP affinity probes, we assessed the global kinome of radioresistant MCF-7/C6 cells and their parental MCF-7 human breast cancer cells. We rigorously quantified 120 kinases, of which 1/3 exhibited significant differences in expression levels or ATP binding affinities. Several kinases involved in cell cycle progression and DNA damage response were found to be overexpressed or hyperactivated, including checkpoint kinase 1 (CHK1), cyclin-dependent kinases 1 and 2 (CDK1 and CDK2), and the catalytic subunit of DNA-dependent protein kinase. The elevated expression of CHK1, CDK1, and CDK2 in MCF-7/C6 cells was further validated by Western blot analysis. Thus, the altered kinome profile of radioresistant MCF-7/C6 cells suggests the involvement of kinases on cell cycle progression and DNA repair in tumor adaptive radioresistance. The unique kinome profiling results also afforded potential effective targets for resensitizing radioresistant cancer cells and counteracting deleterious effects of ionizing radiation exposure.

Poly(ADP-ribose) polymerase 3 (PARP3) is a member of the PARP family enzymes which catalyze the ADP-ribosylation of proteins. PARP3 plays an important role in DNA damage repair and mitotic progression. In this study, we identified, using mass spectrometric techniques, two novel post-translational modification sites in PARP3, α-N-methylation and phosphorylation of serine 461 (S461). We found that the N-terminal α-amino group of PARP3 is heavily methylated in human cells, and N-terminal RCC1 methyltransferase (NRMT) is a key enzyme required for this methylation. We also observed that the phosphorylation level of S461 in PARP3 could be reduced in human cells upon treatment with flavopiridol, a cyclin-dependent kinase inhibitor. Moreover, we demonstrated that S461 phosphorylation, but not α-N-methylation of PARP3, may be involved in the cellular response toward DNA double-strand breaks. These findings provide novel insights into the post-translational regulation of PARP3.

Oxidation of 5-methylcytosine in DNA by ten-eleven translocation (Tet) family of enzymes has been demonstrated to play a significant role in epigenetic regulation in mammals. We found that Tet enzymes also possess the activity of catalyzing the formation of 5-hydroxymethylcytidine (5-hmrC) in RNA in vitro. In addition, the catalytic domains of all three Tet enzymes as well as full-length Tet3 could induce the formation of 5-hmrC in human cells. Moreover, 5-hmrC was present at appreciable levels (∼1 per 5000 5-methylcytidine) in RNA of mammalian cells and tissues. Our results suggest the involvement of this oxidation in RNA biology.

Arsenic is a widespread environmental contaminant. However, the exact molecular mechanisms underlying the carcinogenic effects of arsenic remain incompletely understood. Core histones can be ubiquitinated by RING finger E3 ubiquitin ligases, among which the RNF20-RNF40 heterodimer catalyzes the ubiquitination of histone H2B at lysine 120. This ubiquitination event is important for the formation of open and biochemically accessible chromatin fiber that is conducive for DNA repair. Herein, we found that arsenite could bind directly to the RING finger domains of RNF20 and RNF40 in vitro and in cells, and treatment with arsenite resulted in substantially impaired H2B ubiquitination in multiple cell lines. Exposure to arsenite also diminished the recruitment of BRCA1 and RAD51 to laser-induced DNA double-strand break (DSB) sites, compromised DNA DSB repair in human cells, and rendered cells sensitive toward a radiomimetic agent, neocarzinostatin. Together, the results from the present study revealed, for the first time, that arsenite may exert its carcinogenic effect by targeting cysteine residues in the RING finger domains of histone E3 ubiquitin ligase, thereby altering histone epigenetic mark and compromising DNA DSB repair. Our results also suggest arsenite as a general inhibitor for RING finger E3 ubiquitin ligases.

Thiopurine drugs are widely used as antileukemic drugs and immunosuppressive agents, and 6-thioguanosine triphosphate (SGTP) is a major metabolite for these drugs. Recent studies have suggested that thiopurine drugs may exert their cytotoxic effects partly through binding of SGTP to a GTP-binding protein, Rac1. However, it remains unclear whether SGTP can also bind to other cellular proteins. Here, we introduced an orthogonal approach, encompassing nucleotide-affinity profiling and nucleotide-binding competition assays, to characterize comprehensively SGTP-binding proteins along with the specific binding sites from the entire human proteome. With the simultaneous use of SGTP and GTP affinity probes, we identified 165 SGTP-binding proteins that are involved in several different biological processes. We also examined the binding selectivities of these proteins toward SGTP and GTP, which allowed for the revelation of the relative binding affinities of the two nucleotides toward the nucleotide-binding motif sequence of proteins. Our results suggest that SGTP mainly targets GTPases, with strong binding affinities observed for multiple heterotrimeric G proteins. We also demonstrated that SGTP binds to several cyclin-dependent kinases (CDKs), which may perturb the CDK-mediated phosphorylation and cell cycle progression. Together, this represents the first comprehensive characterization of SGTP-binding property for the entire human proteome. We reason that a similar strategy can be generally employed for the future characterization of the interaction of other modified nucleotides with the global proteome.

Phosphorylation of cellular components catalyzed by kinases plays important roles in cell signaling and proliferation. Quantitative assessment of perturbation in global kinome may provide crucial knowledge for elucidating the mechanisms underlying the cytotoxic effects of environmental toxicants. Here, we utilized an adenosine triphosphate (ATP) affinity probe coupled with stable isotope labeling by amino acids in cell culture (SILAC) to assess quantitatively the arsenite-induced alteration of global kinome in human cells. We constructed a SILAC-compatible kinome library for scheduled multiple-reaction monitoring (MRM) analysis and adopted on-the-fly recalibration of retention time shift, which provided better throughput of the analytical method and enabled the simultaneous quantification of the expression of ∼300 kinases in two LC-MRM runs. With this improved analytical method, we conducted an in-depth quantitative analysis of the perturbation of kinome of GM00637 human skin fibroblast cells induced by arsenite exposure. Several kinases involved in cell cycle progression, including cyclin-dependent kinases (CDK1 and CDK4) and Aurora kinases A, B, and C, were found to be hyperactivated, and the altered expression of CDK1 was further validated by Western analysis. In addition, treatment with a CDK inhibitor, flavopiridol, partially restored the arsenite-induced growth inhibition of human skin fibroblast cells. Thus, sodium arsenite may confer its cytotoxic effect partly through the aberrant activation of CDKs and the resultant perturbation of cell cycle progression. Together, we developed a high-throughput, SILAC-compatible, and MRM-based kinome profiling method and demonstrated that the method is powerful in deciphering the molecular modes of action of a widespread environmental toxicant. The method should be generally applicable for uncovering the cellular pathways triggered by other extracellular stimuli.

•TiO2/MHMSS and ZrAs–Fe3O4@SiO2 were used for sequential phosphopeptides enrichment.•A novel strategy for the identification of phosphosites was established.•We were able to identify 11,579 unique phosphosites using the method.As one of the most important types of post-translational modifications, reversible phosphorylation of proteins plays crucial roles in a large number of biological processes. However, owing to the relatively low abundance and dynamic nature of phosphorylation and the presence of the unphosphorylated peptides in large excess, phosphopeptide enrichment is indispensable in large-scale phosphoproteomic analysis. Metal oxides including titanium dioxide have become prominent affinity materials to enrich phosphopeptides prior to their analysis using liquid chromatography-mass spectrometry (LC-MS). In the current study, we established a novel strategy, which encompassed strong cation exchange chromatography, sequential enrichment of phosphopeptides using titania-coated magnetic mesoporous hollow silica microspheres (TiO2/MHMSS) and zirconium arsenate-modified magnetic nanoparticles (ZrAs–Fe3O4@SiO2), and LC-MS/MS analysis, for the proteome-wide identification of phosphosites of proteins in HL60 cells. In total, we were able to identify 11,579 unique phosphorylation sites in 3432 unique proteins. Additionally, our results suggested that TiO2/MHMSS and ZrAs–Fe3O4@SiO2 are complementary in phosphopeptide enrichment, where the two types of materials displayed preferential binding of peptides carrying multiple and single phosphorylation sites, respectively.

Genomic integrity is constantly challenged by DNA damaging agents such as reactive oxygen species (ROS). Consequently, DNA damage can compromise the fidelity and efficiency of essential DNA metabolic processes, including replication and transcription, which may contribute significantly to the etiology of many human diseases. Here, we review one family of DNA lesions, the epimeric 2-deoxyribose lesions, which arise from the improper chemical repair of the 2-deoxyribose radicals. Unlike most other DNA lesions, the epimeric 2-deoxyribose lesions are indistinguishable from their corresponding unmodified nucleosides in both molecular mass and chemical reactivity. We placed our emphasis of discussion on the formation of these lesions, their impact on the structure and stability of duplex DNA, their biological consequences, their potential therapeutic relevance, and future research directions about these modified nucleosides.

