Detection and characterization of hydroxyl radical adducts by mass spectrometry

The study of the influence of free radicals in the biological process depends primarily on the capacity to detect these reactive species. In this work we have studied the application of mass spectrometry to the identification of hydroxyl radical species. The detection and identification by collisional activation mass-analyzed ion kinetic energy spectrometry (CA-MIKES) of a spin adduct of DMPO with the hydroxyl radical [(DMPO + O) + H]+ (m/z 130) has demonstrated that mass spectrometry can be a powerful tool in the detection and identification of spin adducts of DMPO with hydroxyl radical species. We were also able to detect the capture of secondary free radicals using ethanol by detecting and identifying the corresponding adduct [(DMPO + ethanol) + H]+. Other spin adducts have also been detected and identified. We consider that the use of mass spectrometry is a relevant technique for the detection of free hydroxyl radicals, especially in complex mixtures, since mass spectrometry is able to discriminate these adducts in such situations. Moreover, using this approach, it was possible to identify new spin adducts.     

Detection and identification of carcinogen-peptide adducts by nanoelectrospray tandem mass spectrometry

Nanoelectrospray (nanoES) tandem mass spectrometry was used to examine covalently modified peptides in crude enzymatic digests of human serum albumin (HSA) that had been exposed to either benzo[a]pyrene diol epoxide (B[a]PDE, 1), chrysene diol epoxide (CDE, 2), 5-methylchrysene diol epoxide (5MeCDE, 3), or benzo[g]chrysene diol epoxide (B[g]CDE, 4). The low flow rates of nanoES (～20 nL/min) allowed several MS/MS experiments to be optimized and performed on a single sample with very little sample consumption (～30 min analysis time/µL sample). Initially, nanoES was compared with conventional LC/MS/MS analysis of carcinogen-peptide adducts. For example, nanoES analysis of an unseparated digest of B[a]PDE-treated serum albumin revealed the same peptides (RRHPY and RRHPY-FYAPE) that were previously shown, by LC/MS/MS, to be adducted with B[a]PDE. In addition, nanoES could detect unstable peptide adducts that might not otherwise have been directly observable. Finally, nanoES was shown to be an effective way to screen mixtures of modified and unmodified peptides for which no chromatographic information is available.     

Structural characterization of acetylpyridinium-ethyl pyruvate adducts by electrospray ionization mass spectrometry

The reactions of ethyl pyruvate with acetic anhydride and pyridine were studied by electrospray ionization mass spectrometry (ESI-MS). Ethyl 2-acetoxy-2-pyridiniumpropionate (1) (m/z 238) resulting from the reaction of the acetylpyridinium cation with ethyl pyruvate, and the adduct of ethyl 2-acetoxyacrylate with a pyridinium cation (2), bound together by non-covalent interactions (m/z 238), were identified by ESI-MS for the first time. Structures 1 and 2 cannot be distinguished, probably because one may be converted into the other and vice versa.     

Identification of leucine-enkephalin radical oxidation products by liquid chromatography tandem mass spectrometry

The oxidation of the peptide leucine-enkephalin (YGGFL) induced by the hydroxyl radical (HO*), formed under Fenton-like conditions [Cu (II)/H(2)O(2)], was studied and monitored by LC-MS. The oxidation products identified included products resultant from (a) the insertion of oxygen atoms (1-5), (b) peptide backbone cleavage (short-chain products formed by diamide pathway) and (c) radical-radical crosslinking reactions. In order to identify the modified residues, LC-MS/MS spectra were obtained. The insertion of oxygen atoms into the peptide originated hydroxide, di-hydroxide and/or hydroperoxide derivatives. In addition it was found that the aromatic amino acids are most susceptible to being hydroxylated, while the aliphatic amino acids are more prone to forming hydroperoxides. Oxidation products with double bonds were also identified. The short chain products resulted from the alpha-carbon radical of terminal amino acids (Tyr and Leu). Products resulting from cross-linking reactions between intact carbon-centered peptide radical (with and without one HO group) and a side chain radical (*C(7)H(7)O) were identified. It was found that, although all amino acids residues of the peptide undergo modifications, the N-terminal seems to be prone to oxidative modifications under these conditions.     

