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Taurine is the most abundant free amino acid in the human body. It is found in relatively high concentrations (1-10 mM) in many animal tissues but not in plants. It has been studied since the early 1800s but has not been found to be covalently incorporated into proteins in any animal tissue. Taurine has been found in only one macromolecular complex as a post-transcriptional modification to mitochondrial tRNA. Tubulin is the subunit of microtubules found in all eukaryotic species and almost all eukaryotic cells and subject to numerous post-translational modifications (PTMs). An important PTM on α-tubulin is the removal and re-ligation of the final carboxyl residue, tyrosine. We here demonstrate that taurine can be covalently incorporated at the C-terminal end of alpha-tubulin in avian erythrocytes in a reaction that requires the de-tyrosination PTM and prevents the re-tyrosination PTM. Further, this is, to our knowledge, the first instance of taurine incorporation into a large protein.
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Taurina , Tubulina (Proteína) , Animais , Humanos , Microtúbulos/metabolismo , Processamento de Proteína Pós-Traducional , Taurina/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Tirosina/metabolismoRESUMO
In this study, we have evaluated a low field limit drift tube ion mobility device for ion mobility-mass spectrometry (IM-MS) measurements that uses nitrogen as a bath gas with electrospray ionization on a modified Q-TOF instrument. We have determined reduced mobility (K0) and collision cross section (CCS) values for a group of analyte ions that have been characterized previously in other drift tube IM-MS instruments. Our determinations of CCS for this set of ions as well as for standards are in agreement with published values. Because of their importance in biophysics and pharmaceuticals, we expanded our analysis to investigate the properties of cyclodextrins in this system. We present CCS data for both positively and negatively charged cyclodextrins and, for purposes of comparison, maltodextrose ions. Our results are the first reports of these materials as negative ions.
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Current approaches to protein identification rely heavily on database matching of fragmentation spectra or precursor peptide ions. We have developed a method for MALDI TOF-TOF instrumentation that uses peptide masses and their measurement errors to confirm protein identifications from a first pass MS/MS database search. The method uses MS1-level spectral data that have heretofore been ignored by most search engines. This approach uses the distribution of mass errors of peptide matches in the MS1 spectrum to develop a probability model that is independent of the MS/MS database search identifications. Peptide mass matches can come from both precursor ions that have been fragmented as well as those that are tentatively identified by accurate mass alone. This additional corroboration enables us to confirm protein identifications to MS/MS-based scores that are otherwise considered to be only of moderate quality. Straightforward and easily applicable to current proteomic analyses, this tool termed "ProteinProcessor" provides a robust and invaluable addition to current protein identification tools.
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Algoritmos , Mapeamento de Peptídeos/métodos , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Animais , Bases de Dados de Proteínas , Humanos , Camundongos , Modelos Estatísticos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Protein quantification, identification, and abundance determination are important aspects of proteome characterization and are crucial in understanding biological mechanisms and human diseases. Different strategies are available to quantify proteins using mass spectrometric detection, and most are performed at the peptide level and include both targeted and untargeted methodologies. Discovery-based or untargeted approaches oftentimes use covalent tagging strategies (i.e., iTRAQ, TMT), where reporter ion signals collected in the tandem MS experiment are used for quantification. Herein we investigate the behavior of the iTRAQ 8-plex chemistry using MALDI-TOF/TOF instrumentation. The experimental design and data analysis approach described is simple and straightforward, which allows researchers to optimize data collection and proper analysis within a laboratory. iTRAQ reporter ion signals were normalized within each spectrum to remove peptide biases. An advantage of this approach is that missing reporter ion values can be accepted for purposes of protein identification and quantification without the need for ANOVA analysis. We investigate the distribution of reporter ion peak areas in an equimolar system and a mock biological system and provide recommendations for establishing fold-change cutoff values at the peptide level for iTRAQ data sets. These data provide a unique data set available to the community for informatics training and analysis.