Recently 5-hydroxymethyl-2′-deoxycytidine (5hmdC), 5-formyl-2′-deoxycytidine (5fdC), and 5-carboxyl-2′-deoxycytidine (5cadC) were discovered in mammalian DNA as oxidation products of 5-methyl-2′-deoxycytidine (5mdC) induced by the ten-eleven translocation family of enzymes. These oxidized derivatives of 5mdC may not only act as intermediates of active cytosine demethylation in mammals but also serve as epigenetic marks on their own. It remains unclear how 5hmdC, 5fdC, and 5cadC affect DNA replication in mammalian cells. Here, we examined the effects of the three modified nucleosides on the efficiency and accuracy of DNA replication in HEK293T human kidney epithelial cells. Our results demonstrated that a single, site-specifically incorporated 5fdC or 5cadC conferred modest drops, by approximately 30%, in replication bypass efficiency without inducing detectable mutations in human cells, whereas replicative bypass of 5hmdC is both accurate and efficient. The lack of pronounced perturbation of these oxidized 5mdC derivatives on DNA replication is consistent with their roles in epigenetic regulation of gene expression.

We investigated systematically the effects of Tet-induced oxidation products of 5-methylcytosine on Dnmt1- and DNMT3a-mediated cytosine methylation in synthetic duplex DNA. We found that the replacement of 5-methylcytosine at a CpG site with a 5-hydroxymethylcytosine, 5-formylcytosine, 5-carboxylcytosine or 5-hydroxymethyluracil resulted in altered methylation of cytosine at both the opposite and the neighboring CpG sites. Our results provided important new knowledge about the implications of the 5-methylcytosine oxidation products in maintenance cytosine methylation.

Alkylation and oxidation constitute major routes of DNA damage induced by endogenous and exogenous genotoxic agents. Understanding the biological consequences of DNA lesions often necessitates the availability of oligodeoxyribonucleotide (ODN) substrates harboring these lesions, and sensitive and robust methods for validating the identities of these ODNs. Tandem mass spectrometry is well suited for meeting these latter analytical needs. In the present study, we evaluated how the incorporation of an ethyl group to different positions (i.e., O2, N3, and O4) of thymine and the oxidation of its 5-methyl carbon impact collisionally activated dissociation (CAD) pathways of electrospray-produced deprotonated ions of ODNs harboring these thymine modifications. Unlike an unmodified thymine, which often manifests poor cleavage of the C3′–O3′ bond, the incorporation of an alkyl group to the O2 position and, to a much lesser extent, the O4 position, but not the N3 position of thymine, led to facile cleavage of the C3′–O3′ bond on the 3′ side of the modified thymine. Similar efficient chain cleavage was observed when thymine was oxidized to 5-formyluracil or 5-carboxyluracil, but not 5-hydroxymethyluracil. Additionally, with the support of computational modeling, we revealed that proton affinity and acidity of the modified nucleobases govern the fragmentation of ODNs containing the alkylated and oxidized thymidine derivatives, respectively. These results provided important insights into the effects of thymine modifications on ODN fragmentation.

β-D-glucosyl-5-hydroxymethyluracil (base J) is a hyper-modified nucleobase found in the nuclear DNA of kinetoplastid parasites. With replacement of a fraction of thymine in DNA, J is localized primarily in telomeric regions of all organisms carrying this modified base. The biosynthesis of J occurs in two putative steps: first, a specific thymine in DNA is recognized and converted into 5-hydroxymethyluracil (5-HmU) by J-binding proteins (JBP1 and JBP2); a glucosyl transferase (GT) subsequently glucosylates the 5-HmU to yield J. Although several recent studies revealed the roles of internal J in regulating transcription in kinetoplastids, functions of telomeric J and proteins involved in J synthesis remain elusive. Assessing the functions of base J and understanding fully its biosynthesis necessitate the measurement of its level in cells and organisms. In this study, we reported a reversed-phase HPLC coupled with tandem mass spectrometry (LC-MS/MS) method, together with the use of a surrogate internal standard (β-D-glucosyl-5-hydroxymethyl-2′-deoxycytidine, 5-gHmdC), for the accurate detection of β-D-glucosyl-5-hydroxymethyl-2′-deoxyuridine (dJ) in Trypanosoma brucei DNA. For comparison, we also measured the level of the precursor for dJ synthesis [i.e. 5-hydroxymethyl-2′-deoxyuridine (5-HmdU)]. We found that base J was not detectable in the JBP-null cells whereas it replaced approximately 0.5% thymine in wild-type cells, which was accompanied with a markedly decreased level of 5-HmdU in JBP1/JBP2-null strain relative to the wild-type strain. These results provided direct evidence supporting that JBP proteins play an important role in oxidizing thymidine to form 5-HmdU, which facilitated the generation of dJ. This is the first report about the application of LC-MS/MS for the quantification of base J. The analytical method built a solid foundation for dissecting the molecular mechanisms of J biosynthesis and assessing the biological functions of base J in the future.

Nucleotide-binding proteins play pivotal roles in many cellular processes including cell signaling. However, targeted studies of the subproteome of nucleotide-binding proteins, especially protein kinases and GTP-binding proteins, remain challenging. Here, we report a general strategy in using affinity-labeled chemical probes to enrich, identify, and quantify ATP- and GTP-binding proteins in the entire human proteome. Our results revealed that the ATP/GTP affinity probes facilitated the identification of 100 GTP-binding proteins and 206 kinases with the use of low milligram quantities of lysate of HL-60 cells. In combination with the use of the stable isotope labeling by amino acids in cell culture-based quantitative proteomics method, we assessed the ATP/GTP binding selectivities of nucleotide-binding proteins at the global proteome scale. Our results confirmed known and, more importantly, unveiled new ATP/GTP-binding preferences of hundreds of nucleotide-binding proteins. Additionally, our strategy led to the identification of three and one unique nucleotide-binding motifs for kinases and GTP-binding proteins, respectively, and the characterizations of the nucleotide-binding selectivities of individual motifs. Our strategy for capturing and characterizing ATP/GTP-binding proteins should be generally applicable for those proteins that can interact with other nucleotides.

Interstrand cross-links (ICLs) are highly toxic DNA lesions that block transcription and replication by preventing strand separation. ICL-inducing agents were among the earliest and are still the most widely used forms of chemotherapeutic drugs. Because of the repair of DNA ICLs, the therapeutic efficacy of the DNA cross-linking agents is often reduced by the development of chemoresistance in patients. Thus, it is very important to understand how various DNA ICLs are repaired. Such studies are currently hampered by the lack of an analytical method for monitoring directly the repair of DNA ICLs in cells. Here we report a high-performance liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) method, together with the isotope dilution technique, for assessing the repair of 8-methoxypsoralen (8-MOP)-induced DNA ICLs, as well as monoadducts (MAs), in cultured mammalian cells. We found that, while there were substantial decreases in the levels of ICL and MAs in repair-competent cells 24 h after 8-MOP/UVA treatment, there was little repair of 8-MOP-ICLs and -MAs in xeroderma pigmentosum, complementation group A-deficient human skin fibroblasts and excision repair cross-complementing rodent repair deficiency, complementation group 1-deficient Chinese hamster ovary cells over a 24 h period. This result provided unequivocal evidence supporting the notion that the 8-MOP photoadducts are substrates for nucleotide excision repair in mammalian cells. This is one of the first few reports about the application of LC–MS/MS for assessing the repair of DNA ICLs. The analytical method developed here, when combined with genetic manipulation, will also facilitate the assessment of the roles of other DNA repair pathways in removing these DNA lesions, and the method can also be generally applicable for investigating the repair of other types of DNA ICLs in mammalian cells.

ATP-binding proteins play significant roles in numerous cellular processes. Here, we introduced a novel isotope-coded ATP-affinity probe (ICAP) as an acylating agent to simultaneously enrich and incorporate isotope label to ATP-binding proteins. By taking advantage of the quantitative capability of this isotope-coded probe, we devised an affinity profiling strategy to comprehensively characterize ATP–protein interactions at the entire proteome scale. False-positive identification of ATP-binding sites derived from nonspecific labeling was effectively minimized through the comparison of the labeling behaviors of lysine residues with the use of low and high concentrations of the ICAP reagents. A total of 258 previously known ATP-binding proteins from lysates of HeLa-S3 and Jurkat-T cells were validated with this affinity profiling assay. Additionally, we demonstrated that this novel quantitative ATP-affinity profiling strategy is particularly useful for unveiling previously unrecognized nucleotide-binding sites in ATP-binding proteins. For example, our profiling results revealed K356 as a new ATP-binding site in HSP90. Furthermore, 293 proteins without documented ATP-binding GO were predicted to be ATP-binding proteins on the basis of our quantitative affinity profiling results. We also uncovered, for the first time, the ATP-binding capability of human proliferating cell nuclear antigen (PCNA), identified the lysine residue involved in ATP binding, and validated the protein’s capacity in ATP binding with an independent assay. The ICAP approach described in the present paper should be generally applicable for the quantitative assessment of ATP-binding proteins in proteomic samples from cells and tissues.