Detection of benzopyrene-deoxyguanosine adducts by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A method for the detection of BPDE-d guanosine adducts using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is described and illustrated. The results indicate that MALDI is capable of detecting two other DNA benzopyrene adducts, which are trace products formed during the synthesis of BPDE-d guanosine. This MALDI-TOFMS method offers the potential for the detection of DNA adducts in human tissue using very limited sample purification and preparation.     

High resolution mass spectrometry based profiling of diet-related deoxyribonucleic acid adducts

Exposure of DNA to endo- and exogenous DNA binding chemicals can result in the formation of DNA adducts and is believed to be the first step in chemically induced carcinogenesis. DNA adductomics is a relatively new field of research which studies the formation of known and unknown DNA adducts in DNA due to exposure to genotoxic chemicals. In this study, a new UHPLC-HRMS(/MS)-based DNA adduct detection method was developed and validated. Four targeted DNA adducts, which all have been linked to dietary genotoxicity, were included in the described method; O⁶-methylguanine (O⁶-MeG), O⁶-carboxymethylguanine (O⁶-CMG), pyrimidopurinone (M1G) and methylhydroxypropanoguanine (CroG). As a supplementary tool for DNA adductomics, a DNA adduct database, which currently contains 123 different diet-related DNA adducts, was constructed. By means of the newly developed method and database, all 4 targeted DNA adducts and 32 untargeted DNA adducts could be detected in different DNA samples. The obtained results clearly demonstrate the merit of the described method for both targeted and untargeted DNA adduct detection in vitro and in vivo, whilst the diet-related DNA adduct database can distinctly facilitate data interpretation.     

Structural analysis of drug-DNA adducts by tandem mass spectrometry

The utility of electrospray ionisation (ESI) tandem mass spectrometry (MS/MS) for the characterisation of ligand-oligonucleotide adducts is demonstrated with adducts formed between the oligonucleotide 5'-CACGTG-3' and both a platinating agent, cis-diamminedichloroplatinum(II) (cisplatin), and an alkylating ligand, n-bromohexylphenanthridinium bromide (phenC6Br). We have demonstrated previously that negative ion MS/MS spectra of alkylated oligonucleotides show a highly specific fragmentation pathway that enables the site of binding of the ligand to be readily identified. In comparison, the positive ion ESI-MS/MS spectra reported here also show a single major fragmentation pathway, but the dominant ion is the protonated ligand-base adduct. MS/MS of this ion confirms the site on binding of the ligand to the guanine base. MS/MS spectra of cisplatin adducts show much less specific fragmentation than alkylated adducts, particularly in the negative ion mode. This suggests that the ESI-MS/MS spectra of ligand-DNA adducts are strongly influenced by the extent to which the ligand weakens the glycosidic bond in the residue to which it is bound. For platinating agents, which do not labilise the glycosidic bond, additional experiments involving MS/MS of source-generated product ions were required to enable isomeric adducts to be distinguished.     

Separation and characterization of oxidized isomeric lipid–peptide adducts by ion mobility mass spectrometry

Phospholipids are major components of cell membranes and lipoprotein complexes. They are prone to oxidation by endogenous and exogenous reactive oxygen species yielding a large variety of modified lipids including small aliphatic and phospholipid bound aldehydes and ketones. These carbonyls are strong electrophiles that can modify proteins and, thereby, alter their structures and functions triggering various pathophysiological conditions. The analysis of lipid–protein adducts by liquid chromatography‐MS is challenged by their mixed chemical nature (polar peptide and hydrophobic lipid), low abundance in biological samples, and formation of multiple isomers. Thus, we investigated traveling wave ion mobility mass spectrometry (TWIMS) to analyze lipid–peptide adducts generated by incubating model peptides corresponding to the amphipathic β₁ sheet sequence of apolipoprotein B‐100 with 1‐palmitoyl‐2‐(oxo‐nonanoyl)‐sn‐glycerophosphatidylcholine (PONPC). The complex mixture of peptides, lipids, and peptide–lipid adducts was separated by TWIMS, which was especially important for the identification of two mono‐PONPC‐peptide isomers containing Schiff bases at different lysine residues. Moreover, TWIMS separated structural conformers of one peptide–lipid adduct possessing most likely different orientations of the hydrophobic sn‐1 fatty acyl residue and head group of PONPC, relative to the peptide backbone. Copyright © 2015 John Wiley & Sons, Ltd.     

