RESUMEN
Mass spectrometry is the tool of choice for sequencing peptides and determining the sites of posttranslational modifications; however, this bottom-up approach lacks in providing global information about the modification states of proteins including the number and types of isoforms and their stoichiometry. Recently, various techniques and mass spectrometers, such as high-field Fourier Transform Ion Cyclotron Resonance (FTICR) mass spectrometers, have been developed to study intact proteins (top-down proteomics). While the protein molecular mass and the qualitative and quantitative information about protein isoforms can be revealed by FTICR-MS analysis, their primary structure (including the identification of modifications and their exact locations in the amino acid sequence) can directly be determined using the MS/MS capability offered by the FTICR mass spectrometer. The distinct advantage of top-down methods are that modifications can be determined for a specific protein isoform rather than for peptides belonging to one or several isoforms. In this chapter, we describe different top-down proteomic approaches enabled by high-field (7, 9.4, and 12 T) FTICR mass spectrometers, and their applicability to answer biological and biomedical questions. We also describe the use of the free flow electrophoresis (FFE) to separate proteins prior to top-down mass spectrometric characterization.
Asunto(s)
Ciclotrones , Análisis de Fourier , Espectrometría de Masas/métodos , Proteómica/métodos , Secuencia de Aminoácidos , Animales , Focalización Isoeléctrica , Datos de Secuencia Molecular , Proteínas/análisis , Proteínas/química , Proteínas/aislamiento & purificación , Espectrometría de Masas en TándemRESUMEN
With unmatched mass resolution, mass accuracy, and exceptional detection sensitivity, Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FTICR-MS) has the potential to be a powerful new technique for high-throughput metabolomic analysis. In this study, we examine the properties of an ultrahigh-field 12-Tesla (12T) FTICR-MS for the identification and absolute quantitation of human plasma metabolites, and for the untargeted metabolic fingerprinting of inbred-strain mouse serum by direct infusion (DI). Using internal mass calibration (mass error ≤1 ppm), we determined the rational elemental compositions (incorporating unlimited C, H, N and O, and a maximum of two S, three P, two Na, and one K per formula) of approximately 250 out of 570 metabolite features detected in a 3-min infusion analysis of aqueous extract of human plasma, and were able to identify more than 100 metabolites. Using isotopically-labeled internal standards, we were able to obtain excellent calibration curves for the absolute quantitation of choline with sub-pmol sensitivity, using 500 times less sample than previous LC/MS analyses. Under optimized serum dilution conditions, chemical compounds spiked into mouse serum as metabolite mimics showed a linear response over a 600-fold concentration range. DI/FTICR-MS analysis of serum from 26 mice from 2 inbred strains, with and without acute trichloroethylene (TCE) treatment, gave a relative standard deviation (RSD) of 4.5%. Finally, we extended this method to the metabolomic fingerprinting of serum samples from 49 mice from 5 inbred strains involved in an acute alcohol toxicity study, using both positive and negative electrospray ionization (ESI). Using these samples, we demonstrated the utility of this method for high-throughput metabolomics, with more than 400 metabolites profiled in only 24 h. Our experiments demonstrate that DI/FTICR-MS is well-suited for high-throughput metabolomic analysis.
RESUMEN
A novel algorithm based on Data Self-Recalibration and a subsequent Mixture Mass Fingerprint search (DASER-MMF) has been developed to improve the performance of protein identification from online 1D and 2D-LC-MS/MS experiments conducted on high-resolution mass spectrometers. Recalibration of 40% to 75% of the MS spectra in a human serum dataset is demonstrated with average errors of 0.3 +/- 0.3 ppm, regardless of the original calibration quality. With simple protein mixtures, the MMF search identifies new proteins not found in the MS/MS based search and increases the sequence coverage for identified proteins by six times. The high mass accuracy allows proteins to be identified with as little as three peptide mass hits. When applied to very complex samples, the MMF search shows less dramatic performance improvements. However, refinements such as additional discriminating factors utilized within the search space provide significant gains in protein identification ability and indicate that further enhancements are possible in this realm.
Asunto(s)
Algoritmos , Mapeo Peptídico/estadística & datos numéricos , Proteínas/química , Animales , Proteínas Sanguíneas/química , Bovinos , Cromatografía Liquida , Bases de Datos de Proteínas , Humanos , Espectrometría de Masas en TándemRESUMEN
Electron transfer dissociation (ETD) of proteins is demonstrated in a hybrid quadrupole-hexapole Fourier transform ion cyclotron resonance mass spectrometer (Qh-FTICRMS). Analyte ions are selected in the mass analyzing quadrupole, accumulated in the hexapole linear ion trap, reacted with fluoranthene reagent anions, and then analyzed via an FTICR mass analyzer. The hexapole trap allows for a broad fragment ion mass range and a high ion storage capacity. Using a 3 T FTICRMS, resolutions of 60 000 were achieved with mass accuracies averaging below 1.4 ppm. The high resolution, high mass accuracy ETD spectra provided by FTICR obviates the need for proton transfer reaction (PTR) charge state reduction of ETD product ions when analyzing proteins or large peptides. This is demonstrated with the ETD of ubiquitin and apomyoglobin yielding sequence coverages of 37 and 20%, respectively. We believe this represents the first reported successful combination of ETD and a FTICRMS.