The association of tobacco smoke with decreased cell motility and wound healing is well documented; however, the cellular mechanisms and specific toxic tobacco constituents responsible for this effect are not well understood. Tobacco-specific N-nitrosamines (TSNAs) are among the most important classes of carcinogens found in tobacco products. The TSNA N′-nitrosonornicotine (NNN) is present at relatively high levels in tobacco and its smoke, as well as second- and third-hand smoke. To investigate the cellular pathways that are perturbed upon NNN exposure, we employed a quantitative proteomic approach, utilizing stable isotope labeling by amino acids in cell culture and mass spectrometry, to assess the NNN-induced alteration of protein expression in GM00637 human skin fibroblast cells. With this approach, we were able to quantify 2599 proteins, 191 of which displayed significantly changed expression following NNN exposure. One of the main findings from our proteomic analysis was the down-regulation of six different subunits of myosin, particularly nonmuscle myosin II heavy chain, isoforms A, B, and C. In addition, we found the altered expression of several extracellular matrix proteins and proteins involved in cellular adhesion. Together, our quantitative proteomic results suggested that NNN exposure may interfere with fibroblast motility. An in vitro scratch wound assay result supported that NNN exposure reduced the ability of dermal fibroblast to migrate into the scratched area. The results from the present study offer novel insights into the cellular mechanisms of NNN toxicity and identify NNN as a specific tobacco constituent that contributes to decreased fibroblast migration.

Hexavalent chromium [Cr(VI)] generated during industrial processes is carcinogenic. Although much is known about the deleterious effects caused by reactive oxygen species generated during the reduction of Cr(VI) after its absorption by biological systems, the precise mechanisms underlying Cr(VI) cytotoxicity remain poorly defined. Here, we analyzed, at the global proteome scale, the perturbation of protein expression in GM00637 human skin fibroblast cells upon exposure to potassium dichromate (K2Cr2O7). We were able to quantify ∼4600 unique proteins, among which ∼400 exhibited significant alterations in expression levels upon a 24-h treatment with 0.5 μM K2Cr2O7. Pathway analysis revealed the Cr(VI)-induced perturbation of cholesterol biosynthesis, G-protein signaling, inflammatory response, and selenoprotein pathways. In particular, we discovered that the K2Cr2O7 treatment led to pronouncedly elevated expression of a large number of enzymes involved in de novo cholesterol biosynthesis. Real-time PCR analysis revealed the increased mRNA expression of selected genes involved in cholesterol biosynthesis. Consistently, K2Cr2O7 treatment resulted in marked increases in cellular cholesterol level in multiple cell lines. Moreover, the Cr(VI)-induced growth inhibition of cultured human cells could be rescued by a cholesterol-lowering drug, lovastatin. Together, we demonstrated, for the first time, that Cr(VI) may exert its cytotoxic effect, at least partly, through the up-regulation of enzymes involved in de novo cholesterol biosynthesis and the resultant increase of cholesterol level in cells.

The eukaryotic centromere is an essential chromatin region required for accurate segregation of sister chromatids during cell division. Centromere protein B (CENP-B) is a highly conserved protein which can bind to the 17-bp CENP-B box on the centromeric DNA. In this study, we found that CENP-B could be α-N-methylated in human cells. We also showed that the level of the α-N-methylation was stimulated in cells in response to a variety of extracellular stimuli, including increased cell density, heat shock, and arsenite treatment, although the methylation level was not altered upon metaphase arrest. We identified N-terminal RCC1 methyltransferase (NRMT) as a major enzyme required for the CENP-B methylation. Additionally, we found that chromatin-bound CENP-B was primarily trimethylated and α-N-trimethylation could enhance CENP-B’s binding to CENP-B box in cells. Our study also expands the function of protein α-N-methylation that has been known for decades and whose function remains largely unexplored.

Causal links exist between smoking cigarettes and cancer development. Some genotoxic agents in cigarette smoke are capable of alkylating nucleobases in DNA, and higher levels of ethylated DNA lesions were observed in smokers than in nonsmokers. In this study, we examined comprehensively how the regioisomeric O2-, N3-, and O4-ethylthymidine (O2-, N3-, and O4-EtdT, respectively) perturb DNA replication mediated by purified human DNA polymerases (hPols) η, κ, and ι, yeast DNA polymerase ζ (yPol ζ), and the exonuclease-free Klenow fragment (Kf–) of Escherichia coli DNA polymerase I. Our results showed that hPol η and Kf– could bypass all three lesions and generate full-length replication products, whereas hPol ι stalled after inserting a single nucleotide opposite the lesions. Bypass conducted by hPol κ and yPol ζ differed markedly among the three lesions. Consistent with its known ability to efficiently bypass the minor groove N2-substituted 2′-deoxyguanosine lesions, hPol κ was able to bypass O2-EtdT, though it experienced great difficulty in bypassing N3-EtdT and O4-EtdT. yPol ζ was only modestly blocked by O4-EtdT, but the polymerase was strongly hindered by O2-EtdT and N3-EtdT. LC–MS/MS analysis of the replication products revealed that DNA synthesis opposite O4-EtdT was highly error-prone, with dGMP being preferentially inserted, while the presence of O2-EtdT and N3-EtdT in template DNA directed substantial frequencies of misincorporation of dGMP and, for hPol ι and Kf–, dTMP. Thus, our results suggested that O2-EtdT and N3-EtdT may also contribute to the AT → TA and AT → GC mutations observed in cells and tissues of animals exposed to ethylating agents.

Exposure of aqueous solutions of DNA to X- or γ-rays, which induces the hydroxyl radical as one of the major reactive oxygen species (ROS), can result in the generation of a battery of single-nucleobase and bulky DNA lesions. These include the (5′R) and (5′S) diastereomers of 8,5′-cyclo-2′-deoxyadenosine (cdA) and 8,5′-cyclo-2′-deoxyguanosine (cdG), which were also found to be present at appreciable levels in DNA isolated from mammalian cells and tissues. However, it remains unexplored how efficiently the cdA and cdG can be induced by Fenton-type reagents. By employing HPLC coupled with tandem mass spectrometry (LC-MS/MS/MS) with the use of the isotope-dilution technique, here we demonstrated that treatment of calf thymus DNA with Cu(II) or Fe(II), together with H2O2 and ascorbate, could lead to dose-responsive formation of both the (5′R) and (5′S) diastereomers of cdA and cdG, though the yields of cdG were 2–4 orders of magnitude lower than that of 8-oxo-7,8-dihydro-2′-deoxyguanosine. This result suggests that the Fenton reaction may constitute an important endogenous source for the formation of the cdA and cdG. Additionally, the (5′R) diastereomers of cdA and cdG were induced at markedly higher levels than the (5′S) counterparts. This latter finding, in conjunction with the previous observations of similar or greater levels of the (5′S) than (5′R) diastereomers of the two lesions in mammalian tissues, furnishes an additional line of evidence to support the more efficient repair of the (5′R) diastereomers of the purine cyclonucleosides in mammalian cells.

The tobacco-specific N-nitrosamine, 4-(methylnitrosamino)-1-(3-pyridinyl)-1-butanone (NNK), is a well-known carcinogen. Although the ability of the metabolically activated form of NNK to generate DNA adducts is well established, little is known about the cellular pathways perturbed by NNK in its native state. In this study, we utilized stable isotope labeling by amino acid in cell culture (SILAC), together with mass spectrometry, to assess the perturbation of protein expression in GM00637 human skin fibroblast cells upon NNK exposure. With this approach, we were able to quantify 1412 proteins and 137 of them were with significantly altered expression following NNK exposure, including the up-regulation of all subunits of the 20S proteasome core complex. The up-regulation of the 20S core complex was also reflected by a significant increase in 20S proteasome activities in GM00637, IMR90, and MCF-7 cells upon NNK treatment. Furthermore, the β-adrenergic receptor (β-AR) antagonist propranolol could attenuate significantly the NNK-induced increase in proteasome activity in all the three cell lines, suggesting that up-regulation of the 20S proteasome may be mediated through the β-AR. Additionally, we found that NNK treatment altered the expression levels of other important proteins including mitochondrial proteins, cytoskeleton-associated proteins, and proteins involved in glycolysis and gluconeogenesis. Results from the present study provided novel insights into the cellular mechanisms targeted by NNK.

Reactive oxygen species (ROS), which can be produced during normal aerobic metabolism, can induce the formation of tandem DNA lesions, including 8,5′-cyclo-2′-deoxyadenosine (cyclo-dA) and 8,5′-cyclo-2′-deoxyguanosine (cyclo-dG). Previous studies have shown that cyclo-dA and cyclo-dG accumulate in cells and can block mammalian RNA polymerase II and replicative DNA polymerases. Here, we used primer extension and steady-state kinetic assays to examine the efficiency and fidelity for polymerase η to insert nucleotides opposite, and extend primer past, these cyclopurine lesions. We found that Saccharomyces cerevisiae and human polymerase η inserted 2′-deoxynucleotides opposite cyclo-dA, cyclo-dG and their adjacent 5′ nucleosides at fidelities and efficiencies that were similar to those of their respective undamaged nucleosides. Moreover, the yeast enzyme exhibited similar processivity in DNA synthesis on templates housing a cyclo-dA or cyclo-dG to those carrying an unmodified dA or dG; the human polymerase, however, dissociated from the primer–template complex after inserting one or two additional nucleotides after the lesion. Pol η’s accurate and efficient bypass of cyclo-dA and cyclo-dG indicates that this polymerase is likely responsible for error-free bypass of these lesions, whereas mutagenic bypass of these lesions may involve other translesion synthesis DNA polymerases. Together, our results suggested that pol η may have an additional function in cells, i.e., to alleviate the cellular burden of endogenously induced DNA lesions, including cyclo-dA and cyclo-dG.

