RESUMEN
Although malondialdehyde and methylglyoxal have the same molecular formula, they have different chemistry in forming protein adducts. The major lysine adduct of malondialdehyde in hemoglobin is the N-propenal type, while that of methylglyoxal is N6-(1-carboxyethyl)lysine. This Letter provides evidence that the "methylglyoxal-like" hemoglobin adducts are not derived from malondialdehyde. This Letter also discusses the quantification of malondialdehyde-induced post-translational modifications in human hemoglobin by different mass spectrometry-based methods.
Asunto(s)
Hemoglobinas , Piruvaldehído , Humanos , Piruvaldehído/química , Malondialdehído/química , Hemoglobinas/química , Espectrometría de Masas , Procesamiento Proteico-PostraduccionalRESUMEN
Acrolein is a major component in cigarette smoke and a product of endogenous lipid peroxidation. It is difficult to distinguish human exposure to acrolein from exogenous sources versus endogenous causes, as components in cigarette smoke can stimulate lipid peroxidation in vivo. Therefore, analysis of acrolein-induced DNA and protein adducts by the highly accurate, sensitive, and specific mass spectrometry-based methods is vital to estimate the degree of damage by this IARC Group 2A carcinogen. This Perspective reviews the analyses of acrolein-induced DNA and protein adducts in humans by mass spectrometry focusing on samples accessible for biomonitoring, including DNA from leukocytes and oral cells and abundant proteins from blood, i.e., hemoglobin and serum albumin.
Asunto(s)
Acroleína , Fumar Cigarrillos , Aductos de ADN , Humanos , Acroleína/química , Biomarcadores , Fumar Cigarrillos/efectos adversos , Fumar Cigarrillos/metabolismo , ADN/química , Aductos de ADN/química , Espectrometría de Masas , Proteínas/química , Nicotiana/metabolismoRESUMEN
Malondialdehyde (MDA) is the most abundant α,ß-unsaturated aldehyde generated from endogenous peroxidation of polyunsaturated fatty acids and is present in cigarette smoke. Post-translational modifications of blood hemoglobin can serve as biomarkers for exposure to chemicals. In this study, two types of MDA-induced modifications, the N-propenal and the dihydropyridine (DHP), were identified at multiple sites in human hemoglobin digest by the high-resolution mass spectrometry. The N-propenal and the DHP types of modification led to the increase of 54.0106 and 134.0368 amu, respectively, at the N-terminal and lysine residues. Among the 21 MDA-modified peptides, 14 with dose-response to MDA concentrations were simultaneously quantified in study subjects by the nanoflow liquid chromatography nanoelectrospray ionization tandem mass spectrometry under selected reaction monitoring (nanoLC-NSI-MS/MS-SRM) without prior enrichment. The results showed that the modifications of the N-propenal-type at α-Lys-11, α-Lys-16, α-Lys-61, ß-Lys-8, and ß-Lys-17, as well as the DHP-type at the α-N-terminal valine, are significantly higher in hemoglobin isolated from the blood of smokers than in nonsmoking individuals. This is the first report to identify and quantify multiple sites of MDA-induced modifications in human hemoglobin from peripheral blood. Our results suggest that the MDA-derived modifications on hemoglobin might represent valuable biomarkers for MDA-induced protein damage.
Asunto(s)
Hemoglobinas , Procesamiento Proteico-Postraduccional , Fumadores , Humanos , Biomarcadores/metabolismo , Hemoglobinas/química , Lisina/metabolismo , Malondialdehído , Espectrometría de Masas en TándemRESUMEN
Exposure to acrolein, the smallest α, ß-unsaturated aldehyde, in humans originates from cigarette smoking and other environmental sources, food cooking, and endogenous lipid peroxidation and metabolism. The protein modification caused by acrolein is associated with various diseases, including cancer, cardiovascular, and neurodegenerative diseases. In this study, acrolein-modified human hemoglobin was reduced by sodium borohydride. Thus, three types of modifications, that is, Schiff base, Michael addition, and formyl-dehydropiperidion adducts, were converted to the corresponding stable adducts, leading to mass increases of 40.0313, 58.0419, and 96.0575 Da, respectively. These stable acrolein-modified hemoglobin peptides were identified by nanoflow liquid chromatography coupled to a high-resolution nanoelectrospray ionization tandem mass spectrometry. Among the 26 different types and sites of modifications, 15 of them showed a dose-dependent increase with increasing concentrations of acrolein. To investigate the role of acrolein-induced modifications in smoking and oral cancer, the 15 dose-responsive acrolein-modified peptides, together with three ethylated peptides previously identified, were quantified in oral cancer patients, healthy smokers, and healthy nonsmokers. The results reveal that the relative extents of the Michael-type adduct at α-Lys-16, α-His-50, and ß-Lys-59 are significantly higher in smokers (oral cancer and healthy) than in nonsmokers. Areas under the receiver operating characteristic curve of these peptides range from 0.7500 to 0.9375, indicating the ability to discriminate smokers from nonsmokers. Additionally, these acrolein-modified peptides correlate with three ethylated peptides at the N-termini of α- and ß-globin, as well as ß-His-77, and with the number of cigarettes smoked per day. Therefore, measuring the reduced Michael adducts at α-Lys-16, α-His-50, and ß-Lys-59 of hemoglobin from one drop of blood by this sensitive and specific method may reflect the increase of acrolein exposure due to cigarette smoking.
