Biomedical subjects

David R Goodlett

Publications and source records attributed to David R Goodlett.

At least 37 records · Page 2Linked to original sources

PubMed2005

Identification of phosphorylation sites using microimmobilized metal affinity chromatography.

One of the most important roles that mass spectrometry (MS) has played in the late twentieth and early twenty-first centuries has been to assist in the growth of knowledge of dynamic phosphorylation events. Not only has MS allowed researches to pinpoint the site of phosphorylation, but it has also enabled them to identify the kinase/phosphatase pairs responsible for regulation of a specific modification as well as to follow the functional consequences of the observed phosphorylation events on the biology of the system. For phosphorylation analysis, the important contribution of MS has been critical but not definitive. There are numerous methods that have been applied with success, yet none are generally applicable to all analyses. So, for the time being, researchers in the field must select from a panel of methods to find (de)phosphorylation events. In the work described in this chapter, a collection of integrated methods are presented. A detailed account is provided for phosphorylation capture via on- and off-line immobilized metal affinity chromatography (IMAC). This is followed by a suite of useful strategies for discovery of phosphorylation positioning through sequence determination by phosphate-specific diagnostic ion scans, including precursor and product ion scans, neutral loss scans, and in-source dissociation and post-source decay.

Amino Acid Sequence↗

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↗

PubMed2005

Pancreatic cancer proteome: the proteins that underlie invasion, metastasis, and immunologic escape.

BACKGROUND & AIMS: Pancreatic cancer is a highly lethal disease that has seen little headway in diagnosis and treatment for the past few decades. The effective treatment of pancreatic cancer is critically relying on the diagnosis of the disease at an early stage, which still remains challenging. New experimental approaches, such as quantitative proteomics, have shown great potential for the study of cancer and have opened new opportunities to investigate crucial events underlying pancreatic tumorigenesis and to exploit this knowledge for early detection and better intervention. METHODS: To systematically study protein expression in pancreatic cancer, we used isotope-coded affinity tag technology and tandem mass spectrometry to perform quantitative proteomic profiling of pancreatic cancer tissues and normal pancreas. RESULTS: A total of 656 proteins were identified and quantified in 2 pancreatic cancer samples, of which 151 were differentially expressed in cancer by at least 2-fold. This study revealed numerous proteins that are newly discovered to be associated with pancreatic cancer, providing candidates for future early diagnosis biomarkers and targets for therapy. Several differentially expressed proteins were further validated by tissue microarray immunohistochemistry. Many of the differentially expressed proteins identified are involved in protein-driven interactions between the ductal epithelium and the extracellular matrix that orchestrate tumor growth, migration, angiogenesis, invasion, metastasis, and immunologic escape. CONCLUSIONS: Our study is the first application of isotope-coded affinity tag technology for proteomic analysis of human cancer tissue and has shown the value of this technology in identifying differentially expressed proteins in cancer.

Humans↗

PubMed2005

Proteomic biomarker discovery in cerebrospinal fluid for neurodegenerative diseases.

The clinical diagnosis of major neurodegenerative disorders, e.g. Alzheimer's disease, Parkinson's disease, and dementia with Lewy body, remains unsatisfactory based on current clinical criteria and limited laboratory investigations. The possibility of identifying multiple novel biomarkers for neurodegenerative diseases, those related to disease pathogeneses in particular, has been greatly enhanced with recent advances in genomics, proteomics, and metabolomics. In this chapter, we will be reviewing a few issues related to proteomic identification of proteins in human cerebrospinal fluid (CSF) as well as unique protein markers that could be used for clinical diagnosis of various neurodegenerative diseases or monitoring their progression. Great attention has been directed to practical considerations and limitations of several major aspects of proteomic analysis of human CSF.

Apolipoproteins B↗

PubMed2004

Quantitative mass spectrometry reveals a role for the GTPase Rho1p in actin organization on the peroxisome membrane.

We have combined classical subcellular fractionation with large-scale quantitative mass spectrometry to identify proteins that enrich specifically with peroxisomes of Saccharomyces cerevisiae. In two complementary experiments, isotope-coded affinity tags and tandem mass spectrometry were used to quantify the relative enrichment of proteins during the purification of peroxisomes. Mathematical modeling of the data from 306 quantified proteins led to a prioritized list of 70 candidates whose enrichment scores indicated a high likelihood of them being peroxisomal. Among these proteins, eight novel peroxisome-associated proteins were identified. The top novel peroxisomal candidate was the small GTPase Rho1p. Although Rho1p has been shown to be tethered to membranes of the secretory pathway, we show that it is specifically recruited to peroxisomes upon their induction in a process dependent on its interaction with the peroxisome membrane protein Pex25p. Rho1p regulates the assembly state of actin on the peroxisome membrane, thereby controlling peroxisome membrane dynamics and biogenesis.

