RESUMEN
A quantitative proteomics workflow was implemented that provides extended plasma protein coverage by extensive protein depletion in combination with the sensitivity and breadth of analysis of two-dimensional LC-MS/MS shotgun analysis. Abundant proteins were depleted by a two-stage process using IgY and Supermix depletion columns in series. Samples are then extensively fractionated by two-dimensional chromatography with fractions directly deposited onto MALDI plates. Decoupling sample fractionation from mass spectrometry facilitates a targeted MS/MS precursor selection strategy that maximizes measurement of a consistent set of peptides across experiments. Multiplexed stable isotope labeling provides quantification relative to a common reference sample and ensures an identical set of peptides measured in the set of samples (set of eight) combined in a single experiment. The more extensive protein depletion provided by the addition of the Supermix column did not compromise overall reproducibility of the measurements or the ability to reliably detect changes in protein levels between samples. The implementation of this workflow is presented for a case study aimed at generating molecular signatures for prediction of first heart attack.
Asunto(s)
Proteínas Sanguíneas , Cromatografía de Afinidad/métodos , Proteómica/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Espectrometría de Masas en Tándem/métodos , Anciano , Anciano de 80 o más Años , Biomarcadores , Proteínas Sanguíneas/análisis , Proteínas Sanguíneas/química , Proteínas Sanguíneas/aislamiento & purificación , Femenino , Humanos , Inmunoensayo , Inmunoglobulinas/metabolismo , Marcaje Isotópico , Masculino , Persona de Mediana Edad , Infarto del Miocardio/metabolismo , Reproducibilidad de los ResultadosRESUMEN
Cysteine thiol modifications are increasingly recognized to occur under both physiological and pathophysiological conditions, making their accurate detection, identification, and quantification of growing importance. Among free cysteines, the bulk of modifications occurs on a subset of cysteines that are more reactive. These exist as thiolate anions at physiological pH because of their surrounding electrostatic environment. Reagents with iodoacetamide-active groups can be used to selectively label these reactive thiols with a high degree of selectivity. Thiol adducts can be detected by the failure to label with iodoacetamide or other reagents; restoration of labeling by specific reducing agents (e.g., ascorbate or glutaredoxin) can be used to detect reversible S-nitroso and S-glutathione adducts. These adducts also may be detected with radiolabels and antibodies. S-Glutathiolation in response to physiological stimuli may be detected in cells and tissues with glutathione ester labeled with biotin. Mass spectrometry can identify thiol modifications with precision, and with isotope-coded affinity tags, used to quantify modification of specific thiols. Combinations of these methods increase sensitivity and specificity, and enable quantification and precise identification of thiol modifications that occur under physiological and pathological conditions.
Asunto(s)
Cisteína/metabolismo , Animales , Biotinilación , Glutatión/metabolismo , Yodoacetamida/química , Marcaje Isotópico , Espectrometría de Masas , Nitrosación , Oxidación-Reducción , Procesamiento Proteico-Postraduccional , ProteómicaRESUMEN
p21ras GTPase is the protein product of the most commonly mutated human oncogene and has been identified as a target for reactive oxygen and nitrogen species. Posttranslational modification of reactive thiols, by reversible S-glutathiolation and S-nitrosation, and potentially also by irreversible oxidation, may have significant effects on p21ras activity. Here we used an isotope-coded affinity tag (ICAT) and mass spectrometry to quantitate the reversible and irreversible oxidative posttranslational thiol modifications of p21ras caused by peroxynitrite (ONOO(-)) or glutathione disulfide (GSSG). The activity of p21ras was significantly increased after exposure to GSSG, but not to ONOO(-). The results of LC-MS/MS analysis of tryptic peptides of p21ras treated with ONOO(-) showed that ICAT labeling of Cys(118) was decreased by 47%, whereas Cys(80) was not significantly affected and was thereby shown to be less reactive. The extent of S-glutathiolation of Cys(118) by GSSG was 53%, and that of the terminal cysteines was 85%, as estimated by the decrease in ICAT labeling. The changes in ICAT labeling caused by GSSG were reversible by chemical reduction, but those caused by peroxynitrite were irreversible. The quantitative changes in thiol modification caused by GSSG associated with increased activity demonstrate the potential importance of redox modulation of p21ras.
Asunto(s)
Cisteína/química , Espectrometría de Masas/métodos , Oxidación-Reducción , Oxígeno/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Compuestos de Sulfhidrilo/química , Secuencia de Aminoácidos , Cromatografía Liquida , Radicales Libres , Glutatión/química , Humanos , Datos de Secuencia Molecular , Oxígeno/química , Ácido Peroxinitroso/química , Procesamiento Proteico-Postraduccional , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Helicobacter pylori causes disease manifestations in humans including chronic gastric and peptic ulcers, gastric cancer, and lymphoid tissue lymphoma. Increasing rates of H. pylori clarithromycin resistance has led to higher rates of disease development. Because antibiotic resistance involves modifications of outer membrane proteins (OMP) in other Gram-negative bacteria, this study focuses on identification of H. pylori OMP's using comparative proteomic analyses of clarithromycin-susceptible and -resistant H. pylori strains. Comparative proteomics analyses of isolated sarcosine-insoluble OMP fractions from clarithromycin-susceptible and -resistant H. pylori strains were performed by 1) one dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis protein separation and 2) in-gel digestion of the isolated proteins and mass spectrometry analysis by Matrix Assisted Laser Desorption Ionization-tandem mass spectrometry. Iron-regulated membrane protein, UreaseB, EF-Tu, and putative OMP were down-regulated; HopT (BabB) transmembrane protein, HofC, and OMP31 were up-regulated in clarithromycin-resistant H. pylori. Western blotting and real time PCR, respectively, validated UreaseB subunit and EF-Tu changes at the protein level, and mRNA expression of HofC and HopT. This limited proteomic study provides evidence that alteration of the outer membrane proteins' profile may be a novel mechanism involved in clarithromycin resistance in H. pylori.
