Analysis and validation of the phosphorylated metabolites of two anti-human immunodeficiency virus nucleotides (stavudine and didanosine) by pressure-assisted CE-ESI-MS/MS in cell extracts: sensitivity enhancement by the use of perfluorinated acids and alcohols as coaxial sheath-liquid make-up constituents

A CE method utilizing triple quadrupole electrospray (ES) MS (MS/MS) detection was developed and validated for the simultaneous measurement of nucleoside 5'-triphosphate and 5'-monophosphate anabolites of the anti-HIV (human immunodeficiency virus) didanosine (ddAMP, ddATP) and stavudine (d4TMP, d4TTP), among a pool of 14 endogenous 5'-mono-, di-, and triphosphate nucleosides. These compounds were spiked and extracted from peripheral blood mononuclear cells (PBMCs) which are the sites of HIV replication and drug action. An acetic acid/ammonia buffer (pH 10, ionic strength of 40 mM) was selected as running electrolyte, and the separation was performed by the simultaneous application of a CE voltage of +30 kV and an overimposed pressure of 28 mbar (0.4 psi). The application of pressure assistance was needed to provide stable ES conditions for successful coupling. The coupling was carried out with a modified sheath-flow interface, with one uninterrupted capillary (80 cmx 50 microm id; 192 microm od) in a dimension that fits into the ESI needle to get a stable ion spray. Some CE-MS parameters such as overimposed pressure, sheath-liquid composition, sheath-liquid and sheath-gas flow rates, ES voltage, and the CE capillary position were optimized in order to obtain an optimal sensitivity. The use of perfluorinated alcohols and acids in the coaxial sheath-liquid make-up (2,2,2-trifluoroethanol + 0.2 mM tridecafluoroheptanoic acid) appeared to provide the best MS sensitivity and improve the stability of spray. The linearity of the CE-MS and CE-MS/MS methods was checked under these conditions. Validation parameters such as accuracy, intraday and interday precision, and LOQs were determined in CE-MS/MS mode. Finally, the quantitation of d4T-TP and ddA-TP was validated in this CE-MS/MS system.     

Methods to track single-molecule trajectories

Methods are discussed to track single molecules in planar-supported phospholipid bilayers. Mainly, these methods constitute optimizations for a low signal-to-noise ratio and for the dim optical characteristics of single molecules. Algorithmic modifications to compensate and correct for misidentifications and misassignments are also described. One key advance, which exchanges the typically fragmented reconstruction of the molecules' motion for more complete trajectories, is the incorporation of information about the molecules' past and future positions into the tracking. Although the main point of these methods is to aid in extending methods of object tracking to the single-molecule regime, they may also find use in other situations where the signal-to-noise ratio is low.     

Stabilities and properties of complexes pairing hydroperoxyl radical with monohalomethanes

UMP2/aug-cc-pvdz calculations are used to analyze the interaction between hydroperoxyl radical (HOO) and CH(3)X (X = F, Cl, Br). Two minima are located on the potential energy surface of each complex. The more strongly bound contains a OH...X bond, along with CH...O; only CH...O bonds occur in the less stable minimum. Binding energies of the dominant minimum lie in the range of 20-24 kJ/mol, with X = F the most strongly bound. Analysis of the perturbations of the covalent bond lengths within each subunit caused by complexation, coupled with vibrational frequencies and charge transfers, opens a window into the nature of the interactions.     

Mono and dimeric naphthalene derivatives from the roots of Diospyros assimilis

Three 2-naphthaldehydes, including the novel 4-hydroxy-3,5-dimethoxy-2-naphthaldehyde, the simple naphthoquinone plumbagin and the dimeric naphthoquinones diospyrin and 8'-hydroxyisodiospyrin have been isolated from the roots of Diospyrosassimilis and their structures established by spectroscopic analysis.     

Associahedra, cellular -construction and products of -algebras

The aim of this paper is to construct a functorial tensor product of -algebras or, equivalently, an explicit diagonal for the operad of cellular chains, over the integers, of the Stasheff associahedron. These constructions in fact already appeared (Saneblidze and Umble, 2000 and 2002); we will try to give a more conceptual presentation. We also prove that there does not exist a coassociative diagonal.