Asunto(s)
Fumar Cigarrillos , Hemoglobinas , Neoplasias de la Boca , Humanos , Acroleína/química , Fumar Cigarrillos/efectos adversos , Hemoglobinas/química , Espectrometría de Masas , Péptidos/químicaRESUMEN
Cells are continually exposed to endogenous reactive oxygen, nitrogen, and halogen species, causing damage to biomolecules. Among them, peroxynitrite and hypochlorous acid are not only oxidants but also biological nitrating and chlorinating agents, leading to the formation of 3-nitrotyrosine and 3-chlorotyrosine, respectively, in proteins. 3-Nitrotyrosine has been detected in vivo under several pathophysiological conditions, including breast cancer. Studies show that the concentrations of 3-nitrotyrosine in plasma proteins and platelets were significantly elevated in breast cancer patients. Compared to blood serum albumin, hemoglobin adducts represent biomonitoring of exposure with a longer lifetime. In this study, human hemoglobin was freshly isolated from blood and digested into peptides with trypsin, and the levels of protein adducts, including nitration, nitrosylation, and chlorination of tyrosine as well as oxidation of methionine residues, were simultaneously quantified by nanoflow liquid chromatography nanoelectrospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) with selected reaction monitoring. The results demonstrated that the relative extents of nitration at α-Tyr-42 and ß-Tyr-130, nitrosylation at α-Tyr-24, and chlorination at α-Tyr-24 and ß-Tyr-130 are significantly higher in globin of 25 breast cancer patients compared to those in 25 healthy subjects (p < 0.05). In particular, nitration at α-Tyr-42 and chlorination at α-Tyr-24 showed the area under the receiver operating characteristic curve of >0.8. While the age of the subjects is correlated with the extents of some of these adducts, the body mass index does not have an effect on any of them. Starting with 1 drop of blood, our results indicated that this highly sensitive and specific nanoLC-NSI/MS/MS is useful in investigating the role of reactive nitrogen oxide species and reactive chlorine species in the etiology of breast cancer.
Asunto(s)
Neoplasias de la Mama/metabolismo , Hemoglobinas/metabolismo , Nanotecnología , Neoplasias de la Mama/sangre , Neoplasias de la Mama/patología , Cromatografía Liquida , Femenino , Halogenación , Hemoglobinas/análisis , Hemoglobinas/aislamiento & purificación , Humanos , Persona de Mediana Edad , Oxidación-Reducción , Espectrometría de Masas en TándemRESUMEN
We are exposed to endogenous reactive oxygen species (ROS) produced during normal aerobic metabolism and by the innate immune systems. Excessive production of ROS is critically important in disease onset and progression. Post-translational oxidative modifications of hemoglobin have been used as a surrogate biomarker for monitoring the oxidative stress in vivo. In this study, the Fenton reaction was used as a model to produce ROS and react with human hemoglobin. After trypsin digestion, the types and sites of modifications were characterized by a nanoflow LC-nanoelectrospray ionization high-resolution mass spectrometer. Besides oxidation at certain sites of Met, His, and Tyr, conversion of histidine to aspartate and hydroxyaspartate was identified at four sites. Furthermore, advanced oxidation of histidine to hydroxyaspartate was identified at two sites. Elevated oxidative stress is tightly associated with oral cancer. The relative extent of modification at the identified sites was quantified relative to the native peptide present in the digest as the reference peptide in hemoglobin from 18 oral cancer patients and 15 healthy control subjects. The results revealed that the extents of oxidation at ß-His-77 and ß-Asp-99 of globin were significantly elevated in oral cancer patients compared to healthy subjects, while the extents of conversion of histidine residues at α-His-20, α-His-50, and ß-His-2 to aspartate were significantly decreased. Furthermore, the advanced oxidation of histidine to hydroxyaspartate at α-His-50 and ß-His-2 is also significantly higher in oral cancer patients than in healthy subjects (p < 0.05). To our knowledge, this advanced oxidation of histidine to hydroxyaspartate is a new post-translational protein modification, and it is found in human hemoglobin isolated from blood. This advanced oxidative modification in hemoglobin might be a potential biomarker to assess oxidative stress in oral cancer patients.
Asunto(s)
Hemoglobinas/metabolismo , Neoplasias de la Boca/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Cromatografía Líquida de Alta Presión , Hemoglobinas/análisis , Humanos , Espectrometría de Masas , Neoplasias de la Boca/diagnóstico , Oxidación-Reducción , Especies Reactivas de Oxígeno/análisisRESUMEN
Glutathione is an intracellular antioxidant capable of scavenging free radicals and detoxifying electrophiles from endogenous and exogenous sources via the free thiol group. Post-translational glutathionylation at cysteine residues of proteins can affect the structure and cause a functional change of proteins. Protein glutathionylation has been proven to reflect the cellular redox status. Our previous report indicates that the levels of glutathionylation in hemoglobin from peripheral blood of smokers are significantly higher than in nonsmokers. In this study, a nanoflow liquid chromatography/nanospray ionization triple-stage mass spectrometric (nanoLC/NSI-MS3 ) method with a linear ion trap mass spectrometer was employed to quantify glutathionylated peptides in the trypsin digests of hemoglobin from gastric cancer patients. We compare the extent of glutathionylation in hemoglobin from nonsmoking gastric cancer patients with that from nonsmoking healthy adults. Using a carboxymethylated peptide as the reference peptide, the relative quantification of each glutathionylated peptide was measured as the peak area ratio of the modified peptide versus the sum of the peak areas of the modified and the carboxymethylated parent peptide in the selected reaction monitoring chromatograms. Using this method, we found that the extents of glutathionylation at Cys-104 of the α-globin and Cys-93 of ß-globulin hemoglobin from 10 gastric cancer patients were significantly higher than those from 14 normal individuals with p values <0.0001. Our results suggest the possibility of using the extent of cysteine glutathionylation at ß-93 of hemoglobin as an oxidative stress biomarker candidate for gastric cancer.