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Misturas Complexas/química , Peptídeos/análise , Proteoma/isolamento & purificação , Proteômica/métodos , Coloração e Rotulagem/métodos , Células Hep G2 , Humanos , Íons/química , Proteólise , Proteômica/instrumentação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem , Tripsina/químicaRESUMO
BACKGROUND: The addition of a calibration curve with every run is both time-consuming and expensive for clinical mass spectrometry assays. We present alternative calibration strategies that eliminate the need for a calibration curve except as required by laboratory regulations. METHODS: We measured serum nortriptyline concentrations prospectively in 68 patients on 16 days over a 2-month period using a method employing calibration schemes that relied on the measurement of the response ratio (RR) corrected by the response factor (RF), i.e., a measurement of the RR for an equimolar mixture of the analyte and internal standard. Measurements were taken with contemporaneous RF (cRF) measurements as well as sporadic RF (sRF) measurements. The measurements with these alternative calibration schemes were compared against the clinical results obtained by interpolation on a calibration curve, and those differences were evaluated for analytical and clinical significance. RESULTS: The differences between the values measured by cRF and sRF calibration and interpolation on a calibration curve were not significant (P = 0.088 and P = 0.091, respectively). Both the cRF- and sRF-based calibration results demonstrated a low mean bias against the calibration curve interpolations of 3.69% (95% CI, -15.8% to 23.2%) and 3.11% (95% CI, -16.4% to 22.6%), respectively. When these results were classified as subtherapeutic, therapeutic, or supratherapeutic, there was categorical agreement in 95.6% of the cRF calibration results and 94.1% of the sRF results. CONCLUSIONS: cRF and sRF calibration in a clinically validated liquid chromatography-tandem mass spectrometry assay yields results that are analytically and clinically commensurate to those produced by interpolation with a calibration curve.
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Testes de Química Clínica/normas , Monitoramento de Medicamentos/normas , Nortriptilina/sangue , Testes de Química Clínica/economia , Testes de Química Clínica/estatística & dados numéricos , Monitoramento de Medicamentos/economia , Humanos , Espectrometria de Massas/economiaRESUMO
Smith-Lemli-Opitz syndrome (SLOS) is a malformation syndrome with neurocognitive deficits due to mutations of DHCR7 that impair the reduction of 7-dehydrocholesterol to cholesterol. To investigate the pathological processes underlying the neurocognitive deficits, we compared protein expression in Dhcr7(+/+) and Dhcr7(Delta3-5/Delta3-5) brain tissue. One of the proteins identified was cofilin-1, an actin depolymerizing factor which regulates neuronal dendrite and axon formation. Differential expression of cofilin-1 was due to increased phosphorylation. Phosphorylation of cofilin-1 is regulated by Rho GTPases through Rho-Rock-Limk-Cofilin-1 and Rac/Cdc42-Pak-Limk-Cofilin-1 pathways. Pull-down assays were used to demonstrate increased activation of RhoA, Rac1 and Cdc42 in Dhcr7(Delta3-5/Delta3-5) brains. Consistent with increased activation of these Rho GTPases, we observed increased phosphorylation of both Limk and Pak in mutant brain tissue. Altered Rho/Rac signaling impairs normal dendritic and axonal formation, and mutations in genes encoding regulators and effectors of the Rho GTPases underlie other human mental retardation syndromes. Thus, we hypothesized that aberrant activation of Rho/Rac could have functional consequences for dendrite and axonal growth. In vitro analysis of Dhcr7(Delta3-5/Delta3-5) hippocampal neurons demonstrated both axonal and dendritic abnormalities. Developmental abnormalities of neuronal process formation may contribute to the neurocognitive deficits found in SLOS and may represent a potential target for therapeutic intervention.
