PubMed Health⌕ Search
Home/Archive/PubMed

PubMed · 10825017

Single bead and hard tag decoding using accurate isotopic difference target analysis-encoded combinatorial libraries.

S J Lane·A Pipe
View original source ↗Export metadata ↓

Abstract

A single fully automated generic microbore liquid chromatography electrospray time-of-flight protocol has been developed for the analysis of single beads, single beads with analytical constructs, and isotopically labelled dialkylamine hard tags. The protocol relies upon the incorporation of an isotopic signature into the linker and/or derivatising molecule to give a specific isotopic ratio together with an accurate isotopic difference. These properties facilitate highly specific and sensitive analysis of beads and hard tags using accurate isotopic difference analysis (AIDA) without prior knowledge of the molecular weight. Three open access methods with automated processing have been developed around a core generic approach.

Explore related subjects

AminesAutomationCarbon IsotopesChromatography, LiquidCombinatorial Chemistry TechniquesDansyl CompoundsFluorescenceMass SpectrometryPolysaccharidesJournal Article

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

S J Lane, A Pipe. 2000. Single bead and hard tag decoding using accurate isotopic difference target analysis-encoded combinatorial libraries.. https://doi.org/10.1002/(sici)1097-0231(20000515)14%3A9%3C782%3A%3Aaid-rcm944%3E3.0.co%3B2-d

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2003

Excess electron transfer from an internally conjugated aromatic amine to 5-bromo-2'-deoxyuridine in DNA.

DNA duplexes containing an N,N,N',N'-tetramethyl-1,5-diaminonaphthalene analogue and 5-bromo-2'-deoxyuridine (BrdU) provide a readily accessible system for investigating excess electron transfer in DNA. Photoexcitation of the aromatic amine (lambda > 335 nm) induces reductive electron transfer as observed by strand cleavage adjacent to the BrdU residue. The weak exponential distance dependence (0.3 A-1) of electron transfer determined for this system of mixed dA-T and dG-dC base pairs suggests that thermally activated electron hopping is competitive with proton transfer within the dG.dC radical anion. The UV-dependent transfer of excess electrons and subsequent strand cleavage proceeds equivalently under anaerobic and aerobic conditions and is not sensitive to e-(aq) or hydroxyl radical trapping agents.

Amines↗
PubMed2003

Chemical and kinetic reaction mechanisms of quinohemoprotein amine dehydrogenase from Paracoccus denitrificans.

Quinohemoprotein amine dehydrogenase (QHNDH) possesses a cysteine tryptophylquinone (CTQ) prosthetic group that catalyzes the oxidative deamination of primary amines. In addition to CTQ, two heme c cofactors are present in QHNDH that mediate the transfer of the substrate-derived electrons from CTQ to an external electron acceptor. Steady-state kinetic assays yielded relatively small k(cat) values (<6 s(-1)), and the rate-limiting step appears to be the interprotein electron transfer from heme in QHNDH to the external electron acceptor. Transient kinetic studies of the CTQ-dependent reduction of heme in QHNDH by amine substrates yielded different rate constants for different substrates (72, 190, and 162 s(-1) for methylamine, butylamine, and benzylamine, respectively). Deuterium kinetic isotope effect (KIE) values of 5.3, 3.9, and 8.5 were observed, respectively, for the reactions of methylamine, butylamine, and benzylamine. These results suggest that the abstraction of a proton from the alpha-methylene group of the substrate, which occurs concomitant with CTQ reduction, is the rate-limiting step in the CTQ-dependent reduction of hemes in QHNDH by these amine substrates. In contrast, the reaction of 2-phenylethylamine with QHNDH does not exhibit a significant KIE ((H)k(3)/(D)k(3) = 1.05) and exhibits a much smaller rate constant of 16 s(-1). This suggests that for 2-phenylethylamine, the rate-limiting step in the single-turnover reaction is either hydrolysis of the imine reaction intermediate from CTQ or product release prior to intraprotein electron transfer. Analysis of the products of the reactions of QHNDH with chiral deuterated 2-phenylethylamines demonstrated that the enzyme abstracts the pro-S proton of the substrate in a highly stereospecific manner. Inspection of the crystal structure of phenylhydrazine-inhibited QHNDH suggests that Asp33(gamma) is the residue that performs the proton abstraction. On the basis of these results, kinetic and chemical reaction mechanisms for QHNDH are proposed and discussed in the context of the crystal structure of the enzyme.

Amines↗
PubMed2003

Synergistic effects of epoxy- and amine-silanes on microarray DNA immobilization and hybridization.

Most microarray slides are manufactured or coated with a layer of poly(L-lysine) or with silanes with different chemical functional groups, for the attachment of nucleic acids on to their surfaces. The efficiency with which nucleic acids bind to these surfaces is not high, because they can be washed away, especially in the case of spotting oligonucleotides. In view of this, we have developed a method to increase the binding capacity and efficiency of hybridization of DNA on to derivatized glass surfaces. This makes use of the synergistic effect of two binding interactions between the nucleic acids and the coating chemicals on the surface of the glass slides. The enhanced binding allows the nucleic acids to be bound tightly and to survive stringency washes. When immobilized, DNA exhibits a higher propensity for hybridization on the surface than on slides with only one binding chemical. By varying the silane concentrations, we have shown that maximal DNA oligonucleotide binding on glass surfaces occurs when the percentage composition of both of the surface-coating chemicals falls to 0.2%, which is different from that on binding PCR products. This new mixture-combination approach for nucleic-acid binding allows signals from immobilization and hybridization to have higher signal-to-noise ratios than for other silane-coated methods.

Amines↗