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David R Goodlett

Publications and source records attributed to David R Goodlett.

At least 19 recordsLinked to original sources
PubMed2007

A combined dataset of human cerebrospinal fluid proteins identified by multi-dimensional chromatography and tandem mass spectrometry.

Human cerebrospinal fluid (CSF) is an important source for studying protein biomarkers of age-related neurodegenerative diseases. Before characterizing biomarkers unique to each disease, it is necessary to categorize CSF proteins systematically and extensively. However, the enormous complexity, great dynamic range of protein concentrations, and tremendous protein heterogeneity due to post-translational modification of CSF create significant challenges to the existing proteomics technologies for an in-depth, nonbiased profiling of the human CSF proteome. To circumvent these difficulties, in the last few years, we have utilized several different separation methodologies and mass spectrometric platforms that greatly enhanced the identification coverage and the depth of protein profiling of CSF to characterize CSF proteome. In total, 2594 proteins were identified in well-characterized pooled human CSF samples using stringent proteomics criteria. This report summarizes our efforts to comprehensively characterize the human CSF proteome to date.

Cerebrospinal Fluid Proteins↗
PubMed2007

Comparison of pancreas juice proteins from cancer versus pancreatitis using quantitative proteomic analysis.

OBJECTIVES: Pancreatitis is an inflammatory condition of the pancreas. However, it often shares many molecular features with pancreatic cancer. Biomarkers present in pancreatic cancer frequently occur in the setting of pancreatitis. The efforts to develop diagnostic biomarkers for pancreatic cancer have thus been complicated by the false-positive involvement of pancreatitis. METHODS: In an attempt to develop protein biomarkers for pancreatic cancer, we previously use quantitative proteomics to identify and quantify the proteins from pancreatic cancer juice. Pancreatic juice is a rich source of proteins that are shed by the pancreatic ductal cells. In this study, we used a similar approach to identify and quantify proteins from pancreatitis juice. RESULTS: In total, 72 proteins were identified and quantified in the comparison of pancreatic juice from pancreatitis patients versus pooled normal control juice. Nineteen of the juice proteins were overexpressed, and 8 were underexpressed in pancreatitis juice by at least 2-fold compared with normal pancreatic juice. Of these 27 differentially expressed proteins in pancreatitis, 9 proteins were also differentially expressed in the pancreatic juice from pancreatic cancer patient. CONCLUSIONS: Identification of these differentially expressed proteins from pancreatitis juice provides useful information for future study of specific pancreatitis-associated proteins and to eliminate potential false-positive biomarkers for pancreatic cancer.

Biomarkers, Tumor↗
PubMed2006

Electron capture in spin-trap capped peptides. An experimental example of ergodic dissociation in peptide cation-radicals.

Electron capture dissociation was studied with tetradecapeptides and pentadecapeptides that were capped at N-termini with a 2-(4'-carboxypyrid-2'-yl)-4-carboxamide group (pepy), e.g., pepy-AEQLLQEEQLLQEL-NH(2), pepy-AQEFGEQGQKALKQL-NH(2), and pepy-AQEGSEQAQKFFKQL-NH(2). Doubly and triply protonated peptide cations underwent efficient electron capture in the ion-cyclotron resonance cell to yield charge-reduced species. However, the electron capture was not accompanied by backbone dissociations. When the peptide ions were preheated by absorption of infrared photons close to the dissociation threshold, subsequent electron capture triggered ion dissociations near the remote C-terminus forming mainly (b(11-14) + 1)(+)* fragment ions that were analogous to those produced by infrared multiphoton dissociation alone. Ab initio calculations indicated that the N-1 and N-1' positions in the pepy moiety had topical gas-phase basicities (GB = 923 kJ mol(-1)) that were greater than those of backbone amide groups. Hence, pepy was a likely protonation site in the doubly and triply charged ions. Electron capture in the protonated pepy moiety produced the ground electronic state of the charge-reduced cation-radical with a topical recombination energy, RE = 5.43-5.46 eV, which was greater than that of protonated peptide residues. The hydrogen atom in the charge-reduced pepy moiety was bound by >160 kJ mol(-1), which exceeded the hydrogen atom affinity of the backbone amide groups (21-41 kJ mol(-1)). Thus, the pepy moiety functioned as a stable electron and hydrogen atom trap that did not trigger radical-type dissociations in the peptide backbone that are typical of ECD. Instead, the internal energy gained by electron capture was redistributed over the peptide moiety, and when combined with additional IR excitation, induced proton-driven ion dissociations which occurred at sites that were remote from the site of electron capture. This example of a spin-remote fragmentation provided the first clear-cut experimental example of an ergodic dissociation upon ECD.