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Cofilina 1/metabolismo , Síndrome de Smith-Lemli-Opitz/genética , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Axônios/patologia , Encéfalo/metabolismo , Colesterol/deficiência , Dendritos/patologia , Ativação Enzimática , Quinases Lim/metabolismo , Camundongos , Mutação , Fosforilação , Transdução de Sinais , Síndrome de Smith-Lemli-Opitz/patologiaRESUMO
Smith-Lemli-Opitz syndrome (SLOS) and lathosterolosis are malformation syndromes with cognitive deficits caused by mutations of 7-dehydrocholesterol reductase (DHCR7) and lathosterol 5-desaturase (SC5D), respectively. DHCR7 encodes the last enzyme in the Kandutsch-Russel cholesterol biosynthetic pathway, and impaired DHCR7 activity leads to a deficiency of cholesterol and an accumulation of 7-dehydrocholesterol. SC5D catalyzes the synthesis of 7-dehydrocholesterol from lathosterol. Impaired SC5D activity leads to a similar deficiency of cholesterol but an accumulation of lathosterol. Although the genetic and biochemical causes underlying both syndromes are known, the pathophysiological processes leading to the developmental defects remain unclear. To study the pathophysiological mechanisms underlying SLOS and lathosterolosis neurological symptoms, we performed quantitative proteomics analysis of SLOS and lathosterolosis mouse brain tissue and identified multiple biological pathways affected in Dhcr7(Delta3-5/Delta3-5) and Sc5d(-/-) E18.5 embryos. These include alterations in mevalonate metabolism, apoptosis, glycolysis, oxidative stress, protein biosynthesis, intracellular trafficking, and cytoskeleton. Comparison of proteome alterations in both Dhcr7(Delta3-5/Delta3-5) and Sc5d(-/-) brain tissues helps elucidate whether perturbed protein expression was due to decreased cholesterol or a toxic effect of sterol precursors. Validation of the proteomics results confirmed increased expression of isoprenoid and cholesterol synthetic enzymes. This alteration of isoprenoid synthesis may underlie the altered posttranslational modification of Rab7, a small GTPase that is functionally dependent on prenylation with geranylgeranyl, that we identified and validated in this study. These data suggested that although cholesterol synthesis is impaired in both Dhcr7(Delta3-5/Delta3-5) and Sc5d(-/-) embryonic brain tissues the synthesis of nonsterol isoprenoids may be increased and thus contribute to SLOS and lathosterolosis pathology. This proteomics study has provided insight into the pathophysiological mechanisms of SLOS and lathosterolosis, and understanding these pathophysiological changes will help guide clinical therapy for SLOS and lathosterolosis.
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Colesterol/biossíntese , Redes e Vias Metabólicas/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/deficiência , Proteômica/métodos , Síndrome de Smith-Lemli-Opitz/genética , Síndrome de Smith-Lemli-Opitz/metabolismo , Animais , Encéfalo/enzimologia , Caspase 3/metabolismo , Colesterol/química , Colesterol/metabolismo , Ativação Enzimática , Feminino , Ácido Mevalônico/metabolismo , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Estrutura Molecular , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Síndrome de Smith-Lemli-Opitz/patologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
Isobaric tagging reagents such as isobaric tag for relative and absolute quantitation (iTRAQ) and tandem mass tag (TMT) typically have isotopic impurities that cause significant cross-talk between channels. Here, we present an efficient solution to compensate for channel cross-talk using linear algebra and find that it is between 20× and 120× faster than previous methods. We also find that the effects of channel cross-talk are as important to manage as the effects of ratio compression because of precursor impurities, and we have released an open-source tool to perform both types of calculations.
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Calutrons were developed in the laboratory of E. O. Lawrence at the University of California at Berkeley. They were a modification of the cyclotrons he had invented and used in his Nobel Prize-winning investigations of the atomic nucleus. At the time their construction was undertaken, calutrons represented the only certain means of preparing enriched uranium isotopes for the construction of a fission bomb. The effort was successful enough that every atom of the 42 kg of 235 U used in the first uranium bomb had passed through at least one stage of calutron separation. At peak production, the first stage separators, α tanks, yielded an aggregate 258-g/day 235 U enriched to about 10 at. % from its natural abundance level of 0.72 at. %. The second stage separators, ß tanks, used the 10 at. % material as feedstock and produced a total 204-g/day 235 U enriched to at least 80 at. %. The latter, weapons grade, material was used in fission bombs. Under typical operating conditions, each α tank operated at a uranium beam intensity at the collectors of approximately 20 mA and each ß tank at a beam intensity of approximately 215 mA at the collectors. Bulk separation of isotopes for bomb production ceased in 1945. Since that time calutrons have been used to separate stable isotopes, but on a more limited scale than wartime weapons production. Stable isotope separations since 1960 have taken place using one modified ß tank.
