RESUMEN
Liquid chromatography-mass spectrometry (LC-MS) is a highly sensitive tool for the analysis of polyphenolic compounds in complex food and beverage matrices. However, the high degree of isomerism among polyphenols in general often complicates this approach, especially for identification of novel compounds. Here, we explore the utility of mild acid-catalyzed deuterium (MACD) labeling via electrophilic aromatic substitution as a complementary method for informing polyphenolic compound structure elucidation. To prevent hydrolysis of acid-labile glycosidic linkages, optimal reaction conditions that maximize regioselective hydrogen/deuterium (H/D) exchange of aromatic protons while preserving compound integrity were characterized (60°C, pH 3.0, 72 h). Under these conditions, standard compounds varying in the number and position of hydroxyl, glycosyl, and methyl groups about their aromatic core structure produced distinguishable H/D exchange patterns. The applicability of this method for the analysis of complex mixtures was demonstrated in red wine where the extent of deuterium exchange, together with accurate mass information, led to the putative identification of an unknown compound. The identification was further supported by tandem MS (MS/MS) data, which matched conclusively to the same compound in the Metlin LC-MS/MS library. With the capacity to discriminate between select isomeric forms, MACD labeling provides structural information that complements accurate mass and tandem mass spectral measurements for informing the identification of polyphenolics by MS.
Asunto(s)
Deuterio/química , Espectrometría de Masas/métodos , Polifenoles/análisis , Polifenoles/química , Solventes/química , Catálisis , Estereoisomerismo , Especificidad por Sustrato , Vino/análisisRESUMEN
Polyphenolics are a chemically diverse class of plant specialized metabolites with strong antioxidant properties, and their consumption has been associated with improved human health. Metabolomic analysis of these compounds in both plant and mammalian samples has relied predominantly on liquid chromatography coupled to electrospray ionization mass spectrometry (LC-ESI-MS). Due to variable matrix effects across samples during ionization, the accuracy of this approach for quantifying compounds is greatly improved by incorporating stable isotope-labeled standards into the sample prior to analysis. However, commercially available, stable isotope-labeled, polyphenolic standards are both limited and costly. Here we present a protocol for generating stable isotope-labeled polyphenolics based on their deuteration by mild acid-catalyzed, electrophilic aromatic substitution. Importantly, this protocol is effective for generating stable isotope-labeled standards of many biologically relevant polyphenolics, both aglycones and the various conjugated forms alike.
Asunto(s)
Metaboloma , Metabolómica , Extractos Vegetales/química , Polifenoles/química , Cromatografía Líquida de Alta Presión , Cromatografía Liquida , Deuterio/química , Marcaje Isotópico , Espectrometría de Masas , Plantas/metabolismo , Extracción en Fase SólidaRESUMEN
A rapid analytical procedure was developed to quantify major selective estrogen receptor modulators (SERMs) simultaneously using stable isotope dilution mass spectrometry (SID-LCMS). Two novel isotopically labeled (SIL) analogues of natural SERMs, genistein and daidzein, were synthesized using a H/D exchange reaction mechanism. Computational chemistry coupled with MS and NMR data confirmed the site and mechanism of deuteration. The SIL analogues, which were mono- and dideutero substituted at the ortho positions, exhibited minimal deuterium isotope effects and were stable under the employed sample preparation protocol and MS analysis. An isotopic overlap correction was successfully employed to improve the accuracy and precision of the analytical method. The developed method, which was found to be sensitive, selective, precise and accurate, could be a valuable tool for research focused on determining the bioavailability of individual SERMs.