Asunto(s)
Cisteína/análisis , Glutatión/análisis , Neoplasias Gástricas/sangre , Globinas alfa/química , Globinas beta/química , Adulto , Anciano , Anciano de 80 o más Años , Cromatografía Liquida , Femenino , Humanos , Masculino , Persona de Mediana Edad , Oxidación-Reducción , Espectrometría de Masa por Ionización de Electrospray , Neoplasias Gástricas/química , Adulto JovenRESUMEN
Glyoxal is an oxoaldehyde generated from the degradation of glucose-protein conjugates and from lipid peroxidation in foods and in vivo, and it is also present in the environment (e.g., cigarette smoke). The major endogenous source of glyoxal is glucose autoxidation, and the glyoxal concentrations in plasma are higher in diabetic patients than in nondiabetics. Glyoxal reacts with biomolecules forming covalently modified DNA and protein adducts. We previously developed sensitive and specific assays based on nanoflow liquid chromatography-nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) for quantification of DNA cross-linked adducts (dG-gx-dC and dG-gx-dA) and for hemoglobin adducts derived from glyoxal. In this study, we isolated and analyzed both leukocyte DNA and hemoglobin from the blood of diabetic patients and compared the adduct levels with those from nondiabetic subjects using the modified assays. The results indicated that the extents of glyoxal-induced hemoglobin modifications on α-Lys-11, α-Arg-92, ß-Lys-17, and ß-Lys-66 were statistically higher in diabetic patients than nondiabetics and they correlated with HbA1c significantly. Moreover, the levels of dG-gx-dC in leukocyte DNA correlated positively with the extents of globin modification at α-Lys-11 and ß-Lys-17, while levels of dG-gx-dA correlated with those at α-Lys-11 and α-Arg-92 in nonsmoking subjects. Comparing the levels and the correlation coefficients of these hemoglobin and DNA adducts including or excluding smokers, it appears that smoking is not a major contributor to glyoxal-induced adduction of hemoglobin and leukocyte DNA. To the best of our knowledge, this is one of the few reports of positive correlation between DNA and protein adducts of the same compound (glyoxal) in the blood from the same subjects. Because of the high abundance of hemoglobin in blood, the results indicate that quantification of glyoxal-modified peptides in hemoglobin might serve as a dosimetry for glyoxal and a practical surrogate biomarker for assessing glyoxal-induced DNA damage and its prevention.
Asunto(s)
Reactivos de Enlaces Cruzados/análisis , ADN/sangre , Glioxal/sangre , Hemoglobinas/análisis , Cromatografía Liquida , Reactivos de Enlaces Cruzados/metabolismo , ADN/química , Glioxal/química , Hemoglobinas/metabolismo , Humanos , Estructura Molecular , Espectrometría de Masas en TándemRESUMEN
Methylation of biomolecules is involved in many important biological processes. The contributing methylating agents arise from endogenous and exogenous sources (such as cigarette smoking). Human hemoglobin is easily accessible from blood and has been used as a molecular dosimeter for monitoring chemical exposure. We recently developed a method for characterization and quantification of the extents of methylation and ethylation in hemoglobin by nanoflow liquid chromatography tandem mass spectrometry under the selected reaction monitoring mode. Using this method, the relative extents of methylated and ethylated peptides in hemoglobin were quantified in nonsmoking subjects at various ages in this study. Among the nine methylation sites, we found that the extents of methylation were significantly higher in elderly subjects at the N-terminal and His-20 of α-globin, and at the N-terminal and Glu-26 of ß-globin. Moreover, the extents of methylation at these sites were significantly correlated with the age of the subjects. On the other hand, no statistically significant difference was found in the ethylated peptides. We also examined the stability of methylated and ethylated hemoglobin when stored on dried blood spot cards. The extents of these modifications on hemoglobin are stable for at least 4 weeks stored at room temperature. Our results suggest that age should be considered as a factor when measuring hemoglobin methylation and that dried blood spot is a valuable biomonitoring technique for hemoglobin modifications in epidemiological studies.