Characterization of soluble oligomers produced by electrochemical oxidation of o-phenylenediamine by electrospray ionization sequential mass spectrometry

Soluble species generated during the electropolymerization of o-phenylenediamine (o-PD) on platinum electrodes in aqueous buffers at different pH values were investigated by electrospray ionization ion trap sequential mass spectrometry (ESI-ITMS(n)). The main protonated molecules (MH(+)) detected in the full scan ESI-MS spectra of the electrolytic solutions were isolated in the ion trap and sequentially fragmented (MS(n), with n up to 5) to obtain fragmentation patterns. The latter led to hypotheses as to the molecular structures of the soluble products of o-PD electropolymerization; it appeared that all of them are actually oligomeric species in different oxidation states. In particular, o-PD dimers, trimers and tetramers could be identified and three common structural features were found, namely: the presence of phenazine, 1,4-benzoquinonediimine, and secondary amine (acting as bridges between benzene rings) units. These findings are in agreement with those already reported for the surface structure of the polymeric films formed on the platinum electrodes during o-PD polymerization, thus suggesting that a close relationship exists between the soluble oligomers and the polymer itself.     

超高效液相色谱-串联质谱法鉴定与分析乙醛诱导脱氧核糖核苷酸加合物

采用超高效液相色谱-串联质谱法对两种非对映异构体（6S,8S）1,N²-丙基-2'-脱氧鸟苷（ProdG）和（6R,8R）ProdG加合物进行鉴定与分析。通过色谱保留时间及质谱碎裂方式分析,证明乙醛与2'-脱氧鸟苷（dG）反应可形成ProdG加合物。体外实验表明,乙醛能够诱导脱氧核糖核苷酸（DNA）形成ProdG加合物,并且（6R,8R）ProdG的生成量大于（6S,8S）ProdG的生成量。细胞实验结果显示,乙醛暴露能显著提高人肺胚成纤维细胞（MRC5）基因组DNA中ProdG加合物的水平,且ProdG加合物的水平与乙醛的暴露浓度呈正相关。此外,100 μ mol/L的乙醛暴露使（6R,8R）ProdG的含量从（6.4±0.3） 个/10⁸个碱基增加到（127.2±2.7） 个/10⁸个碱基,上调程度大于（6S,8S）ProdG（从（6.5±0.3） 个/10⁸个碱基增加到（115.3±2.5） 个/10⁸个碱基）。该工作为乙醛暴露所引起的DNA加合物水平上升提供了实验依据。     

Glycosylation characterization of recombinant human erythropoietin produced in glycoengineered Pichia pastoris by mass spectrometry

Glycosylation plays a critical role in the in vivo efficacy of both endogenous and recombinant erythropoietin (EPO). Using mass spectrometry, we characterized the N‐/O‐linked glycosylation of recombinant human EPO (rhEPO) produced in glycoengineered Pichia pastoris and compared with the glycosylation of Chinese hamster ovary (CHO) cell‐derived rhEPO. While the three predicted N‐linked glycosylation sites (Asn24, Asn38 and Asn83) showed complete site occupancy, Pichia‐ and CHO‐derived rhEPO showed distinct differences in the glycan structures with the former containing sialylated bi‐antennary glycoforms and the latter containing a mixture of sialylated bi‐, tri‐ and tetra‐antennary structures. Additionally, the N‐linked glycans from Pichia‐produced rhEPO were similar across all three sites. A low level of O‐linked mannosylation was detected on Pichia‐produced rhEPO at position Ser126, which is also the O‐linked glycosylation site for endogenous human EPO and CHO‐derived rhEPO. In summary, the mass spectrometric analyses revealed that rhEPO derived from glycoengineered Pichia has a highly uniform bi‐antennary N‐linked glycan composition and preserves the orthogonal O‐linked glycosylation site present on endogenous human EPO and CHO‐derived rhEPO. Copyright © 2013 John Wiley & Sons, Ltd.     