Asunto(s)
Espectrometría de Masas/métodos , Moduladores Selectivos de los Receptores de Estrógeno/análisis , Animales , Calibración , Deuterio , Genisteína/análisis , Genisteína/sangre , Hidrólisis , Isoflavonas/análisis , Isoflavonas/sangre , Espectroscopía de Resonancia Magnética , Masculino , Modelos Teóricos , Ratas , Ratas Wistar , Estándares de Referencia , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Proteomic characterization of carbonylated amino acid sites currently relies on confidently matching tandem mass spectra (MS(2)) to peptides within a sequence database. Although effective to some degree, reliable proteomic characterization of carbonylated peptides using this approach remains a challenge needing new, complementary solutions. To this end, we developed a method based on partial (18)O-labeling of reactive carbonyl modifications, which produces a unique isotope signature in mass spectra of carbonylated peptides and enables their detection without reliance on matching MS(2) spectra to a peptide sequence. Key to our method were optimized measures for eliminating trypsin-catalyzed incorporation of (18)O at peptide C-termini, and for stabilizing the incorporated (18)O within the carbonyl modification to prevent its loss during liquid chromatography separation. Applying our method to a rat skeletal muscle homogenate treated with the carbonyl modification 4-hyroxynonenal (4-HNE), we demonstrated its compatibility with solid-phase hydrazide enrichment of carbonylated peptides from complex mixtures. Additionally, we demonstrated the value of (18)O isotope signatures for confirming HNE-modified peptide sequences matched via sequence database searching, and identifying modified peptides missed by MS(2) and/or sequence database searching. Combining our (18)O-labeling method with a customized automated software script, we systematically evaluated for the first time the efficiency of MS(2) and sequence database searching for identifying HNE-modified peptides. We estimated that less than half of the modified peptides selected for MS(2) were successfully identified. Collectively, our method and software should provide valuable new tools for investigators studying protein carbonylation via mass spectrometry-based proteomics.
Asunto(s)
Espectrometría de Masas/métodos , Isótopos de Oxígeno/química , Péptidos/química , Carbonilación Proteica , Proteómica/métodos , Aldehídos/química , Animales , Bases de Datos de Proteínas , Caballos , Concentración de Iones de Hidrógeno , Marcaje Isotópico , Músculo Esquelético/química , Mioglobina/química , Mioglobina/metabolismo , Isótopos de Oxígeno/metabolismo , Péptidos/metabolismo , Ratas , Temperatura , Tropomiosina/química , Tropomiosina/metabolismo , Tripsina/metabolismoRESUMEN
The modification of proteins by the cytotoxic, reactive aldehyde 4-hydroxynonenal (HNE) is known to alter protein function and impair cellular mechanisms. In order to identify susceptible amino acid sites of HNE modification within complex biological mixtures by microcapillary liquid chromatography and linear ion trap tandem mass spectrometry, we have developed a solid-phase capture and release strategy that utilizes reversible hydrazide chemistry to enrich HNE-modified peptides. To maximize the detection of fragment ions diagnostic of HNE modification, both neutral loss-dependent acquisition of MS/MS/MS spectra and the pulsed Q dissociation operation mode were employed. When the solid-phase hydrazide enrichment strategy was applied to a yeast lysate treated with HNE, 125 distinct amino acid sites of HNE modification were mapped on 67 different proteins. The endogenous susceptibility of many of these proteins to HNE modification was demonstrated by analyzing HNE-treated yeast cell cultures with a complementary biotin hydrazide enrichment strategy. Further analysis revealed that the majority of amino acid sites susceptible to HNE modification were histidine residues, with most of these sites being flanked by basic amino acid residues, and predicted to be solvent exposed. These results demonstrate the effectiveness of this novel strategy as a general platform for proteome-scale identification of amino acid sites susceptible to HNE modification from within complex mixtures.
Asunto(s)
Aldehídos/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas/análisis , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Sitios de Unión , Proteínas Fúngicas/análisis , HidrazinasRESUMEN
Protein sequence database searching of tandem mass spectrometry data is commonly employed to identify post-translational modifications (PTMs) to peptides in global proteomic studies. In these studies, the accurate identification of these modified peptides relies on strategies to ensure high-confidence results from sequence database searching in which differential mass shift parameters are employed to identify PTMs to specific amino acids. Using lysine acetylation as an example PTM, we have observed that the inclusion of differential modification information in sequence database searching dramatically increases the potential for false-positive sequence matches to modified peptides, making the confident identification of true sequence matches difficult. In a proof-of-principle study of whole cell yeast lysates, we demonstrate the combination of preparative isoelectric focusing using free-flow electrophoresis, and an adjusted peptide isoelectric point prediction algorithm, as an effective means to increase the confidence of lysine-acetylated peptide identification. These results demonstrate the potential utility of this general strategy for improving the identification of PTMs which cause a shift to the intrinsic isoelectric point of peptides.
