Biomedical subjects

Hookeun Lee

Publications and source records attributed to Hookeun Lee.

17 recordsLinked to original sources

PubMed2005

Quantitative proteomic analysis of myc-induced apoptosis: a direct role for Myc induction of the mitochondrial chloride ion channel, mtCLIC/CLIC4.

Myc is a key regulatory protein in higher eukaryotes controlling important cellular functions such as proliferation, differentiation, and apoptosis. Myc is profoundly involved in the genesis of many human and animal cancers, and the abrogation of Myc-induced apoptosis is a critical event in cancer progression. Because the mechanisms that mediate Myc-induced apoptosis are largely unknown, we analyzed protein expression during Myc-induced apoptosis using an isotope-coded affinity tag quantitative proteomics approach and identified that a proapoptotic mitochondrial chloride ion channel, mtCLIC/CLIC4, is induced by Myc. Myc binds to the mtCLIC gene promoter and activates its transcription. Suppression of mtCLIC expression by RNA interference inhibited Myc-induced apoptosis in response to different stress conditions and abolished the cooperative induction of apoptosis by Myc and Bax. We also found that Myc reduces the expression of Bcl-2 and Bcl-xL and that the apoptosis-inducing stimuli up-regulate Bax expression. These results suggest that up-regulation of mtCLIC, together with a reduction in Bcl-2 and Bcl-xL, sensitizes Myc-expressing cells to the proapoptotic action of Bax.

Apoptosis↗

PubMed2005

Signal maps for mass spectrometry-based comparative proteomics.

Mass spectrometry-based proteomic experiments, in combination with liquid chromatography-based separation, can be used to compare complex biological samples across multiple conditions. These comparisons are usually performed on the level of protein lists generated from individual experiments. Unfortunately given the current technologies, these lists typically cover only a small fraction of the total protein content, making global comparisons extremely limited. Recently approaches have been suggested that are built on the comparison of computationally built feature lists instead of protein identifications. Although these approaches promise to capture a bigger spectrum of the proteins present in a complex mixture, their success is strongly dependent on the correctness of the identified features and the aligned retention times of these features across multiple experiments. In this experimental-computational study, we went one step further and performed the comparisons directly on the signal level. First signal maps were constructed that associate the experimental signals across multiple experiments. Then a feature detection algorithm used this integrated information to identify those features that are discriminating or common across multiple experiments. At the core of our approach is a score function that faithfully recognizes mass spectra from similar peptide mixtures and an algorithm that produces an optimal alignment (time warping) of the liquid chromatography experiments on the basis of raw MS signal, making minimal assumptions on the underlying data. We provide experimental evidence that suggests uniqueness and correctness of the resulting signal maps even on low accuracy mass spectrometers. These maps can be used for a variety of proteomic analyses. Here we illustrate the use of signal maps for the discovery of diagnostic biomarkers. An imple-mentation of our algorithm is available on our Web server.

Algorithms↗

PubMed2005

Increased quantitative proteome coverage with (13)C/(12)C-based, acid-cleavable isotope-coded affinity tag reagent and modified data acquisition scheme.

Quantitative protein profiling using the isotope-coded affinity tag (ICAT) method and tandem mass spectrometry (MS) enables the pair-wise comparison of protein expression levels in biological samples. A new version of the ICAT reagent with an acid-cleavable bond, which allows removal of the biotin moiety prior to MS and which utilizes (13)C substitution for (12)C in the heavy-ICAT reagent rather than (2)H (for (1)H) as in the original reagent, was investigated. We developed and validated an MS data acquisition strategy using this new reagent that results in an increased number of protein identifications per experiment, without losing the accuracy of protein quantification. This was achieved by following a single survey (precursor) ion scan and serial collision induced dissociations (CIDs) of four different precursor ions observed in the prior survey scan. This strategy is common to many high-performance liquid chromatography-electrospray ionization (HPLC-ESI)-MS shotgun proteomic strategies, but heretofore not to ICAT experiments. This advance is possible because the new ICAT reagent uses (13)C as the "heavy" element rather than (2)H, thus, eliminating the slight delay in retention time of ICAT-labeled "light" peptides on a C18-based HPLC separation that occurs with (2)H and (1)H. Analyses using this new scheme of an ICAT-labeled trypsin-digested six protein mixture as well as a tryptic digest of a total yeast lysate, indicated that about two times more proteins were identified in a single analysis, and that there was no loss in accuracy of quantification.

Carbon↗

PubMed2005

Human Plasma PeptideAtlas.

Peptide identifications of high probability from 28 LC-MS/MS human serum and plasma experiments from eight different laboratories, carried out in the context of the HUPO Plasma Proteome Project, were combined and mapped to the EnsEMBL human genome. The 6929 distinct observed peptides were mapped to approximately 960 different proteins. The resulting compendium of peptides and their associated samples, proteins, and genes is made publicly available as a reference for future research on human plasma.