Cations↗
PubMed2006

Lack of in vitro and in vivo recognition of Francisella tularensis subspecies lipopolysaccharide by Toll-like receptors.

Francisella tularensis is an intracellular gram-negative bacterium that is highly infectious and potentially lethal. Several subspecies exist of varying pathogenicity. Infection by only a few organisms is sufficient to cause disease depending on the model system. Lipopolysaccharide (LPS) of gram-negative bacteria is generally recognized by Toll-like receptor 4 (TLR4)/MD-2 and induces a strong proinflammatory response. Examination of human clinical F. tularensis isolates revealed that human virulent type A and type B strains produced lipid A of similar structure to the nonhuman model pathogen of mice, Francisella novicida. F. novicida LPS or lipid A is neither stimulatory nor an antagonist for human and murine cells through TLR4 or TLR2. It does not appear to interact with TLR4 or MD-2, as it is not an antagonist to other stimulatory LPS. Consistent with these observations, aerosolization of F. novicida LPS or whole bacteria induced no inflammatory response in mice. These results suggest that poor innate recognition of F. tularensis allows the bacterium to evade early recognition by the host innate immune system to promote its pathogenesis for mammals.

Animals↗
PubMed2006

Identification of the interactions between cytochrome P450 2E1 and cytochrome b5 by mass spectrometry and site-directed mutagenesis.

The reaction cycles of cytochrome P450s (P450) require input of two electrons. Electrostatic interactions are considered important driving forces in the association of P450s with their redox partners, which in turn facilitates the transfer of the two electrons. In this study, the cross-linking reagent, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), was used to covalently link cytochrome P450 2E1 (CYP2E1) with cytochrome b(5) (b(5)) through the formation of specific amide bonds between complementary charged residue pairs. Cross-linked peptides in the resulting protein complex were distinguished from non-cross-linked peptides using an (18)O-labeling method on the basis that cross-linked peptides incorporate twice as many (18)O atoms as non-cross-linked peptides during proteolysis conducted in (18)O-water. Subsequent tandem mass spectrometric (MS/MS) analysis of the selected cross-linked peptide candidates led to the identification of two intermolecular cross-links, Lys(428)(CYP2E1)-Asp(53)(b(5)) and Lys(434)(CYP2E1)-Glu(56)(b(5)), which provides the first direct experimental evidence for the interacting orientations of a microsomal P450 and its redox partner. The biological importance of the two ion pairs for the CYP2E1-b(5) interaction, and the stimulatory effect of b(5), was confirmed by site-directed mutagenesis. Based on the characterized cross-links, a CYP2E1-b(5) complex model was constructed, leading to improved insights into the protein interaction. The described method is potentially useful for mapping the interactions of various P450 isoforms and their redox partners, because the method is relatively rapid and sensitive, and is capable of suggesting not only protein interacting regions, but also interacting orientations.

Acetaminophen↗
PubMed2006

Pro-CrossLink. Software tool for protein cross-linking and mass spectrometry.

To facilitate structural analysis of proteins and protein-protein interactions, we developed Pro-CrossLink, a suite of software tools consisting of three programs (Figure 1), DetectShift, IdentifyXLink, and AssignXLink. DetectShift was developed to detect ions of cross-linked peptide pairs in a mixture of 18O-labeled peptides obtained from protein proteolytic digests. The selected candidate ions of cross-linked peptide pairs subsequently undergo tandem mass spectrometric (MS/MS) analysis for sequence determination. Based on the masses of candidate ions as well as y- and b-type ions in the tandem mass spectra, IdentifyXLink assigns the candidate ions to cross-linked peptide pairs. For an identified cross-linked peptide pair, AssignXLink generates an extensive fragment ion list, including a-, b-, c-type, x-, y-, z-type, internal, and immonium ions with associated common losses of H2O, NH3, CO, and CO2, and facilitates a precise location of the cross-linked residues. Pro-CrossLink is automated, highly configurable by the user, and applicable to many studies that map low-resolution protein structures and molecular interfaces in protein complexes.

