RESUMEN
Mass spectrometry imaging (MSI) based on matrix-assisted laser desorption/ionization (MALDI) provides information on the identification and spatial distribution of biomolecules. Quantitative analysis, however, has been challenging largely due to heterogeneity in both the size of the matrix crystals and the extraction area. In this work, we present a compartmentalized elastomeric stamp for quantitative MALDI-MSI of adsorbed peptides. Filling the compartments with matrix solution and stamping onto a planar substrate extract and concentrate analytes adsorbed in each compartment into a single analyte-matrix cocrystal over the entire stamped area. Walls between compartments help preserve spatial information on the adsorbates. The mass intensity of the cocrystals directly correlates with the surface coverage of analytes, which enables not only quantitative analysis but estimation of an equilibrium constant for the adsorption. We demonstrate via MALDI-MSI relative quantitation of peptides adsorbed along a microchannel with varying surface coverages.
Asunto(s)
Péptidos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Adsorción , Fluoresceína-5-Isotiocianato/química , Dispositivos Laboratorio en un Chip , Microscopía FluorescenteRESUMEN
Microtubule dynamics is a target for many chemotherapeutic drugs. In order to understand the biochemical effects of paclitaxel on the GTPase activity of tubulin, the status of guanine nucleotides in microtubules was investigated by (31)P cross-polarization magic angle spinning (CPMAS) NMR. Microtubules were freshly prepared in vitro in the presence of paclitaxel and then lyophilized in sucrose buffer for solid-state NMR experiments. A (31)P CPMAS NMR spectrum with the SNR of 25 was successfully acquired from the lyophilized microtubule sample. The broadness of the (31)P spectral lines in the spectrum indicates that the molecular environments around the guanine nucleotides inside tubulin may not be as crystalline as reported by many diffraction studies. Deconvolution of the spectrum into four spectral components was carried out in comparison with the (31)P NMR spectra obtained from five control samples. The spectral analysis suggested that about 13% of the nucleotides were present as GTP and 37% as GDP in the ß-tubulin (E-site) of the microtubules. It was found that most of the GDPs were present as GDP-Pi complex in the microtubules, which seems to be one of the effects of paclitaxel binding.
Asunto(s)
GTP Fosfohidrolasas/química , Nucleótidos de Guanina/química , Espectroscopía de Resonancia Magnética/métodos , Microtúbulos/química , Paclitaxel/química , Moduladores de Tubulina/química , GTP Fosfohidrolasas/análisis , Nucleótidos de Guanina/análisis , Paclitaxel/análisis , Isótopos de Fósforo/química , Unión ProteicaRESUMEN
Diversity of sequence and structure in naturally occurring antimicrobial peptides (AMPs) limits their intensive structure-activity relationship (SAR) study. In contrast, peptidomimetics have several advantages compared to naturally occurring peptide in terms of simple structure, convenient to analog synthesis, rapid elucidation of optimal physiochemical properties and low-cost synthesis. In search of short antimicrobial peptides using peptidomimetics, which provide facile access to identify the key factors involving in the destruction of pathogens through SAR study, a series of simple and short peptidomimetics consisting of multi-Lys residues and lipophilic moiety have been prepared and found to be active against several Gram-negative and Gram-positive bacteria containing methicillin-resistant Staphylococcus aureus (MRSA) without hemolytic activity. Based on the SAR studies, we found that hydrophobicity, +5 charges of multiple Lys residues, hydrocarbon tail lengths and cyclohexyl group were crucial for antimicrobial activity. Furthermore, membrane depolarization, dye leakage, inner membrane permeability and time-killing kinetics revealed that bacterial-killing mechanism of our peptidomimetics is different from the membrane-targeting AMPs (e. g. melittin and SMAP-29) and implied our peptidomimetics might kill bacteria via the intracellular-targeting mechanism as done by buforin-2.