P-glycoprotein (P-gp) is an energy-dependent multidrug efflux pump conferring resistance to cancer chemotherapy. Characterization of the mechanism of drug transport at a molecular level represents an important prerequisite for the design of pump inhibitors, which resensitize cancer cells to standard chemotherapy. In addition, P-glycoprotein plays an important role for early absorption, distribution, metabolism, excretion, and toxicity profiling in drug development. A set of propafenonetype substrate photoaffinity ligands has been used in this study in conjunction with matrix-assisted laser desorption/ionization timeof-flight mass spectrometry to define the substrate binding domain(s) of P-gp in more detail. The highest labeling was observed in transmembrane segments 3, 5, 8, and 11. A homology model for P-gp was generated on the basis of the dimeric crystal structure of Vibrio cholerae MsbA, an essential lipid transporter. Thereafter, the labeling pattern was projected onto the 3D atomic-detail model of P-gp to allow a visualization of the binding domain(s). Labeling is predicted by the model to occur at the two transmembrane domain/transmembrane domain interfaces formed between the amino-and carboxyl-terminal half of P-gp. These interfaces are formed by transmembrane (TM) segments 3 and 11 on one hand and TM segments 5 and 8 on the other hand. Available data on LmrA and AcrB, two bacterial multidrug efflux pumps, suggest that binding at domain interfaces may be a general feature of polyspecific drug efflux pumps.Multidrug resistance represents a serious obstacle to successful cancer chemotherapy. Although multifactorial in etiology, one type of multidrug resistance is associated with the overexpression of energy-dependent membrane-bound pumps, which intercept and efflux drugs before they reach their intracellular target structures. P-glycoprotein (ABCB1) represents a paradigm ATP-dependent efflux pump expressed in human cancer cells. In addition to its expression in cancer cells, P-gp is also physiologically expressed in a number of tissues such as intestinal epithelial cells, at the brush border of renal tubule epithelial cells, the canalicular side of hepatocytes, and in capillary endothelial cells forming the blood-brain barrier. It thus interferes with oral drug absorption and drug delivery to the brain, and it enhances renal and biliary excretion. P-gp has therefore attracted considerable attention as a nontarget in the field of drug development, because for a large number of active compounds, interaction with P-glycoprotein might compromise their future development into a drug. Considerable energy has therefore been devoted to the characterization of molecular features that make compounds P-gp substrates and to the definition of the molecular mechanism of drug transport by P-gp. A number of studies have dealt with the kinetics and thermodynamics of the transport process
The importance of proper conversion of MALDI‐MS signals into the number of polymers with a certain mass increment (by taking into account a nonconstant conversion factor) is demonstrated with calculated Schulz‐Zimm and Poisson distributions. Measurements of two narrow polystyrene standards were used to investigate the influence of measurement conditions (laser intensity, matrix) and data processing (signal conversion, baseline construction). We observed the following effects: (a) slightly broader distributions with DCTB as matrix at a given laser intensity in comparison to those obtained with all‐trans‐retinoic acid, (b) changes in the relative intensities of several peaks in a sample, and (c) a nonlinear baseline in the low molar mass region in most of the cases. Therefore, the implications of an assumed linear baseline instead of the correct one on the results were scrutinized experimentally and by simulations. In all, measurement of narrow distributions turned out to give reasonable results. Based on the assumption that the polymer standards can be described by Poisson distributions, overlap conditions between two distributions are rephrased. An alternative possibility of how mass discrimination could be determined, in principle, is presented, too.magnified image
The performances of several matrices were investigated for the accurate determination of the molecular mass distributions of pullulans by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The ionic liquid matrix (ILM) 2,5-dihydroxybenzoic acid butylamine (DHBB) gave better and more reliable results than the crystalline matrices 2,5-dihydroxybenzoic acid (DHB) and 2,4,6-trihydroxyacetophenone (THAP). With the ILM it was possible to obtain spectra of pullulans up to more than 100 kDa, the highest molar mass reported thus far. Owing to the known advantages of liquid matrices providing better spot-to-spot reproducibility, an almost noise-free spectrum and constant baselines were obtained when working under optimized conditions. In particular, the extent of in-source fragmentation occurring with this group of fragile polymers was considerably and decisively reduced. Thus, a more reliable representation of the true oligomer and polymer distributions is experimentally attainable, especially for distributions with small polydispersity values. The maximum error in the measured distribution associated with fragmentation was estimated by model calculations describing the changes in the polymer distribution upon different probabilities of fragmentation events. These simulation results indicated that the data obtained by MALDI-TOFMS using the liquid DHBB matrix were of high reliability. In particular, the average value of the distributions, M(w), and the polydispersity were obtained with predicted uncertainties of between 3 and 15% depending on the width of the distribution and the mass of the polymers.
Differential 2-DE (DIGE) is a widely applied tool for the quantitative analysis of differentially represented proteins. The method involves covalent minimal labeling of proteins prior to their electrophoretic separation using CyDye DIGE Fluor minimal dyes. This methodology creates two different species per protein, the labeled (approx. 1-2%) and unlabeled (approx. 98-99%) ones, which differ in their molecular masses by 434-464 Da, depending on the attached dye. DIGE followed by automated spot picking according to the CyDye coordinates misses in many instances the exact positions where the maximum amounts of the considered proteins are located. This fact leads to a loss in sensitivity of the subsequent MALDI-MS analyses and results in a reduced reliability of protein identification and sequence coverage. In this paper, the migration differences of labeled and unlabeled species are quantified together with the impact of this effect on the certainty of protein identification and sequence coverage investigating proteins up to 90 kDa.
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