Asunto(s)
Cromatografía Liquida/métodos , Hemoglobinas/análisis , Espectrometría de Masas en Tándem/métodos , Adulto , Anciano , Secuencia de Aminoácidos , Pruebas con Sangre Seca , Femenino , Hemoglobinas/metabolismo , Humanos , Masculino , Metilación , Persona de Mediana Edad , Péptidos/análisis , Estabilidad Proteica , Adulto JovenRESUMEN
Glyoxal (gx) is a bifunctional electrophile capable of cross-linking DNA. Although it is present in foods and from the environment, endogenous formation of glyoxal occurs through metabolism of carbohydrates and oxidation of lipids and nucleic acids. Plasma concentrations of glyoxal are elevated in in diabetes mellitus patients compared to nondiabetics. The most abundant 2'-deoxyribonucleoside adducts cross-linked by glyoxal are dG-gx-dC, dG-gx-dA, and dG-gx-dG. These DNA cross-links can be mutagenic by damaging the integrity of the DNA structure. Herein, we developed a highly sensitive and specific assay for the simultaneous detection and quantification of the dG-gx-dC and dG-gx-dA cross-links based on stable isotope dilution (SID) nanoflow liquid chromatography nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) under the highly selected reaction monitoring mode and using a triple quadrupole mass spectrometer. The entire assay procedure involved addition of the stable isotope standards [15N5]dG-gx-dC and [15N5]dG-gx-dA as internal standards, enzyme hydrolysis to release the cross-links as nucleosides, enrichment by a reversed-phase solid-phase extraction column, and nanoLC-NSI/MS/MS analysis. The detection limit is 0.19 amol for dG-gx-dC and 0.89 amol for dG-gx-dA, which is 400 and 80 times more sensitive, respectively, than capillary LC-NSI/MS/MS assay of these adducts. The lower limit of quantification was 94 and 90 amol for dG-gx-dC and dG-gx-dA, respectively, which is equivalent to 0.056 and 0.065 adducts in 108 normal nucleotides in 50 µg of DNA. In type 2 diabetes mellitus (T2DM) patients (n = 38), the levels of dG-gx-dC and dG-gx-dA in leukocyte DNA were 1.94 ± 1.20 and 2.10 ± 1.77 in 108 normal nucleotides, respectively, which were significantly higher than those in nondiabetics (n = 39: 0.83 ± 0.92 and 1.05 ± 0.99 in 108 normal nucleotides, respectively). Excluding the factor of smoking, an exogenous source of glyoxal, levels of these two cross-linked adducts were found to be significantly higher in nonsmoking T2DM patients than in nonsmoking control subjects. Furthermore, the levels of dG-gx-dC and dG-gx-dA correlated with HbA1c with statistical significance. To our best knowledge, this is the first report of the identification and quantification of glyoxal-derived cross-linked DNA adducts in human leukocyte DNA and their association with T2DM. This SID nanoLC-NSI/MS/MS assay is highly sensitive and specific and it requires only 50 µg of leukocyte DNA isolated from 2-3 mL of blood to accurately quantify these two cross-linked adducts simultaneously. Our assay thus provides a useful biomarker for the evaluation of glyoxal-derived DNA damage.
Asunto(s)
Aductos de ADN/análisis , Aductos de ADN/química , Diabetes Mellitus Tipo 2/patología , Glioxal/química , Técnicas de Dilución del Indicador , Leucocitos/química , Cromatografía Liquida , Humanos , Estructura Molecular , Espectrometría de Masas en TándemRESUMEN
Alkylating agents contained in cigarettes smoke might be related to cancer development. Post-translational protein methylation and ethylation may cause alteration of protein functions. Human hemoglobin (Hb) has been a target for molecular dosimetry because of its easy accessibility. The goal of this study is to investigate the relationship between the levels of methylation and ethylation at specific sites of Hb with smoking. Because of the low extent of modification of Hb isolated from blood, the methylation and ethylation sites were identified in Hb incubated with a methylating agent (methyl methanesulfonate, MMS) and ethylating agent (ethyl methanesulfonate, EMS), respectively, by accurate mass measurements. After trypsin digestion, the modification sites were identified by nanoflow LC-nanospray ionization coupled with high-resolution mass spectrometry. The selected reaction monitoring mode was used to quantify the relative extent of methylation and ethylation in human Hb incubated with MMS and EMS, respectively. Methylation occurred at 9 sites, including 1V, 20H, 50H, 72H of α-globin and 1V, 26E, 66K, 77H, 93C of ß-globin. Ethylation was detected at 11 sites, including 1V, 16K, 50H, 72H, 87H of α-globin and 1V, 17K, 66K, 77H, 92H, 93C of ß-globin. The relative extents of methylation and ethylation were measured in blood samples from 13 smokers and 13 nonsmokers. No statistically significant difference was found in the methylated peptides. On the other hand, the extents of ethylation at α-terminal Val, α-His-50, α-His-87, ß-terminal Val, ß-His-77, and ß-Cys-93 in Hb were significantly higher in smokers than in nonsmokers (p < 0.05). Furthermore, the relative extents of ethylation at these sites were statistically significantly correlated with the number of cigarettes smoked per day. Therefore, this assay, which requires as little as one drop of blood, should be helpful in measuring Hb ethylation as a potential biomarker for assessing the exposure to cigarette smoking.
Asunto(s)
Fumar Cigarrillos/metabolismo , Hemoglobinas/metabolismo , Adulto , Alquilantes/metabolismo , Alquilación , Secuencia de Aminoácidos , Femenino , Hemoglobinas/química , Humanos , Masculino , Metilación , Péptidos/análisis , Péptidos/metabolismo , Espectrometría de Masas en Tándem , Adulto JovenRESUMEN
The post-translational modification (PTM) of proteins by endogenous reactive chlorine, nitrogen, and oxygen species is implicated in certain pathological conditions, including diabetes mellitus. Evidence showed that the extents of modifications on a number of proteins are elevated in diabetic patients. Measuring modification on hemoglobin has been used to monitor the extent of exposure. This study develops an assay for simultaneous quantification of the extent of chlorination, nitration, and oxidation in human hemoglobin and to examine whether the level of any of these modifications is higher in poorly controlled type 2 diabetic mellitus patients. This mass spectrometry-based assay used the bottom-up proteomic strategy. Due to the low amount of endogenous modification, we first characterized the sites of chlorination at tyrosine in hypochlorous acid-treated hemoglobin by an accurate mass spectrometer. The extents of chlorination, nitration, and oxidation of a total of 12 sites and types of modifications in hemoglobin were measured by nanoflow liquid chromatography-nanospray ionization tandem mass spectrometry under the selected reaction monitoring mode. Relative quantification of these PTMs in hemoglobin extracted from blood samples shows that the extents of chlorination at α-Tyr-24, nitration at α-Tyr-42, and oxidation at the three methionine residues are significantly higher in diabetic patients (n = 19) than in nondiabetic individuals (n = 18). After excluding the factor of smoking, chlorination at α-Tyr-24, nitration at α-Tyr-42, and oxidation at the three methionine residues are significantly higher in the nonsmoking diabetic patients (n = 12) than in normal nonsmoking subjects (n = 11). Multiple regression analysis performed on the combined effect of age, body-mass index (BMI), and HbA1c showed that the diabetes factor HbA1c contributes significantly to the extent of chlorination at α-Tyr-24 in nonsmokers. In addition, age contributes to oxidation at α-Met-32 significantly in all subjects and in nonsmokers. These results suggest the potential of using chlorination at α-Tyr-24-containing peptide to evaluate protein damage in nonsmoking type 2 diabetes mellitus.
