Biomedical subjects

Hookeun Lee

Publications and source records attributed to Hookeun Lee.

23 records · Page 2Linked to original sources

PubMed2003

Analysis of whole bacterial cells by flow field-flow fractionation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The purpose of this study is to develop a novel bacterial analysis method by coupling the flow field-flow fractionation (flow FFF) separation technique with detection by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The composition of carrier liquid used for flow FFF was selected based on retention of bacterial cells and compatibility with the MALDI process. The coupling of flow FFF and MALDI-TOF MS was demonstrated for P. putida and E. coli. Fractions of the whole cells were collected after separation by FFF and further analyzed by MALDI-MS. Each fraction, collected over different time intervals, corresponded to different sizes and possibly different growth stages of bacteria. The bacterial analysis by flow FFF/MALDI-TOF MS was completed within 1 h with only preliminary optimization of the process.

Bacillus cereus↗

PubMed2003

Abundance ratio-dependent proteomic analysis by mass spectrometry.

The goal of quantitative proteomics is to determine the identity and relative quantity of each protein present in two or more complex protein samples. Here we describe a novel approach to quantitative proteomics. It is based on a highly accurate algorithm for the automated quantification of chromatographically fractionated, isotope-coded affinity-tagged peptides and MALDI quadrupole time-of-flight tandem mass spectrometry for their identification. The method is capable of detecting and selectively identifying those proteins within a complex mixture that show a difference in relative abundance. We demonstrate the effectiveness and the versatility of this approach in the analysis of a standard protein mixture, protein expression profiling in a human prostate cancer cell line model, and identification of the specific components of the multiprotein transcriptional machinery in S. cerevisiae.

Humans↗

PubMed2003

A microcapillary trap cartridge-microcapillary high-performance liquid chromatography electrospray ionization emitter device capable of peptide tandem mass spectrometry at the attomole level on an ion trap mass spectrometer with automated routine operation.

Reversed-phase microcapillary chromatography (RP-microLC) combined with electrospray ionization tandem mass spectrometry (ESI-MS/MS) is one of two prevailing techniques in proteomic analysis, the other being matrix-assisted laser desorption/ionization (MALDI). Despite the arguably better dynamic range obtainable with ESI, MALDI is increasingly popular due to ease of use, ruggedness and the ability to decouple separation from ionization. By contrast, in order to take advantage of the sensitivity and dynamic range afforded by the concentration-dependent nature of ESI, it is directly coupled to separations that take place in small i.d. RP-microLC columns. This gain in sensitivity often comes at a loss of ruggedness due to clogging of the small i.d. RP-microLC columns, one result of which is limited sample throughput. Here we describe a combined micropre-column-microLC-ESI device that is sensitive, rugged and modular in design allowing facile construction and troubleshooting. Due to low signal-to-noise as little as 1 attomole of a peptide can be selected by data-dependent methods for collision-induced dissociation. Importantly, the resulting tandem mass spectrum is of high enough quality to identify the peptide sequence by a database search against a complex database using SEQUEST. Finally, the device is demonstrated to be rugged as judged by >60 consecutive reversed-phase microLC separations on complex peptide mixtures before chromatographic resolution is degraded.

Angiotensin I↗

PubMed2002

Development of a multiplexed microcapillary liquid chromatography system for high-throughput proteome analysis.

Comprehensive proteome analysis requires the identification (and quantification) of the proteins in samples consisting of thousands of proteins spanning a range of abundance of several orders of magnitude. The currency of proteome analysis by mass spectrometry is the peptides generated by protein proteolysis. The high sample complexity of such samples requires a large separation capacity, which is commonly achieved by fractionation of the mixture followed by further serial separations of each fraction. The sample throughput of proteome analysis is therefore limited by the need to sequentially process large numbers of samples. We have developed a novel four-plexed microcapillary liquid chromatography system for automated, high-throughput separation of complex peptide samples. The system supports the concurrent separation of four different samples by directing identically split solvent-gradient flows into four microcapillary C18 columns. The simple design of the system achieves multiplexed separation without the need for extra solvent pumps. Peak resolution, reproducibility, and parallel separating capacity of the system were investigated using standard peptides. The applicability of the system to high-throughput protein expression profiling was demonstrated in qualitative and quantitative analyses of protein expression in S. cerevisiae grown on two different carbon sources using the isotope-coded affinity tag (ICAT) reagent and matrix-assisted desorption/ionization quadrupole time-of-flight mass spectrometry.

Chromatography, High Pressure Liquid↗

PubMed2002

Approaching complete peroxisome characterization by gas-phase fractionation.

We examined the utility of gas-phase fractionation (GPF) in the m/z dimension to increase proteome coverage and reproducibility of peptide ion selection by direct microliquid chromatography/electrospray ionization-tandem mass spectrometry (microLC/ESI-MS/MS) analysis of the peptides produced by proteolytic digestion of unfractionated proteins from a yeast whole-cell lysate and in a peroxisomal membrane protein fraction derived from isolated yeast peroxisomes. We also investigated GPF in the relative ion intensity dimension and propose denoting the two types of GPF as GPF(m/z) and GPF(RI). Comparison of results of direct nuLC/ESI-MS/MS analysis of the unfractionated mixture of peptides from proteolysis of a yeast whole cell lysate by DD ion selection from 400-1800 m/z in triplicate and GPF(m/z) from 400-800, 800-1200 and 1200-1800 produced the following results: (i) 1.3 x more proteins were identified by GPF(m/z) for an equal amount of effort (i.e., 3 microLC/ESI-MS/MS) and (ii) proteins identified by GPF(m/z) had a lower average codon bias value. Use of GPF(RI) identified more proteins per m/z unit scanned than GPF(m/z) or triplicate analysis over a wide m/z range. After tryptic digestion of all the proteins from a discontinuous Nycodenz gradient fraction known to be enriched with yeast peroxisomal membrane proteins we detected 93% (38/41) of known peroxisomal proteins using GPF(m/z), but only 73% using a standard wide m/z range survey scan.

Chromatography, High Pressure Liquid↗