Actins↗

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

Analysis of alpha-synuclein-associated proteins by quantitative proteomics.

To identify the proteins associated with soluble alpha-synuclein (AS) that might promote AS aggregation, a key event leading to neurodegeneration, we quantitatively compared protein profiles of AS-associated protein complexes in MES cells exposed to rotenone, a pesticide that produces parkinsonism in animals and induces Lewy body (LB)-like inclusions in the remaining dopaminergic neurons, and to vehicle. We identified more than 250 proteins associated with Nonidet P-40 soluble AS, and demonstrated that at least 51 of these proteins displayed significant differences in their relative abundance in AS complexes under conditions where rotenone was cytotoxic and induced formation of cytoplasmic inclusions immunoreactive to anti-AS. Overexpressing one of these proteins, heat shock protein (hsp) 70, not only protected cells from rotenone-mediated cytotoxicity but also decreased soluble AS aggregation. Furthermore, the protection afforded by hsp70 transfection appeared to be related to suppression of rotenone-induced oxidative stress as well as mitochondrial and proteasomal dysfunction.

Amino Acid Sequence↗

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

Proteomic analysis of the intestinal epithelial cell response to enteropathogenic Escherichia coli.

We present the first large scale proteomic analysis of a human cellular response to a pathogen. Enteropathogenic Escherichia coli (EPEC) is an enteric human pathogen responsible for much childhood morbidity and mortality worldwide. EPEC uses a type III secretion system (TTSS) to inject bacterial proteins into the cytosol of intestinal epithelial cells, resulting in diarrhea. We analyzed the host response to TTSS-delivered EPEC effector proteins by infecting polarized intestinal epithelial monolayers with either wild-type or TTSS-deficient EPEC. Host proteins were isolated and subjected to quantitative profiling using isotope-coded affinity tagging (ICAT) combined with electrospray ionization tandem mass spectrometry. We identified over 2000 unique proteins from infected Caco-2 monolayers, of which approximately 13% are expressed differentially in the presence of TTSS-delivered EPEC effector proteins. We validated these data in silico and through immunoblotting and immunofluorescence microscopy. The identified changes extend cytoskeletal observations made in less relevant cell types and generate testable hypotheses with regard to host proteins potentially involved in EPEC-induced diarrhea. These data provide a framework for future biochemical analyses of host-pathogen interactions.

Biological Transport↗

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↗

PubMed2004

Quantitative proteomic analysis of proteins released by neoplastic prostate epithelium.

Prostate cancer is unusual among neoplasms in that it may be diagnosed at a curable stage through detection of a protein in serum, the serine protease prostate-specific antigen (PSA). PSA is secreted by both normal and neoplastic prostate epithelial cells in response to androgenic hormones and has found widespread use in cancer screening. Because PSA screening is controversial due to sensitivity and specificity issues, efforts continue to focus on the identification and characterization of additional markers that may be used for diagnostic and therapeutic purposes. In this study, we report the application of quantitative proteomic techniques that incorporate isotope coded affinity tag reagents and tandem mass spectrometry to comprehensively identify secreted and cell surface proteins from neoplastic prostate epithelium. LNCaP cells, a prostate tumor-derived cell line that secretes PSA in response to androgen exposure, were grown in a low protein-defined media under androgen-stimulated (A+) and -starved (A-) conditions. Proteomic analysis of the media identified in excess of 600 proteins, 524 of which could be quantified. Nine percent of the proteins had A+/A- ratios > 2.0, including PSA, and 2.5% had ratios < 0.5. A subset of these androgen-regulated proteins appeared to be expressed in abundance. Of these, selected mass spectrometry observations were confirmed by Western analysis. The findings suggest that androgen-mediated release of proteins may occur through the activation of proteolytic enzymes rather than exclusively through transcriptional or translational control mechanisms. On the basis of their known functional roles, several of the abundant androgen-regulated proteins may participate in the progression of neoplastic epithelial cell growth and should be considered as potential serum markers of neoplastic prostate diseases.

Biomarkers, Tumor↗

PubMed2004

Lipid raft proteins and their identification in T lymphocytes.