O2- and O4-methylthymidine (O2-MdT and O4-MdT) can be induced in tissues of laboratory animals exposed with N-methyl-N-nitrosourea, a known carcinogen. These two O-methylated DNA adducts have been shown to be poorly repaired and may contribute to the mutations arising from exposure to DNA methylating agents. Here, in vitro replication studies with duplex DNA substrates containing site-specifically incorporated O2-MdT and O4-MdT showed that both lesions blocked DNA synthesis mediated by three different DNA polymerases, including the exonuclease-free Klenow fragment of Escherichia coli DNA polymerase I (Kf–), human DNA polymerase κ (pol κ), and Saccharomyces cerevisiae DNA polymerase η (pol η). Results from steady-state kinetic measurements and LC-MS/MS analysis of primer extension products revealed that Kf– and pol η preferentially incorporated the correct nucleotide (dAMP) opposite O2-MdT, while O4-MdT primarily directed dGMP misincorporation. While steady-state kinetic experiments showed that pol κ-mediated nucleotide insertion opposite O2-MdT and O4-MdT is highly promiscuous, LC-MS/MS analysis of primer extension products demonstrated that pol κ favorably incorporated the incorrect dGMP opposite both lesions. Our results underscored the limitation of the steady-state kinetic assay in determining how DNA lesions compromise DNA replication in vitro. In addition, the results from our study revealed that, if left unrepaired, O-methylated thymidine lesions may constitute important sources of nucleobase substitutions emanating from exposure to alkylating agents.

B3LYP/6-311+G(2d,p)//B3LYP/6-31+G(d) density functional theory calculations were employed to explore the kinetics and thermodynamics of gas-phase N-glycosidic bond cleavage induced by nucleophilic attack of C1′ with a hydroxide ion in 5-substituted 2′-deoxycytidines. The results showed that, among the 5-substituted 2′-deoxycytidine derivatives examined [XdC, where X = H (dC), CH3 (medC), CH2OH (hmdC), CHO (fmdC), COOH (cadC), F (FdC), or Br (BrdC)], fmdC and cadC exhibited the lowest energy barrier and largest exothermicity for N-glycosidic bond cleavage. These results paralleled previously reported nucleobase excision activities of human thymine DNA glycosylase (hTDG) toward duplex DNA substrates harboring a thymine and 5-substituted cytosine derivatives when paired with a guanine. Our study suggests that the inherent chemistry associated with the nucleophilic cleavage of N-glycosidic bond constitutes a major factor contributing to the selectivity of hTDG toward 5-substituted dC derivatives. These findings provided novel insights into the role of TDG in active cytosine demethylation.

The purpose of our study was to develop suitable methods to quantify oxidative DNA lesions in the setting of transition metal-related diseases. Transition metal-driven Fenton reactions constitute an important endogenous source of reactive oxygen species (ROS). In genetic diseases with accumulation of transition metal ions, excessive ROS production causes pathophysiological changes, including DNA damage. Wilson’s disease is an autosomal recessive disorder with copper toxicosis due to deficiency of ATP7B protein needed for excreting copper into bile. The Long-Evans Cinnamon (LEC) rat bears a deletion in Atp7b gene and serves as an excellent model for hepatic Wilson’s disease. We used a sensitive capillary liquid chromatography−electrospray-tandem mass spectrometry (LC−ESI-MS/MS/MS) method in conjunction with the stable isotope-dilution technique to quantify several types of oxidative DNA lesions in the liver and brain of LEC rats. These lesions included 5-formyl-2′-deoxyuridine, 5-hydroxymethyl-2′-deoxyuridine, and the 5′R and 5′S diastereomers of 8,5′-cyclo-2′-deoxyguanosine and 8,5′-cyclo-2′-deoxyadenosine. Moreover, the levels of these DNA lesions in the liver and brain increased with age and correlated with age-dependent regulation of the expression of DNA repair genes in LEC rats. These results provide significant new knowledge for better understanding the implications of oxidative DNA lesions in transition metal-induced diseases, such as Wilson’s disease, as well as in aging and aging-related pathological conditions.

Lovastatin, a member of the statin family of drugs, is widely prescribed for treating hypercholesterolemia. The statin family of drugs, however, also shows promise for cancer treatment and prevention. Although lovastatin is known to be an inhibitor for HMG-CoA reductase, the precise mechanisms underlying the drug’s antiproliferative activity remain unclearly defined. Here we utilized mass spectrometry, in conjunction with stable isotope labeling by amino acids in cell culture (SILAC), to analyze the perturbation of protein expression in HL-60 cells treated with lovastatin. We were able to quantify ∼3200 proteins with both forward and reverse SILAC labeling experiments, among which ∼120 exhibited significant alterations in expression levels upon lovastatin treatment. Apart from confirming the expected inhibition of the cholesterol biosynthesis pathway, our quantitative proteomic results revealed that lovastatin perturbed the estrogen receptor signaling pathway, which was manifested by the diminished expression of estrogen receptor α, steroid receptor RNA activator 1, and other related proteins. Lovastatin also altered glutamate metabolism through down-regulation of glutamine synthetase and γ-glutamylcysteine synthetase. Moreover, lovastatin treatment led to a marked down-regulation of carbonate dehydratase II (a.k.a. carbonic anhydrase II) and perturbed the protein ubiquitination pathway. Together, the results from the present study underscored several new cellular pathways perturbed by lovastatin.

Aberrant cytosine methylation is known to be associated with cancer development. Here, we assessed how common cancer chemotherapeutic agents perturb cytosine methylation in Jurkat-T acute lymphoblastic leukemia cells. We tested six antitumor agents and found that cyclophosphamide induced the most pronounced increase in global DNA cytosine methylation after a 24-h treatment. Long-term treatment with cyclophosphamide led to a time-dependent increase in cytosine methylation level with up to 4 days of treatment, and the extent of cytosine methylation returned to normal level after 8 days. The trend of change in DNA methylation level paralleled that of the expression level of DNMT1 protein, whereas no significant increase in DNMT1 mRNA level was observed. Previous studies showed that the stability of endogenous DNMT1 protein is regulated by lysine methylation through histone lysine methyltransferase Set7 and lysine-specific demethylase 1 (LSD1), with the methylated DNMT1 being the target for proteasomal degradation. We observed that the elevated expression of DNMT1 protein at 4 days of treatment was correlated with the increased expression of LSD1 protein and with the decreased frequency of K142 methylation in DNMT1. Taken together, our results showed that cyclophosphamide perturbed temporarily global cytosine methylation in Jurkat-T cells via regulation of the lysine methylation level in DNMT1.

Humans are exposed to N-nitroso compounds (NOCs) both endogenously and exogenously from a number of environmental sources, and NOCs are both mutagenic and carcinogenic. After metabolic activation, some NOCs can induce carboxymethylation of nucleobases through a diazoacetate intermediate, which could give rise to p53 mutations similar to those seen in human gastrointestinal cancers. It was previously found that the growth of polymerase η-deficient human cells was inhibited by treatment with azaserine, a DNA carboxymethylation agent, suggesting the importance of this polymerase in bypassing the azaserine-induced carboxymethylated DNA lesions. In this study, we examined how carboxymethylated DNA lesions, which included N6-carboxymethyl-2′-deoxyadenosine (N6-CMdA), N4-carboxymethyl-2′-deoxycytidine (N4-CMdC), N3-carboxymethylthymidine (N3-CMdT), and O4-carboxymethylthymidine (O4-CMdT), perturbed the efficiency and fidelity of DNA replication mediated by Saccharomyces cerevisiae polymerase η (pol η). Our results from steady-state kinetic assay showed that pol η could readily bypass and extend past N6-CMdA and incorporated the correct nucleotides opposite the lesion and its neighboring 5′-nucleoside with high efficiency. By contrast, the polymerase could bypass N4-CMdC inefficiently, with substantial misincorporation of dCMP followed by dAMP, though pol η could extend past the lesion with high fidelity and efficiency when dGMP was incorporated opposite the lesion. On the other hand, yeast pol η experienced great difficulty in bypassing O4-CMdT and N3-CMdT, and the polymerase inserted preferentially the incorrect dGMP opposite these two DNA lesions; the extension step, nevertheless, occurred with high fidelity and efficiency when the correct dAMP was opposite the lesion, as opposed to the preferentially incorporated incorrect dGMP. These results suggest that these lesions may contribute significantly to diazoacetate-induced mutations and those in the p53 gene observed in human gastrointestinal tumors.