Blood Proteins↗

PubMed2005

Proteomic identification of potential susceptibility factors in drug-induced liver disease.

Drug-induced liver disease (DILD) causes significant morbidity and mortality and impairs new drug development. Currently, no known criteria can predict whether a drug will cause DILD or what risk factors make an individual susceptible. Although it has been shown in mouse studies that the disruption of key regulatory factors, such as cyclooxygenase-2 (COX-2), interleukin (IL)-6, and IL-10, increased susceptibility to DILD caused by acetaminophen (APAP), no single factor seems to be absolute. As an approach to better understand the multifactorial basis of DILD, we compared the hepatic proteome of mice that are resistant (SJL) and susceptible (C57Bl/6) to APAP-induced liver disease (AILD), using solution-based isotope-coded affinity tag (ICAT) liquid chromatography mass spectrometry. Several novel factors were identified that were more highly expressed in the livers of SJL mice, including those involved in stress response, cell proliferation and tissue regeneration, and protein modification, implicating these proteins as potential hepatoprotective factors. There was also a selective loss of several mitochondrial proteins from the livers of the susceptible C57Bl/6 mice, suggesting that the loss of functional mitochondria may indeed play a role in AILD. These findings indicate that comparative hepatic proteomic analyses of susceptible and resistant mouse strains may provide a global approach for identifying potential risk factors and mechanistic pathways responsible for DILD.

Acetaminophen↗

PubMed2004

Integration with the human genome of peptide sequences obtained by high-throughput mass spectrometry.

A crucial aim upon the completion of the human genome is the verification and functional annotation of all predicted genes and their protein products. Here we describe the mapping of peptides derived from accurate interpretations of protein tandem mass spectrometry (MS) data to eukaryotic genomes and the generation of an expandable resource for integration of data from many diverse proteomics experiments. Furthermore, we demonstrate that peptide identifications obtained from high-throughput proteomics can be integrated on a large scale with the human genome. This resource could serve as an expandable repository for MS-derived proteome information.

Amino Acid Sequence↗

PubMed2004

Quantitative proteomic analysis of metabolic regulation by copper ions in Methylococcus capsulatus (Bath).

Copper ions switch the oxidation of methane by soluble methane monooxygenase to particulate methane monooxygenase in Methylococcus capsulatus (Bath). Toward understanding the change in cellular metabolism related to this transcriptional and metabolic switch, we have undertaken genomic sequencing and quantitative comparative analysis of the proteome in M. capsulatus (Bath) grown under different copper-to-biomass ratios by cleavable isotope-coded affinity tag technology. Of the 682 proteins identified, the expressions of 60 proteins were stimulated by at least 2-fold by copper ions; 68 proteins were down-regulated by 2-fold or more. The 60 proteins overexpressed included the methane and carbohydrate metabolic enzymes, while the 68 proteins suppressed were mainly responsible for cellular signaling processes, indicating a role of copper ions in the expression of the genes associated with the metabolism of the organism downstream of methane oxidation. The study has also provided a complete map of the C1 metabolism pathways in this methanotroph and clarified the interrelationships between them.

Bacterial Proteins↗

PubMed2004

System-based proteomic analysis of the interferon response in human liver cells.

BACKGROUND: Interferons (IFNs) play a critical role in the host antiviral defense and are an essential component of current therapies against hepatitis C virus (HCV), a major cause of liver disease worldwide. To examine liver-specific responses to IFN and begin to elucidate the mechanisms of IFN inhibition of virus replication, we performed a global quantitative proteomic analysis in a human hepatoma cell line (Huh7) in the presence and absence of IFN treatment using the isotope-coded affinity tag (ICAT) method and tandem mass spectrometry (MS/MS). RESULTS: In three subcellular fractions from the Huh7 cells treated with IFN (400 IU/ml, 16 h) or mock-treated, we identified more than 1,364 proteins at a threshold that corresponds to less than 5% false-positive error rate. Among these, 54 were induced by IFN and 24 were repressed by more than two-fold, respectively. These IFN-regulated proteins represented multiple cellular functions including antiviral defense, immune response, cell metabolism, signal transduction, cell growth and cellular organization. To analyze this proteomics dataset, we utilized several systems-biology data-mining tools, including Gene Ontology via the GoMiner program and the Cytoscape bioinformatics platform. CONCLUSIONS: Integration of the quantitative proteomics with global protein interaction data using the Cytoscape platform led to the identification of several novel and liver-specific key regulatory components of the IFN response, which may be important in regulating the interplay between HCV, interferon and the host response to virus infection.

