Analysis of an intact G-protein coupled receptor by MALDI-TOF mass spectrometry: molecular heterogeneity of the tachykinin NK-1 receptor

Integral membrane proteins are among the most challenging targets for biomedical research as most important cellular functions are tied to these proteins. To analyze intrinsically their structure/function, their transduction mechanism, or both, these proteins are commonly expressed in cultured cells as recombinant proteins. However, it is not possible to check whether these recombinant proteins are homogeneously or heterogeneously expressed. Owing to difficulties in their purification, very few mass spectrometry studies have been performed with those proteins and even less with G-protein coupled receptors. Here we have set up a procedure that is highly compatible with MALDI-TOF mass spectrometry to analyze an intact histidine-tagged G-protein coupled, namely, the tachykinin NK-1 receptor expressed in CHO cells, solubilized and purified using cobalt or nickel chelating magnetic beads. The metal-chelating magnetic beads containing the receptor were directly spotted on the MALDI plate for analysis. SDS-PAGE, combined with in-gel digestion analyzed by mass spectrometry, Western blot ((His)6 and FLAG M2 tags), photoaffinity labeling with a radioactive agonist, and Edman sequencing, confirmed the identity of the purified protein as the human tachykinin NK-1 receptor. Mass spectrometry study of both the glycosylated and deglycosylated intact protein forms revealed the existence of several receptor species that is tempting to correlate with the unusual pharmacological behavior of the receptor.     

Charge transport in cellular nanoparticle networks: meandering through nanoscale mazes

The transport of electrons through topologically complex two-dimensional Au nanoparticle networks has been investigated using a combination of low temperature (4.5 K) direct current I(V) measurements and numerical simulations. Intricate, spatially correlated nanostructured networks were formed via spin-casting. The topological complexity of the nanoparticle assemblies produces I(V) curves associated with nonlinearity exponents, zeta approximately 4.0. Simulations based on tunneling transport in sparse and inhomogeneous planar networks are used to elucidate the influence of topology on the value of zeta.     

Copper Transfer from Cu–Aβ to Human Serum Albumin Inhibits Aggregation,Radical Production and Reduces Aβ Toxicity

Amyloid‐β peptides (Aβ) and the protein human serum albumin (HSA) interact in vivo. They are both localised in the blood plasma and in the cerebrospinal fluid. Among other functions, HSA is involved in the transport of the essential metal copper. Complexes between Aβ and copper ions have been proposed to be an aberrant interaction implicated in the development of Alzheimer's disease, where Cu is involved in Aβ aggregation and production of reactive oxygen species (ROS). In the present work, we studied copper‐exchange reaction between Aβ and HSA or the tetrapeptide DAHK (N‐terminal Cu‐binding domain of HSA) and the consequence of this exchange on Aβ‐induced ROS production and cell toxicity. The following results were obtained: 1) HSA and DAHK removed Cu^II from Aβ rapidly and stoichiometrically, 2) HSA and DAHK were able to decrease Cu‐induced aggregation of Aβ, 3) HSA and DAHK suppressed the catalytic HO^. production in vitro and ROS production in neuroblastoma cells generated by Cu–Aβ and ascorbate, 4) HSA and DAHK were able to rescue these cells from the toxicity of Cu–Aβ with ascorbate, 5) DAHK was more potent in ROS suppression and restoration of neuroblastoma cell viability than HSA, in correlation with an easier reduction of Cu^II–HSA than Cu–DAHK by ascorbate, in vitro. Our data suggest that HSA is able to decrease aberrant Cu^II–Aβ interaction. The repercussion of the competition between HSA and Aβ to bind Cu in the blood and brain and its relation to Alzheimer's disease are discussed.     

Design and facile production of recombinant resilin-like polypeptides: gene construction and a rapid protein purification method

Resilin is an elastic protein found in specialized regions of the cuticle of insects, which displays unique resilience and fatigue lifetime properties. As is the case with many elastomeric proteins, including elastin, gliadin and spider silks, resilin contains distinct repetitive domains that appear to confer elastic properties to the protein. Recent work within our laboratory has demonstrated that cloning and expression of exon 1 of the Drosophila melanogaster CG15920 gene, encoding a putative resilin-like protein, results in a recombinant protein that can be photochemically crosslinked to form a highly resilient, elastic biomaterial (Rec1 resilin). The current study describes a recursive cloning strategy for generating synthetic genes encoding multiple copies of consensus polypeptides, based on the repetitive domains within resilin-like genes from D. melanogaster and Anopheles gambiae. A simple non-chromatographic purification method that can be applied to these synthetic proteins and Rec1 is also reported. These methods for the design and purification of resilin-like periodic polypeptides will facilitate the future investigation of structural and functional properties of resilin, and the development of novel highly resilient biomaterials.     

