RESUMEN
A tandem quadrupole time-of-flight (Q-TOF) mass spectrometer has been programmed such that phosphorylated peptides can automatically be discovered and identified in a way similar to that of the use of precursor ion or neutral loss scanning, but without the need to scan the quadrupole mass filter. Instead, the method capitalizes on the innate capability of the Q-TOF to record mass spectra and product ion spectra quickly, with good sensitivity and with good mass accuracy. Alternate mass spectra, with and without fragmentation, are recorded at high and low collision energy with the quadrupole operating in wideband mode. The method of analysis is both compatible with and dependant on liquid chromatography for separation of complex mixtures. The method has been demonstrated by searching for the neutral loss of 98 Da (H3PO4) from phosphoserine and phosphothreonine residues, or for the phosphorylated immonium ion at m/z 216 from phosphotyrosine. The method also incorporates acquisition of the product ion spectrum from any candidate precursor ions, thereby allowing confirmation of the neutral loss or product ion and providing additional sequence information to assist identification of the protein and assign the site of phosphorylation.
Asunto(s)
Proteínas/química , Caseínas/química , Cromatografía Líquida de Alta Presión , Espectrometría de Masas , Fosfopéptidos/química , Fosforilación , Hidrolisados de Proteína/química , Procesamiento Proteico-Postraduccional , TripsinaRESUMEN
The dynamics of enzyme-catalyzed glutathione conjugation was studied by electrospray quadrupole/time-of-flight (Q-TOF) mass spectrometry with a nanospray interface. After incubation of human glutathione S-transferase A1-1 (GT) with glutathione (GSH) and an electrophilic substrate, electrospray indicated the presence of enzyme/product adducts such as [2GT + product], [2GT + GSH' + product], and [2GT + 2 products] as well as [2GT] and [2GT + GSH']. The relative abundance of GT/product adduct ions increased with incubation time. The wide m/z range of detection (m/z 300-5000) allowed the observation of product, suggested to be released from enzyme/product adducts, in the same mass spectrum. The noncovalent complexes of GT/product were completely replaced by GT/inhibitor complexes following the addition of GT inhibitor to the incubation mixture. Furthermore, a collision-activated decomposition analysis of these ion species provided us with useful information to interpret or identify ion species. The results suggest that electrospray Q-TOF mass spectrometry is a powerful approach for studying the dynamics of the enzyme reaction as well as the structure of enzyme complexes at high sensitivity.
Asunto(s)
Dinitroclorobenceno/química , Glutatión Transferasa/metabolismo , Glutatión/análisis , Espectrometría de Masas/métodos , Nitrobenzoatos/química , Dinitroclorobenceno/metabolismo , Inhibidores Enzimáticos/metabolismo , Glutatión/análogos & derivados , Glutatión/metabolismo , Humanos , Isoenzimas , Cinética , Nitrobenzoatos/metabolismo , Unión Proteica/fisiologíaRESUMEN
Development of cancer is a complex process involving multiple changes in gene expression. To unravel these alterations, a proteome approach aimed at the identification of qualitative and quantitative changes in protein composition, including their post-translational modifications, attracts great attention. Our study was focused on the identification of proteins whose amount is altered in the course of malignant transformation of colon mucosa. Proteins extracted from tissue specimens or cell lysates were separated by two-dimensional gel electrophoresis (2-DE). Comparative analyses of 2-DE protein patterns were done using computerized image analysis. Selected proteins exhibiting statistically significant abundance alterations comparing healthy and diseased tissues were identified by mass spectrometry. Globally, we have found 57 proteins that exhibited either a significant decrease or increase in amount in pathological tissues, and 18 of these were annotated by mass spectrometry. The alterations in the expression of nine proteins were common for both precancerous and neoplastic tissues suggesting their role in colon tumorigenesis. The epithelial origin of all identified spots was checked in two cell lines Caco-2 and DLD-1 originating from well-differentiated and poorly differentiated colon carcinoma, respectively.
Asunto(s)
Adenocarcinoma/química , Adenoma/química , Pólipos del Colon/química , Neoplasias Colorrectales/química , Proteínas de Neoplasias/análisis , Proteoma , Adenocarcinoma/genética , Adenocarcinoma/patología , Adenoma/genética , Adenoma/patología , Diferenciación Celular , Colon/química , Pólipos del Colon/genética , Pólipos del Colon/patología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Electroforesis en Gel Bidimensional , Humanos , Procesamiento de Imagen Asistido por Computador , Mucosa Intestinal/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/aislamiento & purificación , Lesiones Precancerosas/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Células Tumorales Cultivadas/químicaRESUMEN
Qualitative and quantitative analyses of urinary nucleosides have diagnostic potential as tumour markers. We have developed separation techniques linked to mass spectrometric detection in order to overcome the problems associated with past identification and quantitation methods. The three methods of analysis utilised were: gas chromatography/mass spectrometry (GC/MS), high-performance liquid chromatography/ion-trap mass spectrometry (HPLC/ITMS) and capillary liquid chromatography/triple quadrupole mass spectrometry (CapLC/TQMS). Here we compare the relative effectiveness of each of the techniques for subsequent application in the systematic study of urinary nucleoside profiles in cancer patients. All three methods proved to be valuable techniques for such urinary nucleoside analyses, and a combination rather than one single choice is concluded as the ideal.
