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Biomedical subjects

Timothy D Veenstra

Publications and source records attributed to Timothy D Veenstra.

At least 109 records · Page 6Linked to original sources
PubMed2003

Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse.

To evaluate the role of oncogenic RAS mutations in pancreatic tumorigenesis, we directed endogenous expression of KRAS(G12D) to progenitor cells of the mouse pancreas. We find that physiological levels of Kras(G12D) induce ductal lesions that recapitulate the full spectrum of human pancreatic intraepithelial neoplasias (PanINs), putative precursors to invasive pancreatic cancer. The PanINs are highly proliferative, show evidence of histological progression, and activate signaling pathways normally quiescent in ductal epithelium, suggesting potential therapeutic and chemopreventive targets for the cognate human condition. At low frequency, these lesions also progress spontaneously to invasive and metastatic adenocarcinomas, establishing PanINs as definitive precursors to the invasive disease. Finally, mice with PanINs have an identifiable serum proteomic signature, suggesting a means of detecting the preinvasive state in patients.

Animals↗
PubMed2003

Cancer diagnosis using proteomic patterns.

The advent of proteomics has brought with it the hope of discovering novel biomarkers that can be used to diagnose diseases, predict susceptibility and monitor progression. Much of this effort has focused upon the mass spectral identification of the thousands of proteins that populate complex biosystems such as serum and tissues. A revolutionary approach in proteomic pattern analysis has emerged as an effective method for the early diagnosis of diseases such as ovarian cancer. Proteomic pattern analysis relies on the pattern of proteins observed and does not rely on the identification of a traceable biomarker. Hundreds of clinical samples per day can be analyzed utilizing this technology, which has the potential to be a novel, highly sensitive diagnostic tool for the early detection of cancer.

Biomarkers, Tumor↗
PubMed2003

Quantitative proteomics employing primary amine affinity tags.

A proteomics-based method using stable isotope labeling to assess the relative abundance of peptides or proteins is described. Bradykinin and carbonic anhydrase were labeled with sulfosuccinimidyl-2-(biotinamido) ethyl-1,3-dithiopropionate, a membrane impermeant reagent that is reactive with primary amines. Specificity of the label to primary amines was demonstrated using tandem mass spectrometry. Also, relative quantitation was achieved by secondary labeling with natural isotopic abundance and stable isotope-labeled methyl iodide. We believe this to be an effective stable isotope-labeling method for quantitative proteomics.

Amines↗
PubMed2003

Anti-angiogenic activity of human endostatin is HIF-1-independent in vitro and sensitive to timing of treatment in a human saphenous vein assay.

Endostatin is a 20-kDa endogenous angiogenesis inhibitor that has recently been shown to inhibit the expression of vascular endothelial growth factor (VEGF), an angiogenic growth factor that is up-regulated by hypoxia via the HIF-1 transcription factor complex. To determine if the anti-angiogenic activity of endostatin involves a modulation of the HIF-1/VEGF pathway in cancer cells, experiments were conducted to establish what effect endostatin has on HIF-1 activity, HIF-1alpha protein production, and cellular localization in prostate cancer cells and endothelial cells. Endothelial cell tube formation was inhibited by endostatin purchased from Calbiochem (San Diego, CA) but not endostatin obtained from EntreMed (Rockville, MD). Subsequent experiments using Calbiochem endostatin showed that it did not alter HIF-1alpha protein production or cellular localization in any of the cell lines tested, nor did it alter HIF-1 transactivational activity in hypoxia. Whether or not this is also true in vivo remains to be determined. Nevertheless, these data suggest that the anti-angiogenic activity of endostatin is independent of the HIF-1/VEGF pathway. Immunocytochemical staining results do not indicate a decreased production of VEGF in Calbiochem endostatin-treated LNCaP or human umbilical vein endothelial cells (HUVEC). Treatment of rat aortic cross sections with human endostatin from Calbiochem resulted in a dose-dependent inhibition of microvessel outgrowth. Importantly, inhibition of vessel outgrowth by Calbiochem endostatin in a human saphenous vein angiogenesis assay required early treatment. In view of this in vitro data, we suggest that clinical trials involving endostatin treatment of late-stage disease may not adequately represent the efficacy of this drug in early-stage cancer.