This review focuses on how membrane lipid rafts have been detected and isolated, mostly from lymphocytes, and their associated proteins identified. These proteins include transmembrane antigens/receptors, GPI-anchored proteins, cytoskeletal proteins, Src-family protein kinases, G-proteins, and other proteins involved in signal transduction. To further understand the biology of lipid rafts, new methodological approaches are needed to help characterize the raft protein component, and changes that occur in this component as a result of cell perturbation. We describe the application of new proteomic approaches to the identification and quantification of raft proteins in T-lymphocytes. Similar approaches, applied to other model cell systems, will provide valuable new insights into both cellular signal transduction and lipid raft biology.

Animals↗

PubMed2004

Proteomic approach to studying Parkinson's disease.

Parkinson's disease is a common age-related neurodegenerative disease characterized pathologically by a loss of dopaminergic neurons in the substantia nigra with resultant depletion of striatal dopamine and presence of Lewy bodies in the remaining neurons. The Lewy body contains numerous functional and structural proteins, including alpha-synuclein and ubiquitin; aggregation of alpha-synuclein is thought to be important in Lewy body formation as well as neurodegeneration, although the detailed mechanisms remain to be defined. Increasing evidence has suggested that mitochondrial dysfunction, increased oxidative stress, and dysfunction of the ubiquitin-proteasome system may be involved in alpha-synuclein aggregation, Lewy body formation, and neurodegeneration. However, how these processes are related to each other is not fully understood, given that there are Parkinsonian animal models as well as human diseases with significant nigral neurodegeneration regardless of whether Lewy bodies form or not. This review summarizes the current related research fields and proposes a proteomic approach to investigate the mechanisms that may dictate alpha-synuclein aggregation, Lewy body formation, and neurodegeneration.

Animals↗

PubMed2003

Proteomic analysis of an extreme halophilic archaeon, Halobacterium sp. NRC-1.

Halobacterium sp. NRC-1 insoluble membrane and soluble cytoplasmic proteins were isolated by ultracentrifugation of whole cell lysate. Using an ion trap mass spectrometer equipped with a C18 trap electrospray ionization emitter/micro-liquid chromatography column, a number of trypsin-generated peptide tags from 426 unique proteins were identified. This represents approximately one-fifth of the theoretical proteome of Halobacterium. Of these, 232 proteins were found only in the soluble fraction, 165 were only in the insoluble membrane fraction, and 29 were in both fractions. There were 72 and 61% previously annotated proteins identified in the soluble and membrane protein fractions, respectively. Interestingly, 57 of previously unannotated proteins found only in Halobacterium NRC-1 were identified. Such proteins could be interesting targets for understanding unique physiology of Halobacterium NRC-1. A group of proteins involved in various metabolic pathways were identified among the expressed proteins, suggesting these pathways were active at the time the cells were collected. This data containing a list of expressed proteins, their cellular locations, and biological functions could be used in future studies to investigate the interaction of the genes and proteins in relation to genetic or environmental perturbations.

Archaeal Proteins↗

PubMed2003

Proteomic analysis of human prostasomes.

BACKGROUND: Prostasomes are secretory particles in human seminal fluid. Other than a microscopic description of these secretory particles and an incomplete two-dimensional gel electrophoresis (2DE) study, little is known about the composition of proteins in prostasomes. METHODS: We employed a direct iterative approach using Gas phase fractionation and microcapillary HPLC-tandem mass spectrometry (microLC-MS/MS) to catalogue the prostasome proteome. RESULTS: We identified 139 proteins that can be divided into the following categories: (1). enzymes (33.8% of total), (2). transport/structural (19.4% of total), (3). GTP proteins (14.4% of total), (4). chaperone proteins (5.8% of total), (5). signal transduction proteins (17.3% of total), and (6). unannotated proteins (9.4% of total). A total of 128 of the 139 proteins have not previously been described as prostasomal. CONCLUSIONS: The proteins identified can be used as reference dataset in future work comparing prostasome proteins between normal and pathological states such as prostate cancer, benign prostatic hyperplasia, prostatitis, and infertility.

Enzymes↗

PubMed2003

The application of new software tools to quantitative protein profiling via isotope-coded affinity tag (ICAT) and tandem mass spectrometry: I. Statistically annotated datasets for peptide sequences and proteins identified via the application of ICAT and tandem mass spectrometry to proteins copurifying with T cell lipid rafts.