Asunto(s)
Nucleósidos/orina , Cromatografía Líquida de Alta Presión , Electroforesis Capilar , Cromatografía de Gases y Espectrometría de Masas , Humanos , Espectrometría de Masas , Neoplasias/orinaRESUMEN
Human glutathione S-transferase A1-1 was observed predominantly as dimeric ions (51 kDa) during electrospray mass spectrometric analysis from aqueous solution at pH 7.4, in keeping with the known dimeric structure in solution. When analyses were performed on solutions of the enzyme containing glutathione (GSH), noncovalent adducts of protein dimer and one or two ligand molecules were observed; each mass increment, which exceeded the mass of GSH alone, was provisionally interpreted to indicate concomitant association of two water molecules per bound GSH. Noncovalent adducts of ligand and protein dimer were similarly observed for oxidized glutathione and for two glutathione inhibitors, both incorporating substituted thiol structures. In these instances, the mass increments exactly matched the ligand masses, suggesting that the apparent concomitant binding of water was associated with the presence in the ligand of a free thiol group. Collisionally activated decomposition during tandem mass spectrometry analyses of noncovalent adducts incorporating protein dimer and ligands yielded initially the denuded dimer; at higher collision energies the monomer and a protein fragment were formed.
Asunto(s)
Glutatión Transferasa/química , Electroquímica , Humanos , Indicadores y Reactivos , Ligandos , Espectrometría de Masas , Peso Molecular , Proteínas Recombinantes/químicaRESUMEN
Oligosaccharides released from several glycoproteins were derivatized with either 4-aminobenzoic acid 2-(diethylamino)ethyl ester (ABDEAE) (Yoshino, K.; et al. Anal. Chem. 1995, 67, 4028-4031) or 2-aminopyridine. The resulting derivatives were analyzed on a nanoflow electrospray ionization (ESI) quadrupole-inlet time-of-flight mass spectrometer using the low-energy collision-induced dissociation technique. In the MS/MS spectra, the oxonium (b or internal series) and y series ions, which are derived from the multiply charged precursor ions, were predominant and were used for the structural readout. Some oxonium ions that were observed in the low-mass region, but that were not found in the PSD analyses (Mo, W.; et al. Anal. Chem. 1998, 70, 4520-4526), rendered a more detailed structural insight. The oxonium ions at m/z 512.2, which are derived from the fucosylated oligosaccharides of immunoglobulin Y and thyroglobulin, were observed, suggesting that fucosylation had occurred proximal to the outer nonreducing terminus. In addition, the data herein show that structural elucidation can be routinely achieved at a low sample concentration. For the case of ABDEAE derivatives, this can be achieved at the 50 fmol/microL level and with the actual sample consumption at the attomole level using nanoflow ESI MS/MS.
Asunto(s)
Oligosacáridos/análisis , Aminopiridinas/química , Secuencia de Carbohidratos , Inmunoglobulinas/análisis , Inmunoglobulinas/química , Espectrometría de Masas/métodos , Datos de Secuencia Molecular , Oligosacáridos/química , Procaína/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Tiroglobulina/análisis , Tiroglobulina/química , Transferrina/análisis , Transferrina/químicaRESUMEN
Polyglycylation is a posttranslational modification specific to tubulin. This modification was originally identified in highly stable microtubules from Paramecium cilia. As many as 34 posttranslationally added glycine residues have been located in the C-terminal domains of Paramecium alpha- and beta-tubulin. In this study, post source decay matrix-assisted laser desorption/ionization mass spectrometry (PSD MALDI MS) and electrospray ionization on a hybrid quadrupole orthogonal time-of-flight tandem mass spectrometer (ESI Q-TOF MS/MS) were both used to demonstrate that a single molecule of beta-tubulin, from either dynamic cytoplasmic microtubules or stable axonemal microtubules, can be glycylated on each of the last four C-terminal glutamate residues Glu437, Glu438, Glu439, and Glu441 in the sequence 427DATAEEEGEFEEEGEQ442. In both dynamic and stable microtubules the most abundant beta-tubulin isoform contains six posttranslationally added glycine residues: two glycine residues on both Glu437 and Glu438 and one glycine residue on both Glu439 and Glu441. The number and relative abundance of glycylated isoforms of beta-tubulin in both cytoplasmic and axonemal microtubules were compared by MALDI MS.1 The abundance of the major glycylated isoforms in axonemal tubulin decreases regularly with glycylation levels from 6 to 19 whereas it drops abruptly in cytoplasmic tubulin with glycylation levels from 6 to 9. However, the polyglycine chains are similarly distributed on the four C-terminal glutamate residues of cytoplasmic and axonemal tubulin. The polyglycylation results in bulky C-terminal domains with negatively charged surfaces, all surrounding the microtubular structure.