Amino Acid Sequence↗
PubMed2006

Proteome analysis of Halobacterium sp. NRC-1 facilitated by the biomodule analysis tool BMSorter.

To better understand the extremely halophilic archaeon Halobacterium species NRC-1, we analyzed its soluble proteome by two-dimensional liquid chromatography coupled to electrospray ionization tandem mass spectrometry. A total of 888 unique proteins were identified with a ProteinProphet probability (P) between 0.9 and 1.0. To evaluate the biochemical activities of the organism, the proteomic data were subjected to a biological network analysis using our BMSorter software. This allowed us to examine the proteins expressed in different biomodules and study the interactions between pertinent biomodules. Interestingly an integrated analysis of the enzymes in the amino acid metabolism and citrate cycle networks suggested that up to eight amino acids may be converted to oxaloacetate, fumarate, or oxoglutarate in the citrate cycle for energy production. In addition, glutamate and aspartate may be interconverted from other amino acids or synthesized from citrate cycle intermediates to meet the high demand for the acidic amino acids that are required to build the highly acidic proteome of the organism. Thus this study demonstrated that proteome analysis can provide useful information and help systems analyses of organisms.

Amino Acids↗
PubMed2006

Quantitative proteomic profiling of pancreatic cancer juice.

Pancreatic juice is an exceptionally rich source of cancer-specific proteins shed from cancerous ductal cells into the pancreatic juice. Quantitative proteomic analysis of the proteins specific to pancreatic cancer juice has not previously been reported. We used isotope-code affinity tag (ICAT) technology and MS/MS to perform quantitative protein profiling of pancreatic juice from pancreatic cancer patients and normal controls. ICAT technology coupled with MS/MS allows the systematic study of the proteome and measures the protein abundance in pancreatic juice with the potential for development of biomarkers. A total of 105 proteins were identified and quantified in the pancreatic juice from a pancreatic cancer patient, of which 30 proteins showed abundance changes of at least twofold in pancreatic cancer juice compared to normal controls. Many of these proteins have been externally validated. This is the first comprehensive study of the pancreatic juice proteome by quantitative global protein profiling, and the study reveals numerous proteins that are shown for the first time to be associated with pancreatic cancer, providing candidates for diagnostic biomarkers. One of the identified proteins, insulin-like growth factor binding protein-2 was further validated by Western blotting to be elevated in pancreatic cancer juice and overexpressed in pancreatic cancer tissue.

Amino Acid Sequence↗
PubMed2006

Comparison of a Salmonella typhimurium proteome defined by shotgun proteomics directly on an LTQ-FT and by proteome pre-fractionation on an LCQ-DUO.

Shotgun proteomics is rapidly becoming one of the most efficient and popular tools to examine protein expression in cells. Numerous laboratories now have a wide array of low- and high-performance mass spectrometry instrumentation necessary to complete proteome-wide projects. Often these laboratories have time and financial constraints that prohibit all projects from being conducted on high-performance state-of-the-art mass spectrometers. Here, we compare shotgun proteomic results using a direct 'lyse, digest and analyse' approach on a high-performance mass spectrometer (i.e. the LTQ-FT) with the results from a much lower-performance instrument (i.e. the LCQ-DUO) where, for the latter, various traditional protein pre-fractionation steps and gas-phase fractionation were used to increase the proteome coverage. Our results demonstrate that shotgun proteomic analyses conducted on the lower-performance LCQ-DUO mass spectrometer could adequately characterize a PhoP constitutive strain of Salmonella typhimurium if proteome pre-fractionation steps and gas-phase fractionation were included.

Chromatography, High Pressure Liquid↗
PubMed2006

Mining the acute respiratory distress syndrome proteome: identification of the insulin-like growth factor (IGF)/IGF-binding protein-3 pathway in acute lung injury.