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Precise and accurate protein quantification is critical to many areas of proteomics. Antibody-based approaches are costly and time-consuming to develop, consequently, there is considerable interest in alternative quantitative methods that are versatile and can be implemented without the considerable delays associated with antibody development and characterization. Approaches based on MS have therefore attracted considerable attention and are now frequently touted as the most practical and powerful of all options. Nevertheless, there are serious limitations associated with quantifying a protein based on tandem mass analysis of one or two peptides generated by either chemical or enzymatic cleavage. In an accompanying Viewpoint article, Molloy and coworkers point out that selectivity is not necessarily guaranteed despite the power of SRM. Here we address an additional concern that can also compromise specificity. In complex mammalian systems, multiple proteins can serve as precursors of a single peptide and consequently, depending on the peptide(s) selected, protein levels may be significantly under- or overestimated.
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Espectrometria de Massas/métodos , Proteínas/análise , Proteômica/métodos , Animais , Humanos , Peptídeos/análise , Peptídeos/química , Proteínas/químicaRESUMO
This paper presents a novel theoretical basis for accurately calculating the isotope cluster of polypeptides. In contrast to previous approaches to this problem, which consider exhaustive or near exhaustive combinations of isotopic species, the program, Neutron Cluster, groups probabilities to yield highly accurate information without elucidating any fine structure within a nominal mass unit. This is a fundamental difference from any previously described algorithm for calculating the isotope cluster. As a result of this difference, the accurate isotope clusters for high molecular weight polypeptides can be calculated rapidly without any pruning. When applied to isotope enriched polypeptides, the algorithm introduces "grouping error", which is described, quantified, and avoided by using probability partitioning.
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Peptídeos/química , Algoritmos , Animais , Análise por Conglomerados , Simulação por Computador , Dineínas do Citoplasma , Dineínas/química , Humanos , Isótopos , Nêutrons , ProbabilidadeRESUMO
Regulated exocytosis enables temporal and spatial control over the secretion of biologically active compounds; however, the mechanism by which Ca2+ modulates different stages of exocytosis is still poorly understood. For an unbiased, top-down proteomic approach, select thiol- reactive reagents were used to investigate this process in release-ready native secretory vesicles. We previously characterized a biphasic effect of these reagents on Ca2+-triggered exocytosis: low doses potentiated Ca2+ sensitivity, whereas high doses inhibited Ca2+ sensitivity and extent of vesicle fusion. Capitalizing on this novel potentiating effect, we have now identified fluorescent thiol- reactive reagents producing the same effects: Lucifer yellow iodoacetamide, monobromobimane, and dibromobimane. Top-down proteomic analyses of fluorescently labeled proteins from total and cholesterol-enriched vesicle membrane fractions using two-dimensional gel electrophoresis coupled with mass spectrometry identified several candidate targets, some of which have been previously linked to the late steps of regulated exocytosis and some of which are novel. Initial validation studies indicate that Rab proteins are involved in the modulation of Ca2+ sensitivity, and thus the efficiency of membrane fusion, which may, in part, be linked to their previously identified upstream roles in vesicle docking.
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We present a data processing approach based on the spectral dot product for evaluating spectral similarity and reproducibility. The method introduces 95% confidence intervals on the spectral dot product to evaluate the strength of spectral correlation; it is the only calculation described to date that accounts for both the non-normal sampling distribution of the dot product and the number of peaks the spectra have in common. These measures of spectral similarity allow for the recursive generation of a consensus spectrum, which incorporates the most consistent features from statistically similar replicate spectra. Taking the spectral dot product and 95% confidence intervals between consensus spectra from different samples yields the similarity between these samples. Applying the data analysis scheme to replicates of brain tubulin CNBr peptides enables a robust comparison of tubulin isotype expression and post-translational modification patterns in rat and cow brains.