Core histones are known to carry a variety of post-translational modifications (PTMs), including acetylation, phosphorylation, methylation and ubiquitination, which play important roles in the epigenetic control of gene expression. The nature and biological functions of these PTMs in histones from plants, animals and budding yeast have been extensively investigated. In contrast, the corresponding studies for fission yeast were mainly focused on histone H3. In the present study, we applied LC–nano-ESI-MS/MS, coupled with multiple protease digestion, to identify PTMs in histones H2A, H2B and H4 from Schizosaccharomyces pombe, the typical model organism of fission yeast. Various protease digestions provided high sequence coverage for PTM mapping, and accurate mass measurement of fragment ions allowed for unambiguous differentiation of acetylation from tri-methylation. Many modification sites conserved in other organisms were identified in S. pombe. In addition, some unique modification sites, including N-terminal acetylation in H2A and H2B as well as K123 acetylation in H2A.β, were observed. Our results provide a comprehensive picture of the PTMs of histones H2A, H2B and H4 in S. pombe, which serves as a foundation for future investigations on the regulation and functions of histone modifications in this important model organism.Graphical abstractWe reported here a mass spectrometric investigation of the post-translational modifications of histones H2A, H2B, and H4 from Schizosaccharomyces pombe.Research highlights▶ PTMs of histones H2A, H2B and H4 from Schizosaccharomyces pombe were mapped by LC-nano-ESI-MS/MS. ▶ Most modification sites were conserved among Schizosaccharomyces pombe and other organisms. ▶ Novel modification sites were found in H2A and H2B.

Thiopurines, including mercaptopurine (MP), 6-thioguanine (SG) and azathioprine, are widely used for the treatment of many human diseases including acute lymphoblastic leukemia (ALL). To exert their cytotoxic effect, these prodrugs need to be metabolically activated to SG nucleotides and incorporated into nucleic acids. SG in DNA can be methylated spontaneously to S6-methylthioguanine (S6mG) in the presence of S-adenosyl-l-methionine. It was proposed that S6mG, owing to its high miscoding potential (pairing preferentially with thymine), may induce cell death by triggering the postreplicative mismatch repair pathway. Understanding the implications of this pathway in the cytotoxic effect of thiopurine drugs necessitates an accurate measurement of the level of S6-methylthio-2′-deoxyguanosine (S6mdG) in DNA of cells treated with thiopurine drugs. Here we developed a sensitive HPLC coupled with tandem mass spectrometry (LC-MS/MS) method and measured the level of 6-thio-2′-deoxyguanosine (SdG) and S6mdG in genomic DNA of four human leukemia cell lines and one human colorectal carcinoma cell line. Our results revealed that, upon treatment with 3 μM SG for 24 h, approximately 10, 7.4, 7, and 3% of guanine was replaced with SG in Jurkat T, HL-60, CCRF-CEM and K-562 cells, respectively. However, only less than 0.02% of SdG was converted to S6mdG in the above cell lines. HCT-116 cells had the lowest level (0.2%) of guanine being replaced with SG in DNA, and approximately 5 out of 104 SG was converted to its methylated counterpart. This is the first report of the simultaneous and accurate quantification of SdG and S6mdG in genomic DNA of cultured human cells treated with SG. In addition, our results suggested that DNA SG might trigger mismatch repair (MMR) pathway without being converted to S6mG.

Graphene was utilized for the first time as a matrix for the analysis of low molecular weight compounds using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Polar compounds including amino acids, polyamines, anticancer drugs, and nucleosides could be successfully analyzed. Additionally, nonpolar compounds including steroids could be detected with high resolution and sensitivity. Compared with a conventional matrix, graphene exhibited a high desorption/ionization efficiency for nonpolar compounds. The graphene matrix functions as a substrate to trap analytes, and it transfers energy to the analytes upon laser irradiation, which allows for the analytes to be readily desorbed/ionized and interference of intrinsic matrix ions to be eliminated. The use of graphene as a matrix avoided the fragmentation of analytes and provided good reproducibility and a high salt tolerance, underscoring the potential application of graphene as a matrix for MALDI MS analysis of practical samples in complex sample matrixes. We also demonstrated that the use of graphene as an adsorbent for the solid-phase extraction of squalene could improve greatly the detection limit. This work not only opens a new field for applications of graphene, but also offers a new technique for high-speed analysis of low molecular weight compounds in areas such as metabolism research and natural product characterization.

Thiopurines are effective immunosuppressants and anticancer agents. However, the long-term use of thiopurines was found to be associated with a significantly increased risk of various types of cancer. To date, the specific mechanism(s) underlying the carcinogenicity associated with thiopurine treatment remain(s) unclear. Herein, we constructed duplex pTGFP-Hha10 shuttle vectors carrying a 6-thioguanine (SG) or S6-methylthioguanine (S6mG) at a unique site and allowed the vectors to propagate in three different human cell lines. Analysis of the replication products revealed that although neither thionucleoside blocked considerably DNA replication in any of the human cell lines, both SG and S6mG were mutagenic, resulting in G→A mutation at frequencies of ∼8% and ∼39%, respectively. Consistent with what was found from our previous study in E. coli cells, our data demonstrated that the mutagenic properties of SG and S6mG provided significant evidence for mutation induction as a potential carcinogenic mechanism associated with chronic thiopurine intervention.

Arsenic is ubiquitously present in the environment; it is a known human carcinogen and paradoxically it is also a successful drug for the clinical remission of acute promyelocytic leukemia (APL). The cellular responses induced by arsenite treatment have been investigated for years; however, the precise mechanisms underlying its cytotoxicity and therapeutic activity remain unclear. Here we report the use of mass spectrometry together with stable isotope labeling by amino acids in cell culture (SILAC) for the comparative study of protein expression in HL-60 cells that were untreated or treated with a clinically relevant concentration of arsenite. Our results revealed that, among the 1067 proteins quantified in both forward and reverse SILAC measurements, 56 had significantly altered levels of expression induced by arsenite treatment. These included the up-regulation of core histones, neutrophil elastase, α-mannosidase as well as the down-regulation of fatty acid synthase and protein phosphatase 1α. We further demonstrated that the arsenite-induced growth inhibition of HL-60 cells could be rescued by treatment with palmitate, the final product of fatty acid synthase, supporting that arsenite exerts its cytotoxic effect, in part, via suppressing the expression of fatty acid synthase and inhibiting the endogenous production of fatty acid. The results from the present study offered important new knowledge for gaining insights into the molecular mechanisms of action of arsenite.

Doxorubicin remains an important part of chemotherapy regimens in the clinic and is considered an effective agent in the treatment of acute lymphoblastic leukemia (ALL). Although the cellular responses induced by doxorubicin treatment have been investigated for years, the precise mechanisms underlying its cytotoxicity and therapeutic activity remain unclear. Here we utilized mass spectrometry, together with stable isotope labeling by amino acids in cell culture (SILAC), to analyze comparatively the protein expression in Jurkat-T cells before and after treatment with a clinically relevant concentration of doxorubicin. We were able to quantify 1066 proteins in Jurkat-T cells with both forward and reverse SILAC measurements, among which 62 were with significantly altered levels of expression induced by doxorubicin treatment. These included the up-regulation of core histones, heterogeneous nuclear ribonucleoproteins, and superoxide dismutase 2 as well as the down-regulation of hydroxymethylglutaryl-CoA synthase and farnesyl diphosphate synthase. The latter two are essential enzymes for cholesterol biosynthesis. We further demonstrated that the doxorubicin-induced growth inhibition of Jurkat-T cells could be rescued by treatment with cholesterol, supporting that doxorubicin exerts its cytotoxic effect, in part, by suppressing the expression of hydroxymethylglutaryl-CoA synthase and farnesyl diphosphate synthase, thereby inhibiting the endogenous production of cholesterol. The results from the present study provide important new knowledge for gaining insights into the molecular mechanisms of action of doxorubicin.

G-quadruplex motifs are known to be present in telomeres of human and other organisms. Recent bioinformatic studies also revealed the widespread existence of these motifs in promoter regions of human genes. Treatment of cultured cells with 5-bromo-2′-deoxyuridine (BrdU) is known to result in the substitution of DNA thymidine with BrdU; such replacement has been shown to sensitize cells to killing induced by UV light. Our previous studies revealed that the exposure of BrdU-carrying duplex DNA or BrdU-treated MCF-7 cells to UVB light could lead to the facile formation of intrastrand cross-link products initiated from BrdU. Here we found that the exposure of BrdU-bearing G-quadruplex DNA to UVA light could also give rise to the efficient formation of the G[8−5]U intrastrand cross-link, where the C8 of guanine in the external G-tetrad is covalently linked with the C5 of its adjacent 3′ uracil in the loop region. In addition, the yield for the cross-link product is dependent on the conformation of the G-quadruplex. Together, the formation of intrastrand cross-link in G-quadruplex motifs may account for the photocytotoxic effect induced by BrdU incorporation, and the BrdU-mediated photo-cross-linking may constitute a useful method for monitoring the different conformations of G-quadruplex folding.