Asunto(s)
Péptidos/metabolismo , Procesamiento Proteico-Postraduccional , Tubulina (Proteína)/metabolismo , Secuencia de Aminoácidos , Animales , Citoplasma/metabolismo , Espectrometría de Masas , Microtúbulos/metabolismo , Datos de Secuencia Molecular , Paramecium tetraurelia , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/metabolismo , Péptidos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Tubulina (Proteína)/químicaRESUMEN
A sensitive protocol for unambiguously and positively identifying O-glycosylation sites in glycopeptides is described, based on beta-elimination of the glycan chain(s) using NH4OH. On glycan elimination, NH3 is incorporated into the amino acid residue(s) to which the glycan(s) had been attached, to yield a modified amino acid residue having a distinct mass. Electrospray ionization collision-induced dissociation tandem mass spectrometry allows the released, modified peptide to be sequenced and the site(s) of the modified amino acid residue(s) to be identified. The protocol has been optimized using a series of structurally related O-glycopeptides, and standard conditions are recommended for handling unknowns. We demonstrate that site determination can be achieved using as little as 1 pmol of starting material.
Asunto(s)
Glicopéptidos/química , Polisacáridos/química , Secuencia de Aminoácidos , Hidróxido de Amonio , Secuencia de Carbohidratos , Glicosilación , Hidróxidos , Espectrometría de Masas , Datos de Secuencia Molecular , Sensibilidad y EspecificidadAsunto(s)
Adenilil Ciclasas/análisis , Espectrometría de Masa Bombardeada por Átomos Veloces/métodos , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Adenilil Ciclasas/química , Adenilil Ciclasas/metabolismo , AMP Cíclico/química , AMP Cíclico/metabolismo , Técnicas In Vitro , Cinética , Especificidad por SustratoRESUMEN
Cyclic AMP-dependent protein kinase is conventionally assayed by measuring the incorporation of radiolabeled phosphate into a histone substrate. Here the assay of the protein kinase is carried out by the positive-ion fast atom bombardment mass spectrometric analysis of the enzyme incubation mixture after the reaction has been terminated. The data obtained are in good agreement with those obtained from the conventional radiometric assay of the same kinase preparation. The inherent advantage of this mass spectrometric assay is the capacity for multiple component monitoring; in addition to the kinase activity, the ability of the enzyme to bind cyclic nucleotides, together with integral ATPase and phosphodiesterase activity, can also be estimated from the same spectra.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/análisis , AMP Cíclico/metabolismo , Espectrometría de Masa Bombardeada por Átomos VelocesRESUMEN
Analysis of urine from cancer patients by capillary gas chromatography/mass spectrometry positively identified 14 urinary nucleosides including several modified nucleosides. Levels of the modified nucleosides 1-methyl-adenosine, 2-methylguanosine, N2,N2-dimethylguanosine and 1-methylinosine as well as the total nucleoside level were elevated in the urine when a malignant tumour was present; the levels of N2,N2-dimethylguanosine were found to correlate with the stage of the cancer.
Asunto(s)
Biomarcadores de Tumor , Cromatografía de Gases y Espectrometría de Masas , Guanosina/análogos & derivados , Inosina/análogos & derivados , Neoplasias/orina , Nucleósidos/orina , Adenosina/análogos & derivados , Adenosina/orina , Femenino , Guanosina/orina , Humanos , Inosina/orina , MasculinoRESUMEN
Continuous-flow fast-atom bombardment mass spectrometry has been developed to directly monitor cyclic nucleotide (substrate) and its product levels from an on-going phosphodiesterase reaction. Analysis of cAMP and cCMP phosphodiesterase incubates have been performed where the temporal evolution of the enzymic reaction is monitored and the effect of enzyme concentration upon the rate of reaction determined. Quantitative data on the enzyme kinetics have been obtained, in the form of Lineweaver-Burke plots, that are shown to correlate well with well-established radiometric methods.
Asunto(s)
AMP Cíclico/análisis , GMP Cíclico/análisis , Espectrometría de Masa Bombardeada por Átomos Veloces , Animales , HumanosRESUMEN
A protein kinase, stimulated by cytidine 3',5'-cyclic monophosphate, is conventionally assayed by monitoring the incorporation of radiolabelled phosphate from adenosine triphosphate into a histone substrate. Here the assay of the protein kinase is carried out by positive-ion fast-atom bombardment mass spectrometric analysis of the enzyme incubation mixture after the reaction has been terminated. The data so obtained show good agreement with data obtained by the conventional radiometric assay: the intrinsic advantage of the mass spectrometric assay is the capacity for multiple component monitoring; the ability of the kinase to bind competing cyclic nucleotides together with integral adenosine triphosphatase (ATPase) and phosphodiesterase activity can also be assessed.