Lipid rafts were prepared according to standard protocols from Jurkat T cells stimulated via T cell receptor/CD28 cross-linking and from control (unstimulated) cells. Co-isolating proteins from the control and stimulated cell preparations were labeled with isotopically normal (d0) and heavy (d8) versions of the same isotope-coded affinity tag (ICAT) reagent, respectively. Samples were combined, proteolyzed, and resultant peptides fractionated via cation exchange chromatography. Cysteine-containing (ICAT-labeled) peptides were recovered via the biotin tag component of the ICAT reagents by avidin-affinity chromatography. On-line micro-capillary liquid chromatography tandem mass spectrometry was performed on both avidin-affinity (ICAT-labeled) and flow-through (unlabeled) fractions. Initial peptide sequence identification was by searching recorded tandem mass spectrometry spectra against a human sequence data base using SEQUEST software. New statistical data modeling algorithms were then applied to the SEQUEST search results. These allowed for discrimination between likely "correct" and "incorrect" peptide assignments, and from these the inferred proteins that they collectively represented, by calculating estimated probabilities that each peptide assignment and subsequent protein identification was a member of the "correct" population. For convenience, the resultant lists of peptide sequences assigned and the proteins to which they corresponded were filtered at an arbitrarily set cut-off of 0.5 (i.e. 50% likely to be "correct") and above and compiled into two separate datasets. In total, these data sets contained 7667 individual peptide identifications, which represented 2669 unique peptide sequences, corresponding to 685 proteins and related protein groups.

Amino Acid Sequence↗

PubMed2003

The application of new software tools to quantitative protein profiling via isotope-coded affinity tag (ICAT) and tandem mass spectrometry: II. Evaluation of tandem mass spectrometry methodologies for large-scale protein analysis, and the application of statistical tools for data analysis and interpretation.

Proteomic approaches to biological research that will prove the most useful and productive require robust, sensitive, and reproducible technologies for both the qualitative and quantitative analysis of complex protein mixtures. Here we applied the isotope-coded affinity tag (ICAT) approach to quantitative protein profiling, in this case proteins that copurified with lipid raft plasma membrane domains isolated from control and stimulated Jurkat human T cells. With the ICAT approach, cysteine residues of the two related protein isolates were covalently labeled with isotopically normal and heavy versions of the same reagent, respectively. Following proteolytic cleavage of combined labeled proteins, peptides were fractionated by multidimensional chromatography and subsequently analyzed via automated tandem mass spectrometry. Individual tandem mass spectrometry spectra were searched against a human sequence database, and a variety of recently developed, publicly available software applications were used to sort, filter, analyze, and compare the results of two repetitions of the same experiment. In particular, robust statistical modeling algorithms were used to assign measures of confidence to both peptide sequences and the proteins from which they were likely derived, identified via the database searches. We show that by applying such statistical tools to the identification of T cell lipid raft-associated proteins, we were able to estimate the accuracy of peptide and protein identifications made. These tools also allow for determination of the false positive rate as a function of user-defined data filtering parameters, thus giving the user significant control over and information about the final output of large-scale proteomic experiments. With the ability to assign probabilities to all identifications, the need for manual verification of results is substantially reduced, thus making the rapid evaluation of large proteomic datasets possible. Finally, by repeating the experiment, information relating to the general reproducibility and validity of this approach to large-scale proteomic analyses was also obtained.

Amino Acid Sequence↗

PubMed2003

Quantitative proteomic analysis indicates increased synthesis of a quinolone by Pseudomonas aeruginosa isolates from cystic fibrosis airways.

The opportunistic bacterial pathogen Pseudomonas aeruginosa colonizes airways of individuals with cystic fibrosis (CF) with resultant chronic destructive lung disease. P. aeruginosa adaptation to the CF airway includes biofilm formation and antibiotic resistance. Isolates from asymptomatic individuals in the first 3 years of life have unique characteristics, suggesting that adaptation occurs before clinical symptoms. One defined early adaptation is expression of a specific proinflammatory lipopolysaccharide (LPS) that is associated with antimicrobial peptide resistance. This CF-specific LPS is induced when P. aeruginosa is grown in medium that is limited for magnesium. Therefore, qualitative and quantitative proteomic approaches were used to define 1,331 P. aeruginosa proteins, of which 145 were differentially expressed on limitation of magnesium. Among proteins induced by low magnesium were enzymes essential for production of 2-heptyl 3-hydroxy 4-quinolone, the Pseudomonas quinolone signal (PQS), which interacts with the homoserine lactone signaling pathway. Measurement of PQS in P. aeruginosa isolates from asymptomatic children with CF indicated that strains with increased synthesis of PQS are present during early colonization of CF patient airways.

Anti-Bacterial Agents↗