Core histones are susceptible to a variety of post-translational modifications (PTMs), among which methylation and acetylation play critical roles in various chromatin-dependent processes. The nature and biological functions of these PTMs have been extensively studied in plants, animals, and yeasts. In contrast, the histone modifications in Neurospora crassa, a convenient model organism for multicellular eukaryotes, remained largely undefined. In this study, we used several mass spectrometric techniques, coupled with HPLC separation and multiple-protease digestion, to identify the methylation and acetylation sites in core histones isolated from Neurospora. Electron transfer dissociation (ETD) was employed to fragment the heavily modified long N-terminal peptides. In addition, accurate mass measurement of fragment ions allowed for unambiguous differentiation of acetylation from trimethylation. Many modification sites conserved in other organisms were identified in Neurospora. In addition, some unique modification sites in histone H2B, including N-terminal α methylation, methylation at K3, and acetylation at K19, K28, and K29, were observed. Our analysis provides a potentially comprehensive picture of methylation and acetylation of core histones in Neurospora, which should serve as a foundation for future studies of the function of histone PTMs in this model organism.

Glyoxal is generated endogenously from the degradation of glucose and the oxidation of carbohydrates, lipids, and the 2-deoxyribose moieties of DNA. Glyoxal is also widely used in industry and is present in cigarette smoke and food. Glyoxal can conjugate with nucleobases and proteins to give advanced glycation end products. N2-Carboxymethyl-2′-deoxyguanosine (N2-CMdG) and the cyclic 1,N2-glyoxal-dG are the major glyoxal adducts formed in DNA. In this study, we first assessed the stabilities of these two adducts. It turned out that 1,N2-glyoxal-dG was very unstable, with more than 70% of the adduct being decomposed to dG upon a 24 h incubation at 37 °C in phosphate-buffered saline. However, N2-CMdG was very stable; less than 0.5% of the lesion was degraded to dG after a 7 day incubation under the same conditions. We further developed a sensitive capillary liquid chromatography−electrospray ionization−tandem mass spectrometry coupled with a stable isotope dilution method and quantified the formation of N2-CMdG in calf thymus DNA and 293T human kidney epithelial cells that were exposed to glyoxal and in calf thymus DNA treated with d-glucose. Our results showed that N2-CMdG was produced at 2−134 lesions per 106 nucleosides in calf thymus DNA when the surrounding glyoxal concentration was increased from 10 to 500 μM and approximately 3−27 lesions per 107 nucleosides, while the d-glucose concentration changed from 2 to 50 mM. Furthermore, N2-CMdG was induced endogenously in 293T human kidney epithelial cells and exposure to glyoxal further stimulated the formation of this lesion; the level of this adduct ranged from 7 to 15 lesions per 108 nucleosides, while the glyoxal concentration increased from 10 μM to 1.25 mM. Collectively, our results suggested that N2-CMdG might serve as a biomarker for glyoxal exposure.

Reactive oxygen species can induce the formation of not only single-nucleobase lesions, which have been extensively studied, but also tandem lesions. Herein, we report a high frequency of formation of a type of tandem lesion, where two commonly observed oxidatively induced single-nucleobase lesions, that is, thymidine glycol (Tg) and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), are vicinal to each other in calf thymus DNA upon exposure to Cu(II)/ascorbate along with H2O2 or γ-rays. We further explored how the tandem lesions perturb the efficiency and fidelity of DNA replication by assessing the replication products formed from the propagation, in Escherichia coli cells, of the single-stranded pYMV1 shuttle vectors containing two tandem lesions [5′-(8-oxodG)-Tg-3′ and 5′-Tg-(8-oxodG)-3′] or an isolated Tg or 8-oxodG. The bypass efficiencies for the two tandem lesions were approximately one-half of those for the two isolated single-nucleobase lesions. The presence of an adjacent Tg could lead to significant increases in G→T transversion at the 8-oxodG site as compared to that of a single 8-oxodG lesion; the frequencies of G→T mutation were approximately 18, 32, and 28% for 8-oxodG that is isolated, in 5′-(8-oxodG)-Tg-3′ and in 5′-Tg-(8-oxodG)-3′, respectively. Moreover, both pol IV and pol V are involved, in part, in bypassing the Tg, either present alone or as part of the tandem lesions, in E. coli cells. Together, our results support that complex lesions could exert greater cytotoxic and mutagenic effects than when the composing individual lesions are present alone.

DNA-binding proteins are very important constituents of proteomes of all species and play crucial roles in transcription, DNA replication, recombination, repair, and other activities associated with DNA. Although a number of DNA-binding proteins have been identified, many proteins involved in gene regulation and DNA repair are likely still unknown because of their dynamic and/or weak interactions with DNA. In this report, we described an approach for the comprehensive identification of DNA-binding proteins with in vivo formaldehyde cross-linking and LC−MS/MS. DNA-binding proteins could be purified via the isolation of DNA−protein complexes and released from the complexes by reversing the cross-linking. By using this method, we were able to identify more than one hundred DNA-binding proteins, such as proteins involved in transcription, gene regulation, DNA replication and repair, and a large number of proteins that are potentially associated with DNA and DNA-binding proteins. This method should be generally applicable to the investigation of other nucleic acid-binding proteins, and hold great potential in the comprehensive study of gene regulation, DNA damage response and repair, as well as many other critical biological processes at proteomic level.

Thiopurines are among the most successful chemotherapeutic agents for treating a number of human diseases including acute lymphoblastic leukemia. The mechanisms through which the thiopurines elicit their cytotoxic effects remain unclear. We postulate that the incorporation of 6-thioguanine into the CpG site may perturb the methyltransferase-mediated cytosine methylation at this site, thereby interfering with the epigenetic pathways of gene regulation. To gain biochemical evidence for this hypothesis, we assessed, by using a restriction enzyme digestion coupled with LC-MS/MS method, the impact of 6-thioguanine on cytosine methylation mediated by two DNA methyltransferases, human DNMT1 and bacterial HpaII. Our results revealed that the incorporation of 6-thioguanine into the CpG site could affect the methylation of the cytosine residue by both methyltransferases and the effect on cytosine methylation is dependent on the position of 6-thioguanine with respect to the cytosine to be methylated. The presence of 6-thioguanine at the methylated CpG site enhanced the DNMT1-mediated methylation of the opposing cytosine in the complementary strand, whereas the presence of 6-thioguanine at the unmethylated CpG site abolished almost completely the methylation of its 5′ adjacent cytosine by both DNMT1 and HpaII. We further demonstrated that the treatment of Jurkat T cells, which were derived from acute lymphoblastic leukemia, with 6-thioguanine could result in an appreciable drop in the level of global cytosine methylation. These results showed that 6-thioguanine, after being incorporated into DNA, may perturb the epigenetic pathway of gene regulation.

Reactive oxygen species can induce the formation of tandem DNA lesions. We recently showed that the treatment of calf thymus DNA with Cu2+/H2O2/ascorbate could result in the efficient formation of a tandem lesion where a 5,6-dihydroxy-5,6-dihydrothymidine (or thymidine glycol) is situated on the 5′ side of an 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG). In the present study, we assessed how the 5′-Tg-(8-oxodG)-3′ and 5′-(8-oxodG)-Tg-3′ tandem lesions are replicated by purified DNA polymerases and how they are recognized by base excision repair enzymes. Our results revealed that the tandem lesions blocked primer extension mediated by the Klenow fragment and yeast polymerase η more readily than when the Tg or 8-oxodG was present alone. The mutagenic properties of Tg or 8-oxodG differed while they were present alone or in tandem. Moreover, the human 8-oxoguanine-DNA glycosylase (hOGG1)-mediated cleavage of 8-oxodG was compromised considerably by the presence of a neighboring 5′ Tg, whereas the presence of Tg as the adjacent 3′ nucleoside enhanced 8-oxodG cleavage by hOGG1. The efficiency for the cleavage of Tg by endonuclease III was not affected by the presence of an adjoining 8-oxodG. These results supported the notion that the replication and repair of tandem single-nucleobase lesions depend on the types of lesions involved and their spatial arrangement.