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Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Tubulina (Proteína)/análise , Animais , Química Encefálica , Bovinos , Intervalos de Confiança , Brometo de Cianogênio/química , Interpretação Estatística de Dados , Processamento Eletrônico de Dados/métodos , Masculino , Peptídeos/química , Isoformas de Proteínas/análise , Isoformas de Proteínas/química , Processamento de Proteína Pós-Traducional , Ratos , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/estatística & dados numéricos , Testículo/química , Tubulina (Proteína)/químicaRESUMO
[This corrects the article DOI: 10.1371/journal.pone.0175478.].
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The effects of laser fluence on ion formation in MALDI were studied using a tandem TOF mass spectrometer with a Nd-YAG laser and alpha-cyano hydrocinnamic acid matrix. Leucine enkephalin ionization and fragmentation were followed as a function of laser fluence ranging from the threshold of ion formation to the maximum available, that is, about 280-930 mJ/mm2. The most notable finding was the appearance of immonium ions at fluence values close to threshold, increasing rapidly and then tapering in intensity with the appearance of typical backbone fragment ions. The data suggest the presence of two distinct environments for ion formation. One is associated with molecular desorption at low values of laser fluence that leads to extensive immonium ion formation. The second becomes dominant at higher fluences, is associated initially with backbone type fragments, but, at the highest values of fluence, progresses to immonium fragments. This second environment is suggestive of ion desorption from large pieces of material ablated from the surface. Arrhenius rate law considerations were used to estimate temperatures associated with the onset of these two processes.
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Encefalina Leucina/química , Lasers , Neurotransmissores/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectrometria de Massas em TandemRESUMO
Presaging the current discipline of Proteomics, Prof Patrick H. O'Farrell recognized the critical need for detailed protein analyses to dissect and thereby understand molecular mechanisms. [...].
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2-Hydroxypropyl-beta-cyclodextrin (HPßCD) has gained recent attention as a potential therapeutic intervention in the treatment of the rare autosomal-recessive, neurodegenerative lysosomal storage disorder Niemann-Pick Disease Type C1 (NPC1). Notably, HPßCD formulations are not comprised of a single molecular species, but instead are complex mixtures of species with differing degrees of hydroxypropylation of the cyclodextrin ring. The degree of substitution is a critical aspect of the complex mixture as it influences binding to other molecules and thus could potentially modulate biological effects. VTS-270 (Kleptose HPB) and Trappsol® Cyclo™ are HPßCD products under investigation as novel treatments for NPC1. The purpose of the present work is to compare these two different products; analyses were based on ion distribution and abundance profiles using mass spectrometry methodology as a means for assessing key molecular distinctions between products. The method incorporated electrospray ionization and analysis with a linear low-field ion mobility quadrupole time-of-flight instrument. We observed that the number of hydroxypropyl groups (the degrees of substitution) are substantially different between the two products and greater in Trappsol Cyclo than in VTS-270. The principal ions of both samples are ammonium adducts. Isotope clusters for each of the major ions show doubly charged homodimers of the ammonium adducts. In addition, both products show doubly charged homodimers from adduction of both a proton and ammonium. Doubly charged heterodimers are also present, but are more intense in Trappsol Cyclo than in VTS-270. Based on the analytical differences observed between VTS-270 and Trappsol Cyclo with respect to the degree of substitution, the composition and fingerprint of the complex mixture, and the impurity profiles, these products cannot be considered to be the same; the potential biological and clinical implications of these differences are not presently known.