Asunto(s)
Antibacterianos/farmacología , Proteínas de la Membrana Bacteriana Externa/análisis , Claritromicina/farmacología , Farmacorresistencia Bacteriana , Helicobacter pylori/química , Helicobacter pylori/efectos de los fármacos , Proteoma/análisis , Proteínas de la Membrana Bacteriana Externa/aislamiento & purificación , Western Blotting , Electroforesis en Gel de Poliacrilamida , Humanos , Proteoma/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
P21ras, the translation product of the most commonly mutated oncogene, is a small guanine nucleotide exchange protein. Oxidant-induced post-translational modifications of p21ras including S-nitrosation and S-glutathiolation have been demonstrated to modulate its activity. Structural characterization of this protein is critical to further understanding of the biological functions of p21ras. In this study, high-resolution and high mass accuracy Fourier transform mass spectrometry was utilized to map, in detail, the post-translational modifications of p21ras (H-ras) exposed to oxidants by combining bottom-up and top-down techniques. For peroxynitrite-treated p21ras, five oxidized methionines, five nitrated tyrosines, and at least two oxidized cysteines (including C118) were identified by "bottom-up" analysis, and the major oxidative modification of C118, Cys118-SO3H, was confirmed by several tandem mass spectrometry experiments. Additionally, "top-down" analysis was conducted on p21ras S-glutathiolated by oxidized glutathione and identified C118 as the major site of glutathiolation among the four surface cysteines. The present study provides a paradigm for an effective and efficient method not only for mapping post-translational modifications of proteins but also for predicting the relative selectivity and specificity of oxidative post-translational modifications, especially using top-down analysis.
Asunto(s)
Análisis de Fourier , Espectrometría de Masas/métodos , Procesamiento Proteico-Postraduccional , Proteínas ras/análisis , Proteínas ras/química , Secuencia de Aminoácidos , Glutatión/química , Glutatión/metabolismo , Humanos , Datos de Secuencia Molecular , Oxidación-Reducción , Ácido Peroxinitroso , Proteínas ras/metabolismoRESUMEN
An approach is described for identifying and quantifying oxidant-sensitive protein thiols using a cysteine-specific, acid-cleavable isotope-coded affinity tag (ICAT) reagent (Applied Biosystems, Foster City, CA). The approach is based on the fact that only free cysteine thiols are susceptible to labeling by the iodoacetamide-based ICAT reagent, and that mass spectrometry can be used to quantitate the relative labeling of free thiols. To validate our approach, creatine kinase with four cysteine residues, one of which is oxidant-sensitive, was chosen as an experimental model. ICAT-labeled peptides derived from creatine kinase were used to evaluate the relative abundance of the free thiols in samples subjected (or not) to treatment with hydrogen peroxide. As predicted, hydrogen peroxide decreased the relative abundance of the unmodified oxidant-sensitive thiol residue of cysteine-283 in creatine kinase, providing proof of principle that an ICAT-based quantitative mass spectrometry approach can be used to identify and quantify oxidation of cysteine thiols. This approach opens an avenue for proteomics studies of the redox state of protein thiols.
Asunto(s)
Marcadores de Afinidad/análisis , Creatina Quinasa/química , Marcaje Isotópico/métodos , Oxidantes/farmacología , Proteoma/análisis , Compuestos de Sulfhidrilo/química , Marcadores de Afinidad/síntesis química , Marcadores de Afinidad/metabolismo , Animales , Cisteína/química , Peróxido de Hidrógeno/farmacología , Yodoacetamida/química , Espectrometría de Masas , Músculo Esquelético/enzimología , Proteoma/química , Proteoma/metabolismo , ConejosRESUMEN
An approach is described for the simultaneous identification and quantitation of oxidant-sensitive cysteine thiols in a complex protein mixture using a thiol-specific, acid-cleavable isotope-coded affinity tag (ICAT) reagent (Applied Biosystems, USA). The approach is based on the fact that only free cysteine thiols are susceptible to labeling by the iodoacetamide-based ICAT, and that mass spectrometry can be used to quantitate the relative labeling of free thiols. Applying this approach, we have identified cysteine thiols of proteins in a rabbit heart membrane fraction that are sensitive to a high concentration of hydrogen peroxide. Previously known and some novel proteins with oxidant-sensitive cysteines were identified. Of the many protein thiols labeled by the ICAT, only relatively few were oxidized more than 50% despite the high concentration of oxidant used, indicating that oxidant-sensitive thiols are relatively rare, and denoting their specificity and potential functional relevance.