Asunto(s)
Cisteína/análisis , Diabetes Mellitus Tipo 2/sangre , Hemoglobinas/química , Metionina/análisis , Nitratos/análisis , Compuestos Nitrosos/análisis , Tirosina/análisis , Adulto , Anciano , Secuencia de Aminoácidos , Cisteína/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Femenino , Hemoglobina Glucada/química , Hemoglobina Glucada/metabolismo , Halogenación , Hemoglobinas/metabolismo , Humanos , Masculino , Metionina/metabolismo , Persona de Mediana Edad , Nitratos/metabolismo , Compuestos Nitrosos/metabolismo , Oxidación-Reducción , Tirosina/metabolismoRESUMEN
Dried blood spot (DBS) is an emerging microsampling technique for the bioanalysis of small molecules, including fatty acids, metabolites, drugs, and toxicants. DBS offers many advantages as a sample format including easy sample collection and cheap sample shipment. Hemoglobin adducts have been recognized as a suitable biomarker for monitoring chemical exposure. We previously reported that certain modified peptides in hemoglobin derived from reactive chlorine, nitrogen, and oxygen species are associated with factors including smoking, diabetes mellitus, and aging. However, the stability of these oxidation-induced modifications of hemoglobin remains unknown and whether they can be formed artifactually during storage of DBS. To answer these questions, globin extracted from the DBS cards was analyzed, and the stability of the modifications was evaluated. After storage of the DBS cards at 4 °C or room temperature up to 7 weeks, we isolated globin from a quarter of the spot every week. The extents of 11 sites and types of post-translational modifications (PTMs), including nitration and nitrosylation of tyrosine and oxidation of cysteine and methionine residues, in human hemoglobin were measured in the trypsin digest by nanoflow liquid chromatography-nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) using selected reaction monitoring. The extents of all these PTMs are stable within 14 days when stored on DBS at room temperature and at 4 °C, while those from direct extraction of fresh blood are stable for at least 8 weeks when stored as an aqueous solution at -20 °C. Extraction of globin from a DBS card is of particular importance for hemolytic blood samples. To our knowledge, this is the first report on the stability of oxidative modifications of hemoglobin on DBSs, which are stable for 14 days under ambient conditions (room temperature, in air). Therefore, it is feasible and convenient to analyze these hemoglobin modifications from DBSs in studies involving large populations.
Asunto(s)
Cromatografía Liquida/métodos , Hemoglobinas/química , Especies de Nitrógeno Reactivo/química , Especies Reactivas de Oxígeno/química , Espectrometría de Masas en Tándem/métodos , Sangre , Hemoglobinas/aislamiento & purificación , HumanosRESUMEN
Glyoxal and methylglyoxal are oxoaldehydes derived from the degradation of glucose-protein conjugates and from lipid peroxidation, and they are also present in the environment. This study investigated the site-specific reaction of glyoxal and methylglyoxal with the amino acid residues on human hemoglobin using a shot-gun proteomic approach with nanoflow liquid chromatography/nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS). In human hemoglobin incubated with glyoxal, modification on 8 different sites, including lysine residues at α-Lys-11, α-Lys-16, α-Lys-56, ß-Lys-17, ß-Lys-66, ß-Lys-144, and arginine residues at α-Arg-92 and ß-Arg-30, was observed using a data-dependent scan. In methylglyoxal-treated hemoglobin, there were specific residues, namely, α-Arg-92, ß-Lys-66, ß-Arg-30, and ß-Lys-144, forming carboxyethylation as well as the dehydrated product hydroimidazolone at α-Arg-92 and ß-Arg-30. These lysine and arginine modifications were confirmed by accurate mass measurement and the MS(2) and MS(3) spectra. The most intensive signal of each modified peptide was used as the precursor ion to perform the product ion scan. The relative extent of modifications was semiquantified simultaneously relative to the native reference peptide by nanoLC-NSI/MS/MS under the selected reaction monitoring (SRM) mode. The extent of these modifications increased dose-dependently with increasing concentrations of glyoxal or methylglyoxal. Six out of the eight modifications induced by glyoxal and three out of the six modifications induced by methylglyoxal were detected in hemoglobin freshly isolated from human blood samples. The relative extent of modification of these post-translational modifications was quantified in poorly controlled type 2 diabetes mellitus patients (n = 20) and in nondiabetic control subjects (n = 21). The results show that the carboxymethylated peptides at α-Lys-16, α-Arg-92, ß-Lys-17, ß-Lys-66, and the peptide at α-Arg-92 with methylglyoxal-derived hydroimidazolone are significantly higher in diabetic patients than in normal individuals (p value <0.05). This report identified and quantified glyoxal- and methylglyoxal-modified hemoglobin peptides in humans and revealed the association of the extent of modifications at specific sites with T2DM. Only one drop (10 µL) of fresh blood is needed for this assay, and only an equivalent of 1 µg of hemoglobin was analyzed by the nanoLC-NSI/MS/MS-SRM system. These results suggest the potential use of these specific post-translational modifications in hemoglobin as feasible biomarker candidates to assess protein damage induced by glyoxal and methylglyoxal.