Core histones are susceptible to a range of post-translational modifications (PTMs), including acetylation, phosphorylation, methylation, and ubiquitination, which play important roles in the epigenetic control of gene expression. Here, we observed an unusual discrepancy between MALDI-MS/MS and ESI-MS/MS on the methylation of trimethyllysine-containing peptides with residues 9–17 from human histone H3 and residues 73–83 from yeast histone H3. It turned out that the discrepancy could be attributed to an unusual methyl group migration from the side chain of trimethyllysine to the C-terminal arginine residue during peptide fragmentation, and this methyl group transfer only occurred for singly charged ions, but not for doubly charged ions. The methyl group transfer argument received its support from the results on the studies of the fragmentation of the ESI- or MALDI-produced singly charged ions of several synthetic trimethyllysine-bearing peptides. The results presented in this study highlighted that caution should be exerted while MS/MS of singly charged ions is employed to interrogate the PTMs of trimethyllysine-containing peptides.Methyl group migration was observed during the fragmentation of MALDI- or ESI-produced singly charged ions of trimethyllysine-containing peptides.Figure optionsDownload full-size imageDownload high-quality image (51 K)Download as PowerPoint slide

The collision-induced dissociation pathways of isomeric cytosine-guanine and cytosine-adenine intrastrand crosslink-containing dinucleoside monophosphates were investigated with the stable isotope-labeled compounds to gain insights into the effects of chemical structure on the fragmentation pathways of these DNA modifications. A Dimroth-like rearrangement, which was reported for protonated 2′-deoxycytidine and involved the switching of the exocyclic N4 with the ring N3 nitrogen atom, was also observed for the cytosine component in the protonated ions of C[5–8]G, C[5–2]A, and C[5–8]A, but not C[5-N2]G or C[5-N6]A. In these two sets of crosslinks, the C5 of cytosine is covalently bonded with its neighboring purine base via a carbon atom on the aromatic ring and an exocyclic nitrogen atom, respectively. On the contrary, the rearrangement could occur for the deprotonated ions of C[5-N2]G, C[5-N6]A, and unmodified cytosine, but not C[5–8]G, C[5–2]A, or C[5–8]A. In addition, ammonia could be lost more readily from C[5-N2]G and C[5-N6]A than from C[5–8]G, C[5–2]A, and C[5–8]A. The results from the present study afforded important guidance for the application of mass spectrometry for the structure elucidation of other intrastrand/interstrand crosslink lesions.

Upon exposure to UVA light, psoralens can induce DNA interstrand cross-links (ICLs), which can block DNA replication and transcription. Among the psoralen derivatives, 8-methoxypsoralen (8-MOP) is conventionally applied for psoriasis therapy, and amotosalen S59 is used to inactivate bacterial and viral pathogens in blood components. In addition to the ICL formation, psoralens also readily form various monoadducts (MAs) with thymidine residues in DNA when exposed to UVA light, and the biological implications for these monoadducts remain unclear. Here, we reported a method that encompassed digestion with a single enzyme (nuclease P1) and LC−MS/MS, for the simultaneous quantification of ICL and MAs induced in human cells exposed with 8-MOP or S59 and UVA light. Our results showed that the yield of ICL induced by S59, which increased from 3.9 to 12.8 lesions/103 nucleotides as the dose of UVA light increased from 0.5 to 10.0 J/cm2, was ∼100 fold more than that induced by 8-MOP. In addition, three and five products were identified as 8-MOP- and S59-MAs, respectively, and the yields of MAs were significantly lower than that for ICL. The yields of the three 8-MOP-MAs were 7.6−2.2, 1.9−9.9, and 7.2−51 per 106 nucleotides and those of the five S59-MAs were 215−19, 106−39, 25−21, 32−146, and 22−26 per 106 nucleotides as the dose of UVA light increased from 0.5 to 10.0 J/cm2. Although the yields of MAs induced by 8-MOP and S59 were lower than those of the respective ICLs under the same exposure conditions, the formation of appreciable amounts of MAs might account for some of the mutations induced by psoralens.

Plasma membrane proteins play critical roles in cell-to-cell recognition, signal transduction and material transport. Because of their accessibility, membrane proteins constitute the major targets for protein-based drugs. Here, we described an approach, which included stable isotope labeling by amino acids in cell culture (SILAC), cell surface biotinylation, affinity peptide purification and LC-MS/MS for the identification and quantification of cell surface membrane proteins. We applied the strategy for the quantitative analysis of membrane proteins expressed by a pair of human melanoma cell lines, WM-115 and WM-266-4, which were derived initially from the primary and metastatic tumor sites of the same individual. We were able to identify more than 100 membrane and membrane-associated proteins from these two cell lines, including cell surface histones. We further confirmed the surface localization of histone H2B and three other proteins by immunocytochemical analysis with confocal microscopy. The contamination from cytoplasmic and other nonmembrane-related sources is greatly reduced by using cell surface biotinylation and affinity purification of biotinylated peptides. We also quantified the relative expression of 62 identified proteins in the two types of melanoma cells. The application to quantitative analysis of membrane proteins of primary and metastatic melanoma cells revealed great potential of the method in the comprehensive identification of tumor progression markers as well as in the discovery of new protein-based therapeutic targets.

The integrity of the human genome is frequently challenged by endogenous and exogenous agents, and reactive oxygen species (ROS) constitute the major endogenous source of DNA damage. ROS-induced single nucleobase lesions have been extensively investigated; however, the formation and biological implications of bulky DNA lesions emanating from ROS exposure remain under-explored. The combination of synthetic organic chemistry and bioanalytical chemistry have led to the discovery of a group of bulky, oxidatively generated DNA lesions. In these lesions, a nucleobase, often a purine base, can be covalently bonded with the 5′ carbon of the 2-deoxyribose of the same nucleoside or its neighboring pyrimidine base to give purine cyclonucleosides and nucleobase−nucleobase intrastrand cross-links, respectively. Biochemical studies demonstrated that these lesions could markedly block DNA replication and transcription and that these lesions are repaired by the nucleotide excision repair (NER) pathway. These bulky, oxidatively induced DNA lesions may contribute significantly to neurological disorders that are associated with deficiency in NER and the natural processes of aging.

DinB, a Y-family DNA polymerase, is conserved among all domains of life; however, its endogenous substrates have not been identified. DinB is known to synthesize accurately across a number of N2-dG lesions. Methylglyoxal (MG) is a common byproduct of the ubiquitous glycolysis pathway and induces the formation of N2-(1-carboxyethyl)-2′-deoxyguanosine (N2-CEdG) as the major stable DNA adduct. Here, we found that N2-CEdG could be detected at a frequency of one lesion per 107 nucleosides in WM-266-4 human melanoma cells, and treatment of these cells with MG or glucose led to a dose-responsive increase in N2-CEdG formation. We further constructed single-stranded M13 shuttle vectors harboring individual diastereomers of N2-CEdG at a specific site and assessed the cytotoxic and mutagenic properties of the lesion in wild-type and bypass polymerase-deficient Escherichia coli cells. Our results revealed that N2-CEdG is weakly mutagenic, and DinB (i.e., polymerase IV) is the major DNA polymerase responsible for bypassing the lesion in vivo. Moreover, steady-state kinetic measurements showed that nucleotide insertion, catalyzed by E. coli pol IV or its human counterpart (i.e., polymerase κ), opposite the N2-CEdG is both accurate and efficient. Taken together, our data support that N2-CEdG, a minor-groove DNA adduct arising from MG, is an important endogenous substrate for DinB DNA polymerase.

The chromosomal high-mobility group A (HMGA) proteins, composed of HMGA1a, HMGA1b and HMGA2, play important roles in the regulation of numerous processes in eukaryotic cells, such as transcriptional regulation, DNA repair, RNA processing, and chromatin remodeling. The biological activities of HMGA1 proteins are highly regulated by their post-translational modifications (PTMs), including acetylation, methylation, and phosphorylation. Recently, it was found that the homeodomain-interacting protein kinase-2 (HIPK2), a newly identified serine/threonine kinase, co-immunoprecipitated with, and phosphorylated, HMGA1 proteins. However, the sites and the biological significance of the phosphorylation have not been elucidated. Here, we found that HIPK2 phosphorylates HMGA1a at Ser-35, Thr-52, and Thr-77, and HMGA1b at Thr-41 and Thr-66. In addition, we demonstrated that cdc2, which is known to phosphorylate HMGA1 proteins, could induce the phosphorylation of HMGA1 proteins at the same Ser/Thr sites. The two kinases, however, exhibited different site preferences for the phosphorylation: The preference for HIPK2 phosphorylation followed the order of Thr-77 > Thr-52 > Ser-35, whereas the order for cdc2 phosphorylation was Thr-52 > Thr-77 > Ser-35. Moreover, we found that the HIPK2-phosphorylated HMGA1a reduced the binding affinity of HMGA1a to human germ line ϵ promoter, and the drop in binding affinity induced by HIPK2 phosphorylation was lower than that introduced by cdc2 phosphorylation, which is consistent with the notion that the second AT-hook in HMGA1a is more important for DNA binding than the third AT-hook.