Assuntos
Doença de Niemann-Pick Tipo C/tratamento farmacológico , beta-Ciclodextrinas/química , beta-Ciclodextrinas/uso terapêutico , 2-Hidroxipropil-beta-Ciclodextrina , Compostos de Amônio/química , Contaminação de Medicamentos , Humanos , Íons/química , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
We introduce the use of a peptide composition lookup table indexed by residual mass and number of amino acids for de novo sequencing of polypeptides. Polypeptides of 1600 Daltons (Da) or more can be sequenced effectively through exhaustive compositional analysis of MS/MS spectra obtained by unimolecular decomposition (without CID) in a MALDI TOF/TOF despite a fragment mass accuracy of 50 mDa. Peaks are referenced against the lookup table to obtain a complete profile of amino acid combinations, and combinations are assembled into series of increasing length. Concatenating the differences between successive entries in compositional series yields peptide sequences that can be scored and ranked according to signal intensity. While the current work involves measurements acquired on MALDI TOF-TOF, such general treatment of the data anticipates extension to other types of mass analyzers.
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Algoritmos , Bases de Dados de Proteínas , Armazenamento e Recuperação da Informação/métodos , Mapeamento de Peptídeos/métodos , Peptídeos/química , Análise de Sequência de Proteína/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sequência de Aminoácidos , Dados de Sequência MolecularRESUMO
INTRODUCTION: Modern lipid analysis requires mass spectrometric techniques, though to date these have been developed and applied primarily to histological serial sections. As such, there has been little emphasis on using mass spectrometry in such a way that the same specimen can yield both chemical and morphological information. Here, we present a mass spectrometric method that enables measurement of lipids from cells on cytospin slides in a way that preserves the cells for subsequent cytomorphologic evaluation. MATERIALS AND METHODS: Standardized cultures of MDA-MB-231, a breast cancer cell line, were prepared as cytospins and subjected to analysis using a Prosolia Flowprobe sampling and ionization source attached to a Thermo Scientific Quadrupole-Orbitrap mass spectrometer. Chemical compositions were deduced with accurate mass measurements and fragmentation of high intensity peaks to further determine chemical structure. After mass spectrometry, the slides were stained and cover-slipped, and the cells were reviewed for cytomorphologic features of breast cancer. These were compared to control slides of the same cellular concentration that had not been subjected to this analysis. RESULTS: Spectra from samples of all cellular concentrations demonstrated characteristic qualitative features that were discovered to represent phosphatidylcholines, phosphatidylglycerols, and phosphatidylserines with fragmentation and accurate mass matching. Cytomorphologic analysis demonstrated excellent preservation of the cells subjected to the Flowprobe analysis. CONCLUSION: Direct extraction, ionization, and identification of lipids is possible from cytologic preparations in such a way that the analyzed material is preserved and useful for subsequent microscopic analysis.
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Hemoglobin (Hb) adducts have long been used as dosimeters of exposure to xenobiotics and endogenously formed reactive metabolites. In this study, hemoglobin chains were separated from each other and their prosthetic heme groups and reacted with 4-oxo-2-nonenal, a major breakdown product of lipid hydroperoxides. The adducts were characterized by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-TOF/MS) analysis of the intact proteins and by a combination of liquid chromatography/electrospray ionization/tandem MS (MS/MS) and MALDI-TOF/MS/MS analysis of the tryptic peptides. Covalent modifications were found on both hemoglobin chains. The location was determined to be on H20 of the alpha-hemoglobin chain and on H(63) of the beta-hemoglobin chain. Molecular modeling revealed that these two residues were two most solvent accessible H residues present in intact Hb. The proposed reaction mechanism is based on that described for the reaction of 4-hydroxy-2-nonenal with proteins. Initial nucleophilic Michael addition is followed by hydration of the resulting aldehyde, cyclization, and two sequential dehydration reactions to give stable furan derivatives. This results in the addition of 136 Da from 4-oxo-2-nonenal to give adducts corresponding to (17)VGAH(.) AGEYGAEALER(31) from alpha-hemoglobin and (62)AH(.) GK(65) from beta-hemoglobin. These hemoglobin modifications can potentially serve as biomarkers of lipid hydroperoxide-mediated macromolecule damage and may reflect an indirect measurement of the potential for DNA damage in vivo.