Charge‐Transfer Interactions: An Efficient Tool for Recycling Bis(oxazoline)‐Copper Complexes in Asymmetric Henry Reactions

An anthracenyl‐modified chiral bis(oxazoline) copper complex has been demonstrated to efficiently promote nitroaldol reactions between structurally varying aldehydes and nitromethane or nitroethane. The catalyst was recovered through formation of a charge transfer complex between the chiral ligand and trinitrofluorenone and its subsequent precipitation with pentane. The efficiency of this procedure was proved through several consecutive catalytic cycles that allowed the sturdy formation of the expected product with a high enantioselectivity. The catalyst′s stability was also put to the test in an original multi‐substrate procedure. Following the same recovery concept, a new heterogeneous procedure was tested for which trinitrofluorenone was covalently linked to a silica support. Asymmetric heterogeneous catalysis was performed under these conditions as one of the few examples demonstrating the potential catalyst recycling in nitroaldol reactions through reversible, non‐covalent interactions.     

Impact of biofouling on diffusive gradient in thin film measurements in water

The technique of diffusive gradient in thin film (DGT) is commonly used to assess metal contamination in natural waters. In this paper, we assess the effect of biofouling on DGT measured labile concentrations in water and investigate whether an additional nuclepore polycarbonate membrane on the surface of DGT devices can limit biofilm growth. Simultaneous field deployments of DGT equipped with and without the additional membrane in a canal receiving wastewater were compared. The effect of the biofilm was also assessed in controlled laboratory experiments, completed by the experimental determination of several metals diffusion coefficients in the hydrogel and membrane systems. The biofilms effect was problematic only from the 10th day of accumulation. Accumulation of some elements is highly biased by the presence of a thick biofilm (Zn, Ni, Cd). The polycarbonate membrane improved the quantification of Cd and Ni but adversely affects the quantification of Cr and Co. A kinetic model is proposed to explain the biofilm role on the DGT measurement. Depending on the metals of interest, it is possible to limit bias due to biofilms by using an additional polycarbonate membrane.     

Risk and protection factors in fatal accidents

This paper aims at addressing the interest and appropriateness of performing accident severity analyses that are limited to fatal accident data. Two methodological issues are specifically discussed, namely the accident-size factors (the number of vehicles in the accident and their level of occupancy) and the comparability of the baseline risk. It is argued that - although these two issues are generally at play in accident severity analyses - their effects on, e.g., the estimation of survival probability, are exacerbated if the analysis is limited to fatal accident data. As a solution, it is recommended to control for these effects by (1) including accident-size indicators in the model, (2) focusing on different sub-groups of road-users while specifying the type of opponent in the model, so as to ensure that comparable baseline risks are worked with. These recommendations are applied in order to investigate risk and protection factors of car occupants involved in fatal accidents using data from a recently set up European Fatal Accident Investigation database (Reed and Morris, 2009). The results confirm that the estimated survival probability is affected by accident-size factors and by type of opponent. The car occupants’ survival chances are negatively associated with their own age and that of their vehicle. The survival chances are also lower when seatbelt is not used. Front damage, as compared to other damaged car areas, appears to be associated with increased survival probability, but mostly in the case in which the accident opponent was another car. The interest of further investigating accident-size factors and opponent effects in fatal accidents is discussed.     

Matrix-assisted laser desorption/ionization-ion mobility separation-mass spectrometry imaging of vinblastine in whole body tissue sections

During early-stage drug development, drug and metabolite distribution studies are carried out in animal tissues using a range of techniques, particularly whole body autoradiography (WBA). While widely employed, WBA has a number of limitations, including the following: expensive synthesis of radiolabeled drugs and analyte specificity and identification. WBA only images the radiolabel. MALDI MSI has been shown previously to be advantageous for imaging the distribution of a range of drugs and metabolites in whole body sections. Ion mobility separation (IMS) adds a further separation step to imaging experiments; demonstrated here is MALDI-IMS-MS whole body imaging of rats dosed at 6 mg/kg i.v. with an anticancer drug, vinblastine and shown is the distribution of the precursor ion m/z 811.4 and several product ions including m/z 793, 751, 733, 719, 691, 649, 524, and 355. The distribution of vinblastine within the ventricles of the brain is also depicted. Clearly demonstrated in these data are the removal of interfering isobaric ions within the images of m/z 811.4 and also of the transition m/z 811-751, resulting in a higher confidence in the imaging data. Within this work, IMS has shown to be advantageous in both MS and MS/MS imaging experiments by separating vinblastine from an endogenous isobaric lipid.