Asunto(s)
Ciclotrones/instrumentación , Mapeo Peptídico/métodos , Proteínas/química , Manejo de Especímenes/instrumentación , Espectrometría de Masa por Ionización de Electrospray/instrumentación , Espectroscopía Infrarroja por Transformada de Fourier/instrumentación , Secuencia de Aminoácidos , Transporte de Electrón , Diseño de Equipo , Análisis de Falla de Equipo , Microquímica/instrumentación , Microquímica/métodos , Datos de Secuencia Molecular , Proteínas/análisis , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectroscopía Infrarroja por Transformada de Fourier/métodosRESUMEN
PURPOSE: Human eye lenses at birth are primarily constructed of 12 distinct crystallins and two truncated crystallins. The molecular weights of these 14 proteins vary between about 20,000 and 30,000 Da. The relative amounts of these molecules and their post-synthetic changes with age are of substantial interest in the study of lens biochemistry and lens pathology. Fourier transform mass spectrometry of unfractionated lens homogenates now permits precise quantitative measurement of the relative amounts of lens crystallins. We report herein the measurement of the 14 crystallins in 10 pairs of lenses from humans between the ages of 2 and 300 days. METHODS: Eye lenses were obtained from human donors of various ages in the first year of life. These lenses were homogenized in 0.02 M phosphate buffer at pH 7.0 with 0.001 M EDTA, desalted by washing over a 3,000 Da filter, and injected directly into the nanospray source of a hybrid Fourier transform ion cyclotron resonance mass spectrometer, Qq-FT(ICR)MS, equipped with a 12 Tesla magnet. The crystallins were quantitatively ionized and mass analyzed in the ICR cell of the mass spectrometer. The detected signals of all of the isotopic and charge state species for each crystallin were normalized and summed to determine the protein quantities. RESULTS: The relative amounts of the 14 crystallins are found to be quite similar from individual to individual at birth. These amounts are in integer ratios to one another that suggest important structural relations within the lens. In two cases, the relative amounts of alphaA- and betaB2-crystallin change proportionally to the logarithm of age during the first year, with alphaA- decreasing and betaB2-crystallin increasing. The changes in alphaA- and betaB2-crystallin are mutually offsetting, with alphaA-crystallin decreasing from 30% to 18% and betaB2-increasing from 12% to 24%. CONCLUSIONS: These observations suggest that the human eye lens at birth is constructed of crystallins in which the numbers of crystallin molecules have regular integral relationships to each other. As the lens develops during the first year, some of these relationships change. While the functional significance of the reciprocal decrease in alphaA- and increase in betaB2-crystallin is not known, betaB2-crystallin may substitute for alphaA-crystallin in the lens structures synthesized during the year after birth. Direct injection FT(ICR)MS of unfractionated lens was found to be an excellent method for the quantitative measurement of lens crystallins.
Asunto(s)
Cristalinas/metabolismo , Análisis de Fourier , Cristalino/metabolismo , Espectrometría de Masas/métodos , Envejecimiento/metabolismo , Humanos , Técnicas In Vitro , Lactante , Recién Nacido , Cadena A de alfa-Cristalina/metabolismo , Cadena B de beta-Cristalina/metabolismoRESUMEN
The stem-loop-binding protein (SLBP) is involved in multiple aspects of histone mRNA metabolism. To characterize the modification status and sites of SLBP, we combined mass spectrometric bottom-up (analysis of peptides) and top-down (analysis of intact proteins) proteomic approaches. Drosophilia SLBP is heavily phosphorylated, containing up to seven phosphoryl groups. Accurate M(r) determination by Fourier transform ion cyclotron resonance (FTICR)-MS and FTICR-MS top-down experiments using a variety of dissociation techniques show there is removal of the initiator methionine and acetylation of the N terminus in the baculovirus-expressed protein, and that T230 is stoichiometrically phosphorylated. T230 is highly conserved; we have determined that this site is also completely phosphorylated in baculovirus-expressed mammalian SLBP and extensively phosphorylated in both Drosophila and mammalian cultured cells. Removal of the phosphoryl group from T230 by either dephosphorylation or mutation results in a 7-fold reduction in the affinity of SLBP for the stem-loop RNA.
Asunto(s)
Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteómica/métodos , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Factores de Escisión y Poliadenilación de ARNm/química , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster/química , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Humanos , Espectrometría de Masas , Datos de Secuencia Molecular , Peso Molecular , Proteínas Nucleares/genética , Conformación de Ácido Nucleico , Fosforilación , Fosfotreonina/metabolismo , Unión Proteica , ARN/química , Proteínas de Unión al ARN/genética , Espectroscopía Infrarroja por Transformada de Fourier , Factores de Escisión y Poliadenilación de ARNm/genéticaRESUMEN
The structures of several powerful inhibitors of hydrolytic enzymes resemble that of the altered substrate in the transition state, except that a hydrogen atom replaces one substituent (typically the leaving group). To test the hypothesis that a water molecule might be present in the gap resulting from this replacement, we examined a transition-state analogue complex formed by Escherichia coli cytidine deaminase by Fourier transform ion cyclotron resonance MS in electrospray mode. Upon nebularization from aqueous solution under conditions (pH 5.6) where the enzyme is active, cytidine deaminase remains dimeric in the vapor phase. In the presence of inhibitor, the enzyme's exact mass can be used to infer the presence at each active site of zinc, 5-fluoro-3,4-dihydrouridine, and a single water molecule.