RESUMEN
A novel tandem quadrupole mass spectrometer is described that enables gaseous collision-induced dissociation (CID) and surface-induced dissociation (SID) experiments. The instrument consists of a commercially available triple quadrupole mass spectrometer connected to an SID region and an additional, orthogonal quadrupole mass analyser. The performance of the instrument was evaluated using leucine-enkephalin, allowing a comparison between CID and SID, and with previous reports of other SID instruments. The reproducibility of SID data was assessed by replicate determinations of the collision energy required for 50% dissociation of leucine-enkephalin; excellent precision was observed (standard deviation of 0.6 eV) though, unexpectedly, the reproducibility of the equivalent figure for CID was superior. Several peptides were analysed using SID in conjunction with liquid secondary-ion mass spectrometry or electrospray; a comparison of the fragmentation of singly protonated peptide ions and the further dissociation of y-type fragments was consistent with the equivalence of the latter fragments to protonated peptides. Few product ions attributable to high-energy cleavages of amino acid side-chains were observed. The SID properties were investigated of a series of peptides differing only in the derivatization of a cysteine residue; similar decomposition efficiencies were observed for all except the cysteic acid analogue, which demonstrated significantly more facile fragmentation.
Asunto(s)
Espectrometría de Masas/instrumentación , Secuencia de Aminoácidos , Angiotensinógeno/química , Bradiquinina/análogos & derivados , Bradiquinina/química , Cisteína/química , Dinorfinas/química , Encefalina Leucina/química , Fibrinopéptido A/química , Espectrometría de Masas/métodos , Datos de Secuencia Molecular , Estructura Molecular , Fragmentos de Péptidos/química , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , TermodinámicaRESUMEN
Hypoxia-inducible factor (HIF) is a transcriptional complex that plays a central role in the regulation of gene expression by oxygen. In oxygenated and iron replete cells, HIF-alpha subunits are rapidly destroyed by a mechanism that involves ubiquitylation by the von Hippel-Lindau tumor suppressor (pVHL) E3 ligase complex. This process is suppressed by hypoxia and iron chelation, allowing transcriptional activation. Here we show that the interaction between human pVHL and a specific domain of the HIF-1alpha subunit is regulated through hydroxylation of a proline residue (HIF-1alpha P564) by an enzyme we have termed HIF-alpha prolyl-hydroxylase (HIF-PH). An absolute requirement for dioxygen as a cosubstrate and iron as cofactor suggests that HIF-PH functions directly as a cellular oxygen sensor.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Hidroxiprolina/metabolismo , Ligasas , Proteínas Nucleares/metabolismo , Oxígeno/fisiología , Procolágeno-Prolina Dioxigenasa/metabolismo , Proteínas/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor , Ubiquitina-Proteína Ligasas , Secuencia de Aminoácidos , Ácido Ascórbico/farmacología , Hipoxia de la Célula , Proteínas de Unión al ADN/química , Deferoxamina/farmacología , Compuestos Ferrosos/farmacología , Humanos , Hidroxilación , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Datos de Secuencia Molecular , Proteínas Nucleares/química , Mutación Puntual , Procolágeno-Prolina Dioxigenasa/antagonistas & inhibidores , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Factores de Transcripción/química , Células Tumorales Cultivadas , Ubiquitinas/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-LindauRESUMEN
The ease of fragmentation of various charge states of protonated polypropylenamine (POPAM) dendrimers is investigated by surface-induced dissociation. Investigated are the protonated diaminobutane propylenamines [DAB(PA)n] DAB(PA)8 (1+ and 2+), DAB(PA)16 (2+ and 3+), and DAB(PA)32 (3+ and 4+). These ions have been proposed to fragment by charge-directed intramolecular nucleophilic substitution (SNi) reactions. Differences in relative fragment ion abundances between charge states can be related to the occupation of different protonation sites. These positions can be rationalized based on estimates of Coulomb energies and gas-phase basicities of the protonation/fragmentation sites. The laboratory collision energies at which the fragment ion current is approximately 50% of the total ion current were found to increase with the size, but to be independent of charge state of the protonated POPAM dendrimers. It is suggested that intramolecular Coulomb repulsion within the multiply protonated POPAM dendrimers selected for activation does not readily result in easier fragmentation, which is in accordance with the proposed fragmentation mechanism.