Asunto(s)
Diabetes Mellitus Tipo 2 , Glioxal/toxicidad , Hemoglobinas/química , Hemoglobinas/efectos de los fármacos , Piruvaldehído/toxicidad , Femenino , Glioxal/química , Humanos , Masculino , Persona de Mediana Edad , Péptidos/análisis , Péptidos/sangre , Piruvaldehído/química , Estándares de Referencia , Fumar , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Protein glutathionylation is an important protein post-translational modification associated with oxidative stress, in which the thiol groups of cysteine residues react with glutathione and form disulfide bonds. Glutathionylation has been shown to affect protein structure and modulate protein function, and is implicated in the regulation of signaling and metabolic pathways. Glutathionylation of human hemoglobin has been known for decades. However, only glutathionylation on Cys-93 of ß-globin has been characterized. In this study, we used nanoflow liquid chromatography-nanospray ionization multistage mass spectrometry (nanoLC-NSI/MS(n)) under a data-dependent scan mode to identify sites of glutathionylation in human hemoglobin by accurate mass measurement and by their MS(2) and MS(3) spectra. After human hemoglobin was incubated with oxidized glutathione, alkylated and trypsin digested, all three cysteine residues, i.e., α-Cys-104, ß-Cys-93, and ß-Cys-112, were found to be glutathionylated. Glutathionylation at these sites was also detected in hemoglobin freshly isolated from human blood samples by the consecutive reaction monitoring at the MS(3) scan stage, and the extent of modification of each site was quantified relative to the alkylated parent peptide in the selected reaction monitoring (SRM) chromatograms. The peak area ratio of glutathionylated and alkylated parent peptides under MS(3) and MS(2)-SRM, respectively, represents the relative extent of glutathionylation. The extents of glutathionylation at α-Cys-104 and ß-Cys-93 in hemoglobin of 20 smokers were significantly higher than those in 20 nonsmokers with a p value of 0.0008 and <0.0001, respectively. Moreover, there are statistically significant correlations between the extent of glutathionylation at α-Cys-104 and ß-Cys-93 and the number of cigarettes smoked per day as well as smoking index. This assay is highly specific and sensitive as it requires as little as 2 µg of hemoglobin isolated from one drop of blood. These results suggest that this assay might be feasible in measuring the extent of glutathionylation in hemoglobin as a low-invasive biomarker of oxidative stress. To our knowledge, this is the first report identifying all three cysteine residues being glutathionylated in human hemoglobin and quantifying the extent of glutathionylation at the peptide level using multistage mass spectrometry.
Asunto(s)
Disulfuro de Glutatión/metabolismo , Hemoglobinas/metabolismo , Fumar/metabolismo , Adolescente , Adulto , Secuencia de Aminoácidos , Cisteína/análisis , Cisteína/metabolismo , Femenino , Disulfuro de Glutatión/análisis , Hemoglobinas/química , Humanos , Masculino , Espectrometría de Masas , Datos de Secuencia Molecular , Péptidos/química , Péptidos/metabolismo , Fumar/sangre , Espectrometría de Masa por Ionización de Electrospray , Adulto JovenRESUMEN
Nitric oxide (NO) is essential for normal physiology, but excessive production of NO during inflammatory processes can damage the neighboring tissues. Reactive nitrogen oxide species (RNOx), including peroxynitrite (ONOO(-)), are powerful nitrating agents. Biological protein nitration is involved in several disease states, including inflammatory diseases, and it is evident by detection of 3-nitrotyrosine (3NT) in inflamed tissues. In this study, we identified peroxynitrite-induced post-translational modifications (PTMs) in human hemoglobin by accurate mass measurement as well as by the MS(2) and MS(3) spectra. Nitration on Tyr-24, Tyr-42 (α-globin), and Tyr-130 (ß-globin) as well as nitrosation on Tyr-24 (α-globin) were identified. Also characterized were oxidation of all three methionine residues, α-Met-32, α-Met-76, and ß-Met-55 to the sulfoxide, as well as cysteine oxidation determined as sulfinic acid on α-Cys-104 and sulfonic acid on α-Cys-104, ß-Cys-93, and ß-Cys-112. These modifications are detected in hemoglobin freshly isolated from human blood and the extents of modifications were semiquantified relative to the reference peptides by nanoflow liquid chromatography-nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) under the selected reaction monitoring (SRM) mode. The results showed a statistically significant positive correlation between cigarette smoking and the extents of tyrosine nitration at α-Tyr-24 and at α-Tyr-42. To our knowledge, this is the first report on identification and quantification of multiple PTMs in hemoglobin from human blood and association of a specific 3NT-containing peptide with cigarette smoking. This highly sensitive and specific assay only requires hemoglobin isolated from one drop (â¼10 µL) of blood. Thus, measurement of these PTMs in hemoglobin might be feasible for assessing nitrative stress in vivo.