Cell Line, Tumor↗

PubMed2004

A dataset of human liver proteins identified by protein profiling via isotope-coded affinity tag (ICAT) and tandem mass spectrometry.

Proteins from human liver carcinoma Huh7 cells, representing transformed liver cells, and cultured primary human fetal hepatocytes (HFH) and human HH4 hepatocytes, representing nontransformed liver cells, were extracted and processed for proteome analysis. Proteins from stimulated cells (interferon-alpha treatment for the Huh7 and HFH cells and induction of hepatitis C virus [HCV] proteins for the HH4 cells) and corresponding control cells were labeled with light and heavy cleavable ICAT reagents, respectively. The labeled samples were combined, trypsinized, and subject to cation-exchange and avidin-affinity chromatographies. The resulting cysteine-containing peptides were analyzed by microcapillary LC-MS/MS. The MS/MS spectra were initially analyzed by searching the human International Protein Index database using the SEQUEST software (1). Subsequently, new statistical algorithms were applied to the collective SEQUEST search results of each experiment. First, the PeptideProphet software (2) was applied to discriminate true assignments of MS/MS spectra to peptide sequences from false assignments, to assign a probability value for each identified peptide, and to compute the sensitivity and error rate for the assignment of spectra to sequences in each experiment. Second, the ProteinProphet software (3) was used to infer the protein identifications and to compute probabilities that a protein had been correctly identified, based on the available peptide sequence evidence. The resulting protein lists were filtered by a ProteinProphet probability score p > or = 0.5, which corresponded to an error rate of less than 5%. A total of 1,296, 1,430, and 1,476 proteins or related protein groups were identified in three subdatasets from the Huh7, HFH, and HH4 cells, respectively. In total, these subdatasets contained 2,486 unique protein identifications from human liver cells. An increase of the threshold to p > or = 0.9 (corresponding to an error rate of less than 1%) resulted in 2,159 unique protein identifications (1,146, 1,235, and 1,318 for the Huh7, HFH, and HH4 cells, respectively).

Algorithms↗

PubMed2004

A tool to visualize and evaluate data obtained by liquid chromatography-electrospray ionization-mass spectrometry.

We present a software tool for visualizing data obtained from analyzing complex peptide mixtures by liquid chromatography (LC) electrospray ionization (ESI) mass spectrometry (MS). The data are represented as a two-dimensional density plot. For experiments employing collision-induced dissociation (CID), links are embedded in the image to the CID spectra and the corresponding peptide sequences that are represented by the respective feature. The image provides an intuitive method to evaluate sample quality and the performance of an LC-ESI-MS system and can be used to optimize experimental conditions. Local patterns of the image can also be used to identify chemical contaminants and specific peptide features. Therefore, this software tool may have broad application in MS-based proteomics.

Chromatography, Liquid↗

PubMed2004

Optimization of reversed-phase microcapillary liquid chromatography for quantitative proteomics.

Currently, the field of shotgun proteomics relies primarily on the separation of peptides by reversed-phase microcapillary chromatography (RP-microLC) combined with either electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALDI) and tandem mass spectrometry (MS/MS) for protein identification as well as quantification. For this purpose we herein describe construction of a RP-microLC-ESI column-emitter along with optimized microLC conditions for using the device to quantify pair-wise changes in protein expression via the isotope coded affinity tag (ICAT) method that also maximize peak capacity. These optimized RP-microLC parameters required a balance be reached between the disparate needs of quantification which requires good peak shape and identification (i.e. proteome coverage) of proteins via peptide collision induced dissociation (CID) which requires peak capacity be maximized. A complex biological sample from a study of murine acetaminophen toxicity in hepatocyes was chosen for method development because of the high level complexity, but the biological results are not the focus of this manuscript.

Amino Acid Sequence↗

PubMed2004

Gene expression analyzed by high-resolution state array analysis and quantitative proteomics: response of yeast to mating pheromone.

The transcriptome provides the database from which a cell assembles its collection of proteins. Translation of individual mRNA species into their encoded proteins is regulated, producing discrepancies between mRNA and protein levels. Using a new modeling approach to data analysis, a striking diversity is revealed in association of the transcriptome with the translational machinery. Each mRNA has its own pattern of ribosome loading, a circumstance that provides an extraordinary dynamic range of regulation, above and beyond actual transcript levels. Using this approach together with quantitative proteomics, we explored the immediate changes in gene expression in response to activation of a mitogen-activated protein kinase pathway in yeast by mating pheromone. Interestingly, in 26% of those transcripts where the predicted protein synthesis rate changed by at least 3-fold, more than half of these changes resulted from altered translational efficiencies. These observations underscore that analysis of transcript level, albeit extremely important, is insufficient by itself to describe completely the phenotypes of cells under different conditions.

Computational Biology↗

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↗