High mobility group (HMG) A1 proteins are subject to a number of post-translational modifications, which may regulate their function in gene transcription and other cellular processes. We examined, by using mass spectrometry, the acetylation of HMGA1a and HMGA1b proteins induced by histone acetyltransferases p300 and PCAF in vitro and in PC-3 human prostate cancer cells in vivo. It turned out that five lysine residues in HMGA1a, i. e., Lys-14, Lys-64, Lys-66, Lys-70, and Lys-73, could be acetylated by both p300 and PCAF. We further quantified the level of acetylation by analyzing, with LC-MS/MS, the proteolytic peptides of the in vitro or in vivo acetylated HMGA1 proteins where the unmodified lysine residues were chemically derivatized with a perdeuterated acetyl group. Quantification results revealed that p300 and PCAF exhibited different site preferences for the acetylation; the preference of p300 acetylation followed the order of Lys-64∼Lys-70 > Lys-66 > Lys-14∼Lys73, whereas the selectivity of PCAF acetylation followed the sequence of Lys-70∼Lys-73 > Lys-64∼Lys-66 > Lys-14. HMGA1b was acetylated in a very similar fashion as HMGA1a. We also demonstrated that C-terminal phosphorylation of HMGA1 proteins did not affect the in vitro acetylation of the two proteins by either p300 or PCAF. Moreover, we examined the acetylation of lysine residues in HMGA1a and HMGA1b isolated from PC-3 human prostate cancer cells. Our results showed that all the above five lysine residues were also acetylated in vivo, with Lys-64, Lys-66 and Lys-70 in HMGA1a exhibiting higher levels of acetylation than Lys-14 and Lys-73.

Quantitative assessment of post-translational modifications in proteins by mass spectrometry often requires the consideration of the alteration in ionization efficiency of peptides induced by the modification. Herein, we introduced a method to measure the relative ionization efficiencies of peptides using specifically designed unlabeled peptides. In our design, the peptide under study, in either the unmodified or modified form, is linked with an internal standard peptide via an enzyme cleavage site; thus, after enzymatic digestion, we could obtain readily a 1:1 ratio between the peptide under investigation and the internal standard peptide. The relative ionization efficiencies of the modified and unmodified peptides can then be calculated from the modification-induced change in the ratio of relative abundances of the ion of the peptide of interest over that of the internal standard peptide. We demonstrated the usefulness of the method by assessing the change in ionization efficiencies of four peptides introduced by phosphorylation.

Recent studies revealed that the 3′-terminal nucleotides in plant microRNAs were methylated on the ribose at the 2′ or 3′ hydroxyl groups. Here we examined the fragmentation of the electrospray-produced [M + H]+ and [M − H]− ions of 2′- and 3′-O-methylated ribonucleosides. It turned out that the predominant fragmentation pathway for the [M + H]+ ions of ribose-methylated nucleosides was the neutral loss of the methylated ribose, which made it impossible to distinguish 2′-O-methylation from 3′-O-methylation by positive-ion MS/MS. However, characteristic fragment ions, resulting from the cleavage through the ribose rings, were produced for the [M − H]− ions of each pair of ribose-methylated nucleosides. In this respect, the neutral loss of a 90-Da fragment (C3H6O3) was observed for 2′-O-methylated cytidine, guanosine and adenosine, but not for their 3′-O-methylated counterparts. On the other hand, the neutral loss of a 60-Da fragment (C2H4O2) was found for 3′-O-methyluridine, but not for 2′-O-methyluridine.

We examined the collisionally activated dissociation (CAD) pathways of protonated 2′-deoxycytidine (dC), 5-formyl-2′-deoxycytidine (5-FmdC), 5-hydroxy-2′-deoxycytidine (5-OHdC), 5-hydroxymethyl-2′-deoxycytidine (5-HmdC), and their corresponding stable isotope-labeled compounds to gain insights into the effects of modifications on the fragmentation pathways of the pyrimidine bases. Multi-stage MS (MSn) results showed that protonated cytosine, its 5-hydroxyl- and 5-hydroxymethyl-substituted derivatives, but not its 5-formyl-substituted analog, could undergo Dimroth-like rearrangement in the gas-phase. The elimination of HNCO was one of the major fragmentation pathways observed for the protonated ions of all dC derivatives except for 5-hydroxymethylcytosine, which underwent this loss only after a H2O molecule had been eliminated. In addition, the protonated cytosine and 5-hydroxycytosine can undergo a facile elimination of NH3 molecule. This loss, however, was not observed for protonated 5-hydroxymethylcytosine, 5-formylcytosine, and their uracil analogs. Taken together, our study demonstrated that modifications could alter markedly the CAD patterns of the protonated pyrimidine bases. The results from this study provided a basis for the identifications of other modified pyrimidine bases/nucleosides by tandem mass spectrometry.

High mobility group (HMG) A1 proteins are subject to a number of post-translational modifications, which may regulate their function in gene transcription and other cellular processes. We examined, by using mass spectrometry, the acetylation of HMGA1a and HMGA1b proteins induced by histone acetyltransferases p300 and PCAF in vitro and in PC-3 human prostate cancer cells in vivo. It turned out that five lysine residues in HMGA1a, i.e., Lys-14, Lys-64, Lys-66, Lys-70, and Lys-73, could be acetylated by both p300 and PCAF. We further quantified the level of acetylation by analyzing, with LC-MS/MS, the proteolytic peptides of the in vitro or in vivo acetylated HMGA1 proteins where the unmodified lysine residues were chemically derivatized with a perdeuterated acetyl group. Quantification results revealed that p300 and PCAF exhibited different site preferences for the acetylation; the preference of p300 acetylation followed the order of Lys-64∼Lys-70 > Lys-66 > Lys-14∼Lys73, whereas the selectivity of PCAF acetylation followed the sequence of Lys-70∼Lys-73 > Lys-64∼Lys-66 > Lys-14. HMGA1b was acetylated in a very similar fashion as HMGA1a. We also demonstrated that C-terminal phosphorylation of HMGA1 proteins did not affect the in vitro acetylation of the two proteins by either p300 or PCAF. Moreover, we examined the acetylation of lysine residues in HMGA1a and HMGA1b isolated from PC-3 human prostate cancer cells. Our results showed that all the above five lysine residues were also acetylated in vivo, with Lys-64, Lys-66 and Lys-70 in HMGA1a exhibiting higher levels of acetylation than Lys-14 and Lys-73.

Quantitative assessment of post-translational modifications in proteins by mass spectrometry often requires the consideration of the alteration in ionization efficiency of peptides induced by the modification. Herein, we introduced a method to measure the relative ionization efficiencies of peptides using specifically designed unlabeled peptides. In our design, the peptide under study, in either the unmodified or modified form, is linked with an internal standard peptide via an enzyme cleavage site; thus, after enzymatic digestion, we could obtain readily a 1:1 ratio between the peptide under investigation and the internal standard peptide. The relative ionization efficiencies of the modified and unmodified peptides can then be calculated from the modification-induced change in the ratio of relative abundances of the ion of the peptide of interest over that of the internal standard peptide. We demonstrated the usefulness of the method by assessing the change in ionization efficiencies of four peptides introduced by phosphorylation.

The collision-induced dissociation pathways of isomeric cytosine-guanine and cytosine-adenine intrastrand crosslink-containing dinucleoside monophosphates were investigated with the stable isotope-labeled compounds to gain insights into the effects of chemical structure on the fragmentation pathways of these DNA modifications. A Dimroth-like rearrangement, which was reported for protonated 2′-deoxycytidine and involved the switching of the exocyclic N4 with the ring N3 nitrogen atom, was also observed for the cytosine component in the protonated ions of C[5–8]G, C[5–2]A, and C[5–8]A, but not C[5-N2]G or C[5-N6]A. In these two sets of crosslinks, the C5 of cytosine is covalently bonded with its neighboring purine base via a carbon atom on the aromatic ring and an exocyclic nitrogen atom, respectively. On the contrary, the rearrangement could occur for the deprotonated ions of C[5-N2]G, C[5-N6]A, and unmodified cytosine, but not C[5–8]G, C[5–2]A, or C[5–8]A. In addition, ammonia could be lost more readily from C[5-N2]G and C[5-N6]A than from C[5–8]G, C[5–2]A, and C[5–8]A. The results from the present study afforded important guidance for the application of mass spectrometry for the structure elucidation of other intrastrand/interstrand crosslink lesions.Collisional activation of isotope-labeled cytosine-adenine and cytosine-guanine crosslinks revealed structure-dependent neutral losses of ammonia and cleavages arising from Dimroth-like rearrangements.Download high-res image (101KB)Download full-size image

Exogenous and endogenous sources of chemical species can react, directly or after metabolic activation, with DNA to yield DNA adducts. If not repaired, DNA adducts may compromise cellular functions by blocking DNA replication and/or inducing mutations. Unambiguous identification of the structures and accurate measurements of the levels of DNA adducts in cellular and tissue DNA constitute the first and important step towards understanding the biological consequences of these adducts. The advances in mass spectrometry (MS) instrumentation in the past 2–3 decades have rendered MS an important tool for structure elucidation, quantification, and revelation of the biological consequences of DNA adducts. In this review, we summarized the development of MS techniques on these fronts for DNA adduct analysis. We placed our emphasis of discussion on sample preparation, the combination of MS with gas chromatography- or liquid chromatography (LC)-based separation techniques for the quantitative measurement of DNA adducts, and the use of LC-MS along with molecular biology tools for understanding the human health consequences of DNA adducts. The applications of mass spectrometry-based DNA adduct analysis for predicting the therapeutic outcome of anti-cancer agents, for monitoring the human exposure to endogenous and environmental genotoxic agents, and for DNA repair studies were also discussed.