Asunto(s)
Focalización Isoeléctrica/métodos , Lisina/química , Péptidos/química , Péptidos/metabolismo , Procesamiento Proteico-Postraduccional , Acetilación , Secuencia de Aminoácidos , Bases de Datos de Proteínas , Punto Isoeléctrico , Datos de Secuencia Molecular , Péptidos/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Revolutionary advances in biological mass spectrometry (MS) have provided a basic tool to make possible comprehensive proteomic analysis. Traditionally, two-dimensional gel electrophoresis has been used as a separation method coupled with MS to facilitate analysis of complex protein mixtures. Despite the utility of this method, the many challenges of comprehensive proteomic analysis has motivated the development of gel-free MS-based strategies to obtain information not accessible using two-dimensional gel separations. These advanced strategies have enabled researchers to dig deeper into complex proteomes, gaining insights into the composition, quantitative response, covalent modifications and macromolecular interactions of proteins that collectively drive cellular function. This review describes the current state of gel-free, high throughput proteomic strategies using MS, including (i) the separation approaches commonly used for complex mixture analysis; (ii) strategies for large-scale quantitative analysis; (iii) analysis of post-translational modifications; and (iv) recent advances and future directions. The use of these strategies to make new discoveries at the proteome level into the effects of disease or other cellular perturbations is discussed in a variety of contexts, providing information on the potential of these tools in electromagnetic field research.
Asunto(s)
Espectrometría de Masas/métodos , Proteoma/química , Proteoma/metabolismo , Proteómica/métodos , Animales , Electroforesis , Humanos , Isótopos , Espectrometría de Masas/instrumentación , Procesamiento Proteico-Postraduccional , Proteómica/instrumentaciónRESUMEN
BACKGROUND: Limited data are available on carcinogen uptake in smokers who reduce their smoking. To determine whether reducing the number of cigarettes smoked per day would lead to a corresponding reduction in carcinogen uptake, we measured levels of metabolites of the tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in the urine of smokers who reduced their smoking for up to 26 weeks. METHODS: We recruited 153 smokers, of whom 151 were randomly assigned to a reduction group or a waitlist group. In the reduction group of 102 smokers, we measured the metabolites 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its glucuronides (NNAL-Gluc) at two baseline times and at weeks 4, 6, 8, 12, and 26 after baseline. Smokers were then expected to reduce their number of cigarettes per day by 25% in weeks 0-2, 50% in weeks 2-4, and 75% in weeks 4-6 and to maintain the reduced level through week 26. In the waitlist group of 49 smokers, four baseline measurements over 7 weeks were made to assess the longitudinal stability of the metabolite measurements, and then the smokers began the reduction program. All statistical tests were two-sided. RESULTS: For waitlist and reduction groups results were comparable. Statistically significant reductions in the lung carcinogen metabolites were observed at most intervals as smokers reduced the number of cigarettes smoked each day. However, the observed decreases were generally modest, always proportionally less than the reductions in cigarettes smoked per day, and sometimes transient. For example, among the 65 individuals in the reduction group who reduced cigarettes per day by 40% or more during weeks 4-12 after baseline, mean decreases in cigarettes per day were 53% (week 4), 74% (week 6), 75% (week 8), and 74% (week 12); whereas the corresponding mean reductions in NNAL plus NNAL-Gluc were 29%, 33%, 37%, and 29%. (P<.001 for all NNAL plus NNAL-Gluc values) CONCLUSIONS: Statistically significant reductions in levels of urinary metabolites of a tobacco-specific lung carcinogen were achieved by reduction in smoking, but for most smokers, reductions were modest and transient.