To obtain a more complete protein profile of the airspace milieu in acute respiratory distress syndrome (ARDS) and to identify new mediators, we analyzed bronchoalveolar lavage fluid (BALF) by shotgun proteomics. Using BALF from three patients, we identified a total of 870 different proteins, a nearly 10-fold increase from previous reports. Among the proteins identified were known markers of lung injury, such as surfactant, proteases, and serum proteins. We also identified several biologically interesting proteins not previously identified in patients with ARDS, including insulin-like growth factor-binding protein-3 (IGFBP-3). Because of the known role of IGFBP-3 in regulating cell survival, we measured IGFBP-3 levels by enzyme-linked immunosorbent assay in ARDS BALF. Normal controls had low levels of IGFBP-3, whereas patients with early ARDS had a significant increase in IGFBP-3. The IGF pathway, acting through the type 1 IGF-receptor, repressed apoptosis of lung fibroblasts but not lung epithelial cells. Furthermore, depletion of IGF in ARDS BALF led to enhanced fibroblast apoptosis. Our data suggest that the IGFBP-3/IGF pathway is involved in the pathogenesis of lung injury, illustrating the power of shotgun proteomics to catalog proteins present in complex biological fluids, such as BALF, from which hypotheses can be developed and tested.

Adult↗
PubMed2005

Stromal mesenchyme cell genes of the human prostate and bladder.

BACKGROUND: Stromal mesenchyme cells play an important role in epithelial differentiation and likely in cancer as well. Induction of epithelial differentiation is organ-specific, and the genes responsible could be identified through a comparative genomic analysis of the stromal cells from two different organs. These genes might be aberrantly expressed in cancer since cancer could be viewed as due to a defect in stromal signaling. We propose to identify the prostate stromal genes by analysis of differentially expressed genes between prostate and bladder stromal cells, and to examine their expression in prostate cancer. METHODS: Immunohistochemistry using antibodies to cluster designation (CD) cell surface antigens was first used to characterize the stromas of the prostate and bladder. Stromal cells were prepared from either prostate or bladder tissue for cell culture. RNA was isolated from the cultured cells and analyzed by DNA microarrays. Expression of candidate genes in normal prostate and prostate cancer was examined by RT-PCR. RESULTS: The bladder stroma was phenotypically different from that of the prostate. Most notable was the presence of a layer of CD13+ cells adjacent to the urothelium. This structural feature was also seen in the mouse bladder. The prostate stroma was uniformly CD13-. A number of differentially expressed genes between prostate and bladder stromal cells were identified. One prostate gene, proenkephalin (PENK), was of interest because it encodes a hormone. Secreted proteins such as hormones and bioactive peptides are known to mediate cell-cell signaling. Prostate stromal expression of PENK was verified by an antibody raised against a PENK peptide, by RT-PCR analysis of laser-capture microdissected stromal cells, and by database analysis. Gene expression analysis showed that PENK expression was down-regulated in prostate cancer. CONCLUSION: Our findings show that the histologically similar stromas of the prostate and bladder are phenotypically different, and express organ-specific genes. The importance of these genes in epithelial development is suggested by their abnormal expression in cancer. Among the candidates is the hormone PENK and the down-regulation of PENK expression in cancer suggests a possible association with cancer development.

Cells, Cultured↗
PubMed2005

Evidence for the presence of disease-perturbed networks in prostate cancer cells by genomic and proteomic analyses: a systems approach to disease.

Prostate cancer is initially responsive to androgen ablation therapy and progresses to androgen-unresponsive states that are refractory to treatment. The mechanism of this transition is unknown. A systems approach to disease begins with the quantitative delineation of the informational elements (mRNAs and proteins) in various disease states. We employed two recently developed high-throughput technologies, massively parallel signature sequencing (MPSS) and isotope-coded affinity tag, to gain a comprehensive picture of the changes in mRNA levels and more restricted analysis of protein levels, respectively, during the transition from androgen-dependent LNCaP (model for early-stage prostate cancer) to androgen-independent CL1 cells (model for late-stage prostate cancer). We sequenced >5 million MPSS signatures, obtained >142,000 tandem mass spectra, and built comprehensive MPSS and proteomic databases. The integrated mRNA and protein expression data revealed underlying functional differences between androgen-dependent and androgen-independent prostate cancer cells. The high sensitivity of MPSS enabled us to identify virtually all of the expressed transcripts and to quantify the changes in gene expression between these two cell states, including functionally important low-abundance mRNAs, such as those encoding transcription factors and signal transduction molecules. These data enable us to map the differences onto extant physiologic networks, creating perturbation networks that reflect prostate cancer progression. We found 37 BioCarta and 14 Kyoto Encyclopedia of Genes and Genomes pathways that are up-regulated and 23 BioCarta and 22 Kyoto Encyclopedia of Genes and Genomes pathways that are down-regulated in LNCaP cells versus CL1 cells. Our efforts represent a significant step toward a systems approach to understanding prostate cancer progression.

