Biomedical subjects

Kevin Killeen

Publications and source records attributed to Kevin Killeen.

5 recordsLinked to original sources

PubMed2006

A strategy for annotating the human milk glycome.

Oligosaccharides in human milk represent a group of bioactive molecules that have evolved to be an abundant and diverse component of human milk, even though they have no direct nutritive value to the infant. A recent hypothesis proposes that they could be substrates for the development of the intestinal microflora and the mucosal immune system. The inability to determine the exact composition of these oligosaccharides limits research and the ability to understand their biological functions. Oligosaccharides isolated from the lipids and proteins of individual human milk samples were analyzed by a combination of techniques including microchip liquid chromatography mass spectrometry (HPLC-Chip/MS) and matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FT ICR MS). Accurate mass measurements obtained using an orthogonal time-of-flight (o-TOF) mass spectrometry provided oligosaccharide composition for approximately 200 individual molecular species. Comparison of HPLC-Chip/MS profiles from five different women revealed variations in milk oligosaccharide compositions. HPLC-Chip/MS profiling provides a method for routinely identifying milk oligosaccharides. Tandem MS in combination with exoglycosidase digestion provides unambiguous differentiation of structural isomers.

Chromatography, High Pressure Liquid↗

PubMed2006

Integrated microfluidic devices with enhanced separation performance: application to phosphoproteome analyses of differentiated cell model systems.

This work reports on the application of a microfluidic device integrating nanoscale LC to nanoelectrospray MS (nano-LC-chip-MS) for the analysis of complex protein digests. Peak profile analyses of more than 700 peptide ions, reproducibly detected across replicate nano-LC-chip-MS runs (n = 5), indicated that the system provided RSD values of 0.24% on retention time, +/- 30 ppm on m/z measurement and +/- 30% variation on intensity over three orders of magnitude. RP adsorbant media with different alkyl chains and particle size packed in both trapping and separation channels were investigated to improve the chromatographic performance of this system. A two-fold improvement in chromatographic peak capacity was achieved using microfluidic devices comprising a 5 mircrom C3 trap with 2.5 microm C18 trap separation channel compared to the traditional 5 microm C18 stationary phase. Enhanced sample selectivity for the identification of phosphopeptides was obtained by combining immobilized metal affinity media prior to peptide separation on the RP microfluidic device. This system was evaluated in the context of differential phosphoproteome analyses to identify changes in signaling events and protein expression of human monocytes following the administration of phorbol ester.

Animals↗

PubMed2005

Microfluidic chip for peptide analysis with an integrated HPLC column, sample enrichment column, and nanoelectrospray tip.

Current nano-LC/MS systems require the use of an enrichment column, a separation column, a nanospray tip, and the fittings needed to connect these parts together. In this paper, we present a microfabricated approach to nano-LC, which integrates these components on a single LC chip, eliminating the need for conventional LC connections. The chip was fabricated by laminating polyimide films with laser-ablated channels, ports, and frit structures. The enrichment and separation columns were packed using conventional reversed-phase chromatography particles. A face-seal rotary valve provided a means for switching between sample loading and separation configurations with minimum dead and delay volumes while allowing high-pressure operation. The LC chip and valve assembly were mounted within a custom electrospray source on an ion-trap mass spectrometer. The overall system performance was demonstrated through reversed-phase gradient separations of tryptic protein digests at flow rates between 100 and 400 nL/min. Microfluidic integration of the nano-LC components enabled separations with subfemtomole detection sensitivity, minimal carryover, and robust and stable electrospray throughout the LC solvent gradient.

Chromatography, High Pressure Liquid↗

PubMed2005

Nanoliquid chromatography-mass spectrometry of oligosaccharides employing graphitized carbon chromatography on microchip with a high-accuracy mass analyzer.

The nanoLC separations of oligosaccharides using microchip-based columns are described. Mixtures of alditols from mucins and human milk are separated on graphitized carbon. The nanoLC-MS device showed high mass accuracy for the oligosaccharides ranging between 1 and 6 ppm on routine analyses. The high mass accuracy readily allowed identification of oligosaccharide peaks and the determination of their compositions. High retention time reproducibility was exhibited by the microchip LC. Little variation was observed for standard sample either alone or in a complex heterogeneous mixture. The nanoLC-MS exhibits excellent capabilities in profiling mixtures of oligosaccharides.

Carbon↗

PubMed2004

An experimental study of chromatographic dynamics in open and packed non-cylindrical conduits.

A systematic investigation of the influence of the perimeter shape of open and particle packed fused silica capillaries on chromatographic properties such as resistance to flow and dispersion of solutes propelled through these channels has been conducted. Verifications of these uncommon experiments with existing theoretical treatments are presented and the insights transferred to a novel polymer chip design with integrated facilities for complex separations. A comparison of the chromatographic performance of a real life proteomics sample on this chip with a capillary column of "similar" dimensions is presented.

Chromatography, High Pressure Liquid↗