Structural characterization of carcinogen-modified oligodeoxynucleotide adducts using matrix-assisted laser desorption/ionization mass spectrometry

The aim of this study was to determine the chemical structure of in vitro 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-modified oligodeoxynucleotides (ODNs) by exonuclease digestion and matrix-assisted laser desorption/ionization mass spectrometry. A single-stranded 11-mer ODN, 5'-d(CCATCGCTACC), was reacted with N-acetoxy-PhIP, resulting in the formation of one major and eight minor PhIP-ODN adducts. A 10 min treatment of the major and one minor PhIP-ODN adduct with a 3'-exonuclease, bovine intestinal mucosa phosphodiesterase (BIMP), and a 5'-exonuclease, bovine spleen phosphodiesterase, results in inhibition of the primary exonuclease activity at deoxyguanosine (dG) producing 5'-d(CCATCG(PhIP)) and 5'-d(G(PhIP)CTACC) product ions, respectively. Post-source decay (PSD) of these enzymatic end products identifies dG as the sole modification site in two 11-mer ODN-PhIP adducts. PSD of the minor PhIP-ODN adduct digestion end product, 5'-d(CCATCG(PhIP)), also reveals that the PhIP adducted guanine moiety is in an oxidized form. Prolonged treatment of the PhIP-ODN adducts at 37 degrees C with BIMP induces a non-specific, or endonuclease, enzymatic activity culminating in the formation of deoxyguanosine 5'-monophosphate-PhIP (5'-dGMP-PhIP). The PSD fragmentation pattern of the 5'-dGMP-PhIP [M + H](+) ion of the major adduct confirms PhIP binds to the C-8 position of dG. For the minor adduct, PSD results suggest that PhIP binds to the C-8 position of an oxidized guanine, supporting the hypothesis that this adduct arises from oxidative degradation, resulting in a spirobisguanidino structure.     

Identification of ethyl pyruvate and dihydrocinchonidine adducts by electrospray ionization mass spectrometry

Several ethyl pyruvate and dihydrocinchonidine adducts, formed by non-covalent interactions with alkali cations, have been identified for the first time using electrospray ionization mass and tandem mass spectrometry. This type of adduct may have an important role in asymmetric reactions of pyruvates in the presence of cinchonas.     

Antioxidant activity of olive phenols: mechanistic investigation and characterization of oxidation products by mass spectrometry

In this work, the antioxidant activity of olive phenols is first characterized by their stoichiometries n(tot)(number of radicals trapped per antioxidant molecule) and their rate constants for the first H-atom abstraction k(1) by the stable radical DPPH. It appears that oleuropein, hydroxytyrosol and caffeic acid have the largest k(1) values, whereas dihydrocaffeic acid, an intestinal metabolite of caffeic acid, is the best antioxidant in terms of n(tot). For phenols with a catechol moiety n(tot) is higher than two, implying an antioxidant effect of their primarily formed oxidation products. A HPLC-MS analysis of the main products formed in the AAPH-induced oxidation of olive phenols reveals the presence of dimers and trimers. With hydroxytyrosol and dihydrocaffeic acid, oligomerization can take place with the addition of water molecules.The antioxidant activity of olive phenols is then evaluated by their ability to inhibit the AAPH-induced peroxidation of linoleic acid in SDS micelles. It is shown that olive phenols and quercetin act as retardants rather than chain breakers like alpha-tocopherol. From a detailed mechanistic investigation, it appears that the inhibition of lipid peroxidation by olive phenols can be satisfactorily interpreted by assuming that they essentially reduce the AAPH-derived initiating radicals. Overall, olive phenols prove to be efficient scavengers of hydrophilic peroxyl radicals with a long lasting antioxidant effect owing to the residual activity of some of their oxidation products.     

Product analysis of caffeic acid oxidation by on-line electrochemistry/electrospray ionization mass spectrometry

On-line electrochemistry/electrospray ionization mass spectrometry (EC/ESI-MS) was developed using a microflow electrolytic cell. This technique was applied to electrochemical oxidation of caffeic acid (CAF) which is known to be a highly antioxidative agent. Effects of electrolytic potentials on ion intensities of product ions and on electrolytic currents were examined at different pHs. Dimer products were detected at electrolytic potentials of E = 0.7 V (vs. Ag/AgCl) and trimer products at 1.0 V at pH 9. Dimer products were distinguished from hydrogen-bonded complexes by MS/MS experiments. Hydrogen/deuterium exchange experiments determined the number of hydroxyl and carboxyl groups in the Dimers formed by electrolysis. The mechanism of oxidative polymerization of CAF is discussed with speculation as to the structure of the dimer product.     

