Biomedical subjects

Alistair Sterling

Publications and source records attributed to Alistair Sterling.

4 recordsLinked to original sources

PubMed2004

Fully automated chip-based mass spectrometry for complex carbohydrate system analysis.

Carbohydrates represent a major class of biopolymers, which occur in nature either as oligosaccharides or glycoconjugates, in which the sugar moiety is linked to proteins or lipids. The significance of mass spectrometry for highly sensitive analysis of complex carbohydrates increased after the introduction of the electrospray ionization and matrix assisted laser desorption/ionization methods and the possibility of tandem MS for sequencing of single molecular species in complex mixtures. Rapid and sensitive characterization of carbohydrates in biological systems by automated nanoscale liquid delivery and chip-based electrospray interface techniques have not been developed so far. In this contribution, the implementation and optimization of a fully automated chip-based nanoelectrospray assembly (NanoMate system), operating in the negative ion mode, in combination with QTOF-tandem MS for mapping/sequencing and computer-assisted structure assignment for carbohydrate components in complex mixtures is presented.

Automation↗

PubMed2004

Fully-automated chip-based nanoelectrospray tandem mass spectrometry of gangliosides from human cerebellum.

The introduction of chip-based electrospray (ESI) ion sources into biological mass spectrometry (MS) addressed the fundamental issue of how to analyze minute amounts of complex biological systems. The automation of sample delivery into the MS combined with the chip-based ESI allows for high quality bioanalysis in a high-throughput fashion. These advantages have already been demonstrated in proteomics, direct screening of drugs and drug discovery. As part of our continuing effort to implement automated chip-based mass spectrometry into the field of complex carbohydrate analysis, we hereby report the development of a chipESI MS and MS/MS methodology for the screening of gangliosides. A strategy to characterize a complex ganglioside mixture from human cerebellar tissue, by automated ESIchip-quadrupole time-of-flight (QTOF) MS and MS/MS is presented here. The feasibility of this method, and the general experimental requirements for automated chipESI MS analysis of these carbohydrate species is described.

Adult↗

PubMed2003

Use of a microchip device coupled with mass spectrometry for ligand screening of a multi-protein target.

Nanoflow electrospray mass spectrometry has been applied previously to investigate noncovalent protein-protein and protein-ligand interactions. Here we evaluate a commercial microchip device for this application. We show that the microchip can be used to obtain mass spectra of the noncovalent tetramer transthyretin. The device showed a 10-fold increase in signal stability compared with a nanoflow capillary and a high level of nozzle-to-nozzle reproducibility. Binding of the natural ligand thyroxine was clearly observed, and a range of small molecules proposed as inhibitors of transthyretin amyloidosis were shown to be effective in stabilizing the tetramer. We propose that measuring the ability of small molecules to stabilize protein complexes using this automated microchip technology will enable high-throughput screening of multi-protein complexes by mass spectrometry.

Ligands↗

PubMed2003

Automated nano-electrospray mass spectrometry for protein-ligand screening by noncovalent interaction applied to human H-FABP and A-FABP.

A method for ligand screening by automated nano-electrospray ionization mass spectrometry (nano-ESI/MS) is described. The core of the system consisted of a chip-based platform for automated sample delivery from a 96-well plate and subsequent analysis based on noncovalent interactions. Human fatty acid binding protein, H-FABP (heart) and A-FABP (adipose), with small potential ligands was analyzed. The technique has been compared with a previously reported method based on nuclear magnetic resonance (NMR), and excellent correlation with the found hits was obtained. In the current MS screening method, the cycle time per sample was 1.1 min, which is approximately 50 times faster than NMR for single compounds and approximately 5 times faster for compound mixtures. High reproducibility was achieved, and the protein consumption was in the range of 88 to 100 picomoles per sample. Futhermore, a novel protocol for preparation of A-FABP without the natural ligand is presented. The described screening approach is suitable for ligand screening very early in the drug discovery process before conventional high-throughput screens (HTS) are developed and/or used as a secondary screening for ligands identified by HTS.

Adipocytes↗