     

Anomalous temperature-isotope dependence in proton-coupled electron transfer

Motivated by the experiments of Hodgkiss et al. [J. Phys. Chem. (submitted)] on electron transfer (ET) through a H-bonding interface, we present a new theoretical model for proton-coupled electron transfer (PCET) in the condensed phase, that does not involve real proton transfer. These experiments, which directly probe the joint T-isotope effects in coupled charge transfer reactions, show anomalous T dependence in k(H)k(D), where k(H) and k(D) are the ET rates through the H-bonding interface with H-bonded protons and deuterons, respectively. We address the anomalous T dependence of the k(H)k(D) in our model by attributing the modulation of the electron tunneling dynamics to bath-induced fluctuations in the proton coordinate, so that the mechanism for coupled charge transfer might be better termed vibrationally assisted ET rather than PCET. We argue that such a mechanism may be relevant to understanding traditional PCET processes, i.e., those in which protons undergo a transfer from donor to acceptor during the course of ET, provided there is an appropriate time scale separating both coupled charge transfers. Likewise, it may also be useful in understanding long-range ET in proteins, where tunneling pathways between redox cofactors often pass through H-bonded amino acid residues, or other systems with sufficiently decoupled proton and electron donating functionalities.     

Determination of microcystins in water using integrated solid-phase microextraction with microbore high-performance liquid chromatography-electrospray quadruple time-of-flight mass spectrometry

The development of a technique combining solid-phase microextraction (SPME) with microbore high-performance liquid chromatography (micro-HPLC)-tandem quadrupole time-of-flight (QTOF) mass spectrometry (MS) for determination of dissolved microcystins in water is reported. Several important parameters affecting the efficiency of SPME extraction of microcystins are investigated. A microbore C18 column HPLC coupled with tandem QTOF-MS with information-dependent acquisition (IDA) is developed to effectively analyze microcystins in microliter volumes of SPME extracts. The micro-HPLC-QTOF-MS with IDA technique provides comprehensive information, including a survey chromatogram (total ion chromatogram), full scan mass spectrum, and product ion scan mass spectra at different collision energies for individual analytes, which allows for both identification and quantitation in the same run. Linear calibration curves of microcystin standard [microcystin (MC)-arginine (R)R] 1-100 microg/L and of microcystin standard [MC-leucine (L)R] 1-250 microg/L are obtained with a correlation coefficient of 0.996. The combination of SPME with HPLC-QTOF-MS and IDA offers limits of detection of 0.6 pg for MC-RR and 1.6 pg for MC-LR. Analysis of spiked lake-water samples shows a recovery of > 86% for MC-RR and > 70% for MC-LR. This technique requires small sample volumes, minimizes the use of organic solvents, and provides sensitive and information-rich analysis of unknown samples.     

Investigating Ugi/Passerini Multicomponent Reactions for the Site-Selective Conjugation of Native Trastuzumab**

Site-selective modification of proteins has been the object of intense studies over the past decades, especially in the therapeutic field. Prominent results have been obtained with recombinant proteins, for which site-specific conjugation is made possible by the incorporation of particular amino acid residues or peptide sequences. In parallel, methods for the site-selective and site-specific conjugation of native and natural proteins are starting to thrive, allowing the controlled functionalization of various types of amino acid residues. Pursuing the efforts in this field, we planned to develop a new type of site-selective method, aiming at the simultaneous conjugation of two amino acid residues. We reasoned that this should give higher chances of developing a site-selective strategy compared to the great majority of existing methods that solely target a single residue. We opted for the Ugi four-centre three-component reaction to implement this idea, with the aim of conjugating the side-chain amine and carboxylate groups of two neighbouring lysine and aspartate/glutamate. Herein, we show that this strategy can give access to valuable antibody conjugates bearing several different payloads; furthermore, the approach limits the potential conjugation sites to only six on the model antibody trastuzumab.