RESUMEN
The bacterium responsible for causing tuberculosis is increasing its resistance to antibiotics resulting in new multidrug-resistant Mycobacterium tuberculosis (MDR-TB) and extensively drug-resistant M. tuberculosis (XDR-TB) strains. In this study, several analytical techniques including NMR, FT-ICR, MALDI-MS, and LCMS are used to study different aspects of the Copperpolyethylene glycol (PEG)Amikacin complex. The Cu(II) cation and the aggregate formed by PEG serve as a carrier for the antibiotic. Several CuPEGAmikacin complex variations were tested against NIH-NIAID cell lines containing both resistant and nonresistant strains of M. tuberculosis.
Asunto(s)
Amicacina/química , Complejos de Coordinación/química , Cobre/química , Polietilenglicoles/química , Complejos de Coordinación/farmacología , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Espectroscopía de Resonancia Magnética , Pruebas de Sensibilidad Microbiana , Conformación Molecular , Mycobacterium tuberculosis/efectos de los fármacosRESUMEN
The bacterium responsible for tuberculosis is increasing its resistance to antibiotics resulting in new multidrug-resistant Mycobacterium tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB). In this study, several analytical techniques including NMR, FT-ICR, MALDI-MS, LC-MS and UV/Vis are used to study the copper-Rifampicin-Polyethylene glycol (PEG-3350) complex. The copper (II) cation is a carrier for the antibiotic Rifampicin as well as nutrients for the bacterium. The NIH-NIAID cell line containing several Tb strains (including antibiotic resistant strains) is tested against seven copper-PEG-RIF complex variations.
Asunto(s)
Antituberculosos/química , Cobre/química , Polietilenglicoles/química , Rifampin/química , Antituberculosos/análisis , Antituberculosos/farmacología , Espectroscopía de Resonancia Magnética , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/efectos de los fármacos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Placental neurokinin B appears to be post-translationally modified by phosphocholine (PC) attached to the aspartyl side chain at residue 4 of the mature peptide. Corticotrophin releasing factor (CRF) was found to be expressed by the rat placenta with the main secreted forms being phosphocholinated proCRF+/- one or two polysaccharide moieties. A combination of high-pressure liquid chromatography (HPLC) and two-site immunometric analysis suggested that PC was also attached to the placental precursors of adrenocorticotrophin, hemokinin, activin and follistatin. However, the fully processed forms of rat placental activin and CRF were free of PC. Formerly, the parasitic filarial nematodes have used PC as a post-translational modification, attached via the polysaccharide moiety of certain secretory glycoproteins to attenuate the host immune system allowing parasite survival, but it is the PC group itself which endows the carrier with the biological activity. The fact that treatment of proCRF peptides with phospholipase C but not endoglycosidase destroyed PC immunoreactivity suggested a simpler mode of attachment of PC to placental peptides than that used by nematodes. Thus, it is possible that by analogy the placenta uses its secreted phosphocholinated hormones to modulate the mother's immune system and help protect the placenta from rejection.
Asunto(s)
Hormona Liberadora de Corticotropina/metabolismo , Péptidos/metabolismo , Fosforilcolina/metabolismo , Placenta/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Anticuerpos/metabolismo , Hormona Liberadora de Corticotropina/química , Femenino , Humanos , Neuroquinina B/química , Neuroquinina B/metabolismo , Péptidos/química , Ratas , Ratas WistarRESUMEN
Protonated peptides derived from proline-rich proteins (PRP) are often difficult to sequence by standard collision-induced dissociation (CID) mass spectrometry (MS) due to preferential amide bond cleavage N-terminal to proline. In connection with bovine spongiform encephalopathy regulations, proteolytic products derived from the PRP collagen have been suggested as markers for contamination of animal feedstuffs with processed animal protein (Fernandez Ocaña, M. et al., Analyst 2004, 129, 111-115). Herein, we report the identification of these marker peptides using the strategy of C-terminal sequencing by CID MS from their sodium and lithium adducts. Upon fragmentation a new cationized peptide was produced that is one C-terminal amino acid shorter in length. This dissociation pathway allowed for the facile identification of the C-terminal residue by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry. Each newly formed cationized peptide was further fragmented by up to seven stages of electrospray ionization ion trap MS. Proline-rich C-terminal sequence tags were established which permitted successful database identification of collagen alpha type I proteins.
Asunto(s)
Gelatina/química , Fragmentos de Péptidos/análisis , Prolina/química , Análisis de Secuencia de Proteína/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Secuencia de Aminoácidos , Animales , Biomarcadores , Bovinos , Gelatina/genética , Fragmentos de Péptidos/genética , PorcinosRESUMEN
The use of a second electrospray nebuliser has proved to be highly successful for exact mass measurement during high-performance liquid chromatography/Fourier transform ion cyclotron resonance mass spectrometry (HPLC/FTICRMS). Much improved accuracy and precision of mass measurement were afforded by the introduction of the internal calibration solution, thus overcoming space charge issues due to the lack of control over relative ion abundances of the species eluting from the HPLC column. Further, issues of suppression of ionisation, observed when using a T-piece method, are addressed and this simple system has significant benefits over other more elaborate approaches providing data that compares very favourably with these other approaches. The technique is robust, flexible and transferable and can be used in conjunction with HPLC, infusion or flow injection analysis (FIA) to provide constant internal calibration signals to allow routine, accurate and precise mass measurements to be recorded.