Cell Line, Tumor↗
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

Large-scale evaluation of quantitative reproducibility and proteome coverage using acid cleavable isotope coded affinity tag mass spectrometry for proteomic profiling.

Strategies employing non-gel based methods for quantitative proteomic profiling such as isotope coded affinity tags coupled with mass spectrometry (ICAT-MS) are gaining attention as alternatives to two-dimensional gel electrophoresis (2-DE). We have conducted a large-scale investigation to determine the degree of reproducibility and depth of proteome coverage of a typical ICAT-MS experiment by measuring protein changes in Escherichia coli treated with triclosan, an inhibitor of fatty acid biosynthesis. The entire ICAT-MS experiment was conducted on four independent occasions where more than 24 000 peptides were quantitated using an ion-trap mass spectrometer. Our results demonstrated that quantitatively, the technique provided good reproducibility (median coefficient of variation of ratios was 18.6%), and on average identified more than 450 unique proteins per experiment. However, the method was strongly biased to detect acidic proteins (pI < 7), under-represented small proteins (<10 kDa) and failed to show clear superiority over 2-DE methods in monitoring hydrophobic proteins from cell lysates.

Anti-Infective Agents, Local↗
PubMed2005

Characterization of microsomal fraction proteome in human lymphoblasts reveals the down-regulation of galectin-1 by interleukin-12.

T helper cells (Th) are divided into Th1 and Th2 subsets based upon their cytokine profiles and function. Naïve Th cells differentiate into Th1 and Th2 subsets depending on the antigens, costimulatory molecules, and cytokines they encounter. Cytokine interleukin (IL)-12 enhances the generation of Th1 lymphocytes and inhibits the production of Th2 subset. Many genes involved in Th cell differentiation have already been identified at transcriptomic level in microarray studies. In this study, isotope coded affinity tag labeling combined with chromatographic techniques and tandem mass spectrometry was used to find IL-12 regulated proteins in the microsomal fraction of Th cells. A total of 380 and 275 proteins were initially identified and quantitated in two experiments. After the high-confidence protein identifications were restricted to those where at least two different peptides were identified per protein, and these confirmed by manual inspection of the tandem mass spectra, 147 proteins remained. Of these high-confidence protein identifications 41 had at least 1.5-fold change in expression between IL-12 treated and nontreated cells. Among the differentially regulated proteins were galectin-1 (gal-1) and CD7, and their down-regulation was further corroborated with Western blotting and flow cytometry, respectively. Gal-1 and CD7 are known to interact with each other, and regulate immunity through influencing apoptosis and cytokine production. Our data indicate that IL-12 down-regulates the expression of both gal-1 and CD7 in the microsomal fraction of peripheral blood mononuclear cells and cord blood CD4(+) cells. The down-regulation of these proteins is likely to have a role in specific Th cell selection and cytokine environment creation.

Antigens, CD7↗
PubMed2005

Quantitative proteomic analysis of age-related changes in human cerebrospinal fluid.

Identification of cerebrospinal fluid (CSF) biomarkers of the common age-related neurodegenerative diseases would be of great value to clinicians because of the difficulties in differential diagnoses of these diseases in clinical practice. Proteins are one class of potential biomarkers currently under investigation in the hope that different ensembles of proteins will aid in the diagnosis of these diseases, as well as in the assessment of progression and response to therapy. However, before undertaking a rational approach to CSF protein biomarkers of age-related neurodegeneration, we must first systematically identify CSF proteins and determine whether their levels change with normal aging. In this study, we used a powerful shotgun proteomic method, two-dimensional microcapillary liquid chromatography electrospray ionization tandem mass spectrometry, to identify proteins in human CSF. Additionally, using pooled CSF samples, we quantitatively compared the CSF proteome of younger adults with that of older adults using isotope-coded affinity tags (ICAT). From these studies we identified more than 300 proteins in CSF and found that there were 30 proteins with >20% change in concentrations between older and younger individuals. Finally, we validated changes in concentration for two of these proteins using Western blots in CSF from a separate set of individuals. These data not only expand substantially our current knowledge regarding human CSF proteins, but also supply the necessary information to appropriately interpret protein biomarkers of age-related neurodegenerative diseases.

Adult↗