Asunto(s)
Hemoglobinas/química , Especies de Nitrógeno Reactivo/química , Fumar , Espectrometría de Masa por Ionización de Electrospray , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Cisteína/química , Hemoglobinas/metabolismo , Humanos , Metionina/química , Datos de Secuencia Molecular , Oxidación-Reducción , Péptidos/análisis , Ácido Peroxinitroso/química , Procesamiento Proteico-Postraduccional , Ácidos Sulfínicos/química , Tirosina/análogos & derivados , Tirosina/químicaRESUMEN
Studies showed that levels of ethylated DNA adducts in certain tissues and urine are higher in smokers than in nonsmokers. Because cigarette smoking is a major risk factor of various cancers, DNA ethylation might play an important role in cigarette smoke-induced cancer formation. Among the ethylated DNA adducts, O(2)-ethylthymidine (O(2)-edT) and O(4)-ethylthymidine (O(4)-edT) are poorly repaired and are accumulated in the body. In addition, O(4)-edT possesses promutagenic properties. In this study, we have developed a highly sensitive, accurate, and quantitative assay for simultaneous detection and quantification of O(2)-edT, N(3)-edT (N(3)-ethylthymidine), and O(4)-edT adducts by isotope dilution nanoflow liquid chromatography-nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS). Under the highly selected reaction monitoring (H-SRM) mode, the detection limit of O(2)-edT, N(3)-edT, and O(4)-edT injected on-column was 5.0, 10, and 10 fg, respectively. The quantification limit for the entire assay was 50, 100, and 100 fg of O(2)-edT, N(3)-edT, and O(4)-edT, respectively, corresponding to 1.1, 2.3, and 2.3 adducts in 10(9) normal nucleotides, respectively, starting with 50 µg of DNA (from 1.5-2.0 mL of blood). Levels of O(2)-edT, N(3)-edT, and O(4)-edT in 20 smokers' leukocyte DNA were 44.8 ± 52.0, 41.1 ± 43.8, 48.3 ± 53.9 in 10(8) normal nucleotides, while those in 20 nonsmokers were 0.19 ± 0.87, 4.1 ± 13.3, and 1.0 ± 2.9, respectively. Levels of O(2)-edT, N(3)-edT, and O(4)-edT in human leukocyte DNA are all significantly higher in smokers than in nonsmokers, with pvalues of 0.0004, 0.0009, and 0.0004, respectively. Furthermore, levels of O(2)-edT show a statistically significant association (γ = 0.4789, p = 0.0327) with the smoking index in smokers. In the 40 leukocyte DNA samples, the extremely significant statistical correlations (p < 0.0001) are observed between levels of O(2)-edT and O(4)-edT (γ = 0.9896), between levels of O(2)-edT and N(3)-edT (γ = 0.9840), and between levels of N(3)-edT and O(4)-edT (γ = 0.9901). To our knowledge, this is the first mass spectrometry-based assay for ethylated thymidine adducts. Using this assay, the three ethylated thymidine adducts were detected and quantified for the first time. Therefore, this highly sensitive, specific, and accurate assay should be clinically feasible for simultaneous quantification of the three ethylated thymidine adducts as potential biomarkers for exposure to ethylating agents and for cancer risk assessment.
Asunto(s)
Cromatografía Líquida de Alta Presión , Leucocitos/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Timidina/análogos & derivados , Isótopos de Carbono/química , ADN/aislamiento & purificación , ADN/metabolismo , Aductos de ADN/análisis , Humanos , Marcaje Isotópico , Nanotecnología , Fumar , Timidina/análisisRESUMEN
Exocyclic DNA adducts, including 1,N(2)-propano-2'-deoxyguanosine derived from acrolein (AdG) and crotonaldehyde (CdG) and the three lipid peroxidation-related etheno adducts 1,N(6)-etheno-2'-deoxyadenosine (εdAdo), 3,N(4)-etheno-2'-deoxycytidine (εdCyt), and 1,N(2)-etheno-2'-deoxyguanosine (1,N(2)-εdGuo), play an important role in cancer formation and they are associated with oxidative-stress-induced DNA damage. Saliva is an easily accessible and available biological fluid and a potential target of noninvasive biomarkers. In this study, a highly sensitive and specific assay based on isotope dilution nanoflow LC-nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) is developed for simultaneous detection and quantification of these five adducts in human salivary DNA. The levels of AdG, CdG, εdAdo, εdCyd, and 1,N(2)-εdGuo, measured in 27 human salivary DNA samples from healthy volunteers, were determined as 104 ± 50, 7.6 ± 12, 99 ± 50, 72 ± 49, 391 ± 198 (mean ± SD) in 10(8) normal nucleotides, respectively, starting with 25 µg of DNA isolated from an average of 3 mL of saliva. Statistically significant correlations were found between levels of εdAdo and εdCyd (γ = 0.8007, p < 0.0001), between levels of εdAdo and 1,N(2)-εdGuo (γ = 0.6778, p = 0.0001), between levels of εdCyd and 1,N(2)-εdGuo (γ = 0.5643, p = 0.0022), between levels of AdG and 1,N(2)-εdGuo (γ = 0.5756, p = 0.0017), and between levels of AdG and εdAdo (γ = 0.3969, p = 0.0404). Only 5 µg of DNA sample was analyzed for simultaneous quantification of these adducts. The easy accessibility and availability of saliva and the requirement for the small amount of DNA samples make this nanoLC-NSI/MS/MS assay clinically feasible in assessing the possibility of measuring 1,N(2)-propano-2'-deoxyguanosine and etheno adducts levels in human salivary DNA as noninvasive biomarkers for DNA damage resulting from oxidative stress and for evaluating their roles in cancer formation and prevention.