Detection of transient radical cations in electron transfer-initiated Diels-Alder reactions by electrospray ionization mass spectrometry

The coupling of a simple microreactor to an atmospheric pressure ion source, such as electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI), allows the investigation of reactions in solution by mass spectrometry. The tris(p-bromophenyl)aminium hexachloroantimonate (1(*)(+)SbCl(6)(-))-initiated reactions of phenylvinylsulfide (2) and cyclopentadiene (3) and of trans-anethole (5) and isoprene (6) and the dimerization of 1,3-cyclohexadiene (8) to give the respective Diels-Alder products were studied. These preparatively interesting reactions proceed as radical cation chain reactions via the transient radical cations of the respective dienophiles and of the respective Diels-Alder addition products. These radical cations could be detected directly and characterized unambiguously in the reacting solution by ESI-MS-MS. The identity was confirmed by comparison with MS-MS spectra of the authentic radical cations obtained by APCI-MS and by CID experiments of the corresponding molecular ions generated by EI-MS. In addition, substrates and products could be monitored easily in the reacting solution by APCI-MS.     

ZnS nanomaterial characterization by MALDI-TOF mass spectrometry

We present a methodology for mass and size dispersity analysis by MALDI-TOF mass spectrometry of lyothermally grown 2.5-3.7 nm ZnS nanocrystals having a Zn blende crystal structure. These results correlate with information obtained by TEM and absorption spectroscopy. The use of MS methods to probe size and size dispersity provides a convenient method to rapidly analyze II-VI materials at the nanoscale. We believe these results represent the first mass spectrometric analysis of size and size dispersities on II-VI nanocrystals.     

Identification of oxidation products of solanesol produced during air sampling for tobacco smoke by electrospray mass spectrometry and HPLC

Solanesol, a 45-carbon, trisesquiterpenoid alcohol found in tobacco leaves and tobacco smoke, has been used as a quantitative marker for tobacco smoke for years. However, solanesol appears to be unreliable as a quantitative marker for tobacco smoke during environmental air sampling because it can be degraded substantially when present as a component of tobacco smoke and by as much as 100% when present as pure solanesol on fortified filters during air sampling. Since there is strong evidence that ozone is the agent responsible for the degradation, solanesol appears to be unreliable as a quantitative marker during indoor air sampling when indoor levels of ozone are greater than about 15 ppb. The degree of loss of pure solanesol is directly proportional to the concentration of ozone and the length of the sampling period and depends on the type of 37 mm membrane filter used for air sampling (PTFE or quartz fiber). While the degree of loss of solanesol is inversely proportional to the relative humidity of the air at a sampling rate of 1.7 L min(-1), the degree of loss is virtually independent of relative humidity at a lower sampling rate; i.e., 0.25 L min(-1). A curve of loss of solanesol on a filter versus concentration of ozone from an ozone generator is virtually identical to a curve segment based on atmospheric ozone under the same conditions of air sampling. Oxidation of solanesol by ozone to approximately 25 to 60% completion produces at least three series of products for a total of at least 26 compounds: (1) isoprenoid acetones, (2)omega-hydroxyisoprenoid acetaldehydes, and (3) isoprenoid oxoaldehydes. All products in each series were tentatively identified as their derivatives with 2-(p-aminophenyl)ethanol (APE) by electrospray mass spectrometry (ES-MS). Ten ozonation products were detected as their 2,4-dinitrophenylhydrazine derivatives by HPLC at 360 nm: 4-oxopentanal and nine isoprenoid acetones (acetone, 6-methyl-5-hepten-2-one, geranylacetone, farnesylacetone, tetraprenylacetone, geranylfarnesylacetone, farnesylfarnesylacetone, farnesylgeranylgeranylacetone and bombiprenone.