Asunto(s)
Aductos de ADN/análisis , Nanotecnología/métodos , Glándulas Salivales/metabolismo , Cromatografía Liquida , Aductos de ADN/química , Humanos , Técnicas de Dilución del Indicador , Valores de Referencia , Espectrometría de Masas en TándemRESUMEN
Etheno DNA adducts are promutagenic DNA lesions derived from exogenous industrial chemicals, as well as endogenous sources including lipid peroxidation. Furthermore, levels of etheno adducts in tissue DNA are elevated in cancer-prone tissues. In this study, we have developed a highly sensitive and specific stable isotope dilution nanoflow LC-nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) assay for simultaneous detection and accurate quantification of 1,N(6)-etheno-2'-deoxyadenosine (epsilondAdo), 3,N(4)-etheno-2'-deoxycytidine (epsilondCyt), and 1,N(2)-etheno-2'-deoxyguanosine (1,N(2)-epsilondGuo) in tissue DNA. Typically, [(13)C(1),(15)N(2)]epsilondAdo, [(15)N(3])epsilondCyd, and [(13)C(1),(15)N(2)]1,N(2)-epsilondGuo were added to calf thymus, human placenta, or human leukocyte DNA as internal standards, and the mixture was subjected to enzyme hydrolysis to form the nucleosides. The etheno adducts in DNA hydrolysate were enriched by a reversed phase solid-phase extraction column before analysis by nanoLC-NSI/MS/MS under the highly selective reaction monitoring (H-SRM) mode. This nanoLC-NSI/MS/MS assay achieved attomole-level sensitivity with the detection limit of 0.73, 160, and 34 amol for the respective standard epsilondAdo, epsilondCyd, and 1,N(2)-epsilondGuo injected on-column, while the quantification limit for the entire assay was 0.18, 4.0, and 3.4 fmol, respectively. The levels of epsilondAdo, epsilondCyd, and 1,N(2)-epsilondGuo in human placental DNA were 28.2, 44.1, and 8.5 adducts in 10(8) normal nucleosides, respectively. The levels of epsilondAdo, epsilondCyd, and 1,N(2)-epsilondGuo in 11 human leukocyte DNA samples were 16.2 +/- 5.2, 11.1 +/- 5.8, and 8.6 +/- 9.1 (mean +/- S.D.) in 10(8) normal nucleotides, respectively, starting from 30 mug of DNA or 1-1.5 mL of blood, and all the relative standard deviations were within 10%. An aliquot equivalent to 6 mug of DNA hydrolysate was used for analysis by this nanoLC-NSI/MS/MS. Thus, this highly sensitive and specific nanoLC-NSI/MS/MS method is suitable for accurate quantification of the three lipid peroxidation-derived etheno DNA adducts as noninvasive biomarkers in clinical studies for cancer risk assessment and for evaluation of preventive agents.
Asunto(s)
Cromatografía Liquida/métodos , Aductos de ADN/análisis , Aductos de ADN/metabolismo , Peroxidación de Lípido , Nanotecnología/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Animales , Biomarcadores/análisis , Biomarcadores/metabolismo , Calibración , Bovinos , Aductos de ADN/sangre , Aductos de ADN/genética , Daño del ADN , Femenino , Humanos , Isótopos , Leucocitos/metabolismo , Ratas , Reproducibilidad de los Resultados , Factores de TiempoRESUMEN
Humans are exposed to acrolein and crotonaldehyde due to environmental pollution and endogenous lipid peroxidation. These aldehydes react with the 2'-deoxyguanosine moiety of DNA, forming the exocyclic 1,N2-propano-2'-deoxyguanosine adducts AdG and CdG. These adducts are mutagenic lesions, and they play an important role in cancer and neurodegenerative diseases. In this study, a highly sensitive and quantitative assay was developed for simultaneous detection and quantification of AdG and CdG isomers in human placenta and leukocyte DNA by isotope dilution nanoflow LC with nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS). The on-column detection limits (S/N > or = 3) of AdG and CdG were 15 and 8.9 amol, respectively. The quantification limits of AdG and CdG for the entire assay were 619 and 297 amol, respectively, corresponding to 9.8 and 4.7 adducts in 10(9) normal nucleotides, respectively, starting with 20 microg of DNA. Different enzyme hydrolysis methods were compared, and the optimal hydrolysis conditions were employed for the assay. Levels of AdG and CdG in human placental DNA (20 microg) were 108 and 26 in 10(8) normal nucleotides, respectively, with the respective relative standard deviation (RSD) of 2.6% and 3.1% (n = 3). Levels of AdG and CdG in 9 human leukocyte DNA samples were 78 +/- 23 (mean +/- SD) and 6.2 +/- 3.8 (mean +/- SD) in 10(8) normal nucleotides, respectively, starting from 30 microg of DNA. Using this assay, only 4-6 microg of DNA sample was subjected to this nanoLC-NSI/MS/MS system for analysis. Only 1-1.5 mL of blood is needed for measuring AdG and CdG levels in leukocyte DNA. Thus, it is clinically feasible using this highly sensitive assay to investigate the potential of using these adducts as noninvasive biomarkers for DNA damage resulting from acrolein and crotonaldehyde and to study their roles in cancer development and prevention.