Angiogenesis Inhibitors↗
PubMed2003

Serum protein fingerprinting.

The core technologies in the rapidly expanding field of proteomics have matured to the point where quantitative measurements of thousands of proteins can be conducted, enabling truly global measurements of protein expression. This advent has brought with it the hope of discovering novel biomarkers that promise a renaissance in clinical medicine. To meet this need, many proteomic studies have focused on the identification and subsequent comparative analysis of the thousands of proteins that populate complex biological systems such as serum and tissues. A novel application of mass spectrometry has been in proteomic pattern analysis, which has emerged as an effective method for the early diagnosis of diseases. In stark contrast to 'classical' proteomics, proteomic pattern analysis relies on the pattern of proteins observed, rather than on the discrete identification of a protein. Proteomic pattern technology allows hundreds of clinical samples to be analyzed per day and promises to be a novel, highly sensitive predictive clinical tool to improve diagnostic and prognostic medicine.

Blood Proteins↗
PubMed2002

Low-energy collision-induced dissociation fragmentation analysis of cysteinyl-modified peptides.

The development of methods to chemically modify and isolate cysteinyl-residue-containing peptides (Cys-peptides) for LC-MS/MS analysis has generated considerable interest in the field of proteomics. Methods using isotope-coded affinity tags (ICAT) and (+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine (iodoacetyl-PEO-biotin) employ similar Cys-modifying reagents that contain a thiolate-specific biotin group to modify and isolate Cys-containing peptides in conjunction with immobilized avidin. For these strategies to be effective on a proteome-wide level, the presence of the ICAT or acetyl-PEO-biotin tag should not interfere with the efficiency of induced dissociation in MS/MS experiments or with the identification of the modified Cys-peptides by automated database searching algorithms. We have compared the collision-induced dissociation (CID) fragmentation patterns of peptides labeled with iodoacetyl-PEO-biotin and the ICAT reagent to those of the unmodified peptides. CID of Cys-peptides modified with either reagent resulted in the formation of ions attributed to the modified Cys-peptides as well as those unique to the labeling reagent. As demonstrated by analyzing acetyl-PEO-biotin labeled peptides from ribonuclease A and the ICAT-labeled proteome of Deinococcus radiodurans, the presence of these label-specific product ions provides a useful identifier to discern whether a peptide has been modified with the Cys-specific reagent, especially when a number of peptides analyzed using these methods do not contain a modified Cys residue, and to differentiate identical Cys-peptides labeled with either ICAT-d0 or ICAT-d8.

Animals↗
PubMed2002

The SELDI-TOF MS approach to proteomics: protein profiling and biomarker identification.

The need for methods to identify disease biomarkers is underscored by the survival-rate of patients diagnosed at early stages of cancer progression. Surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) is a novel approach to biomarker discovery that combines two powerful techniques: chromatography and mass spectrometry. One of the key features of SELDI-TOF MS is its ability to provide a rapid protein expression profile from a variety of biological and clinical samples. It has been used for biomarker identification as well as the study of protein-protein, and protein-DNA interaction. The versatility of SELDI-TOF MS has allowed its use in projects ranging from the identification of potential diagnostic markers for prostate, bladder, breast, and ovarian cancers and Alzheimer's disease, to the study of biomolecular interactions and the characterization of posttranslational modifications. In this minireview we discuss the application of SELDI-TOF MS to protein biomarker discovery and profiling.

Biomarkers, Tumor↗
PubMed2002

Phosphoprotein isotope-coded affinity tags: application to the enrichment and identification of low-abundance phosphoproteins.

The use of a phosphoprotein isotope-coded affinity tag (PhIAT), which employs differential isotopic labeling and biotinylation, has been shown capable of enriching and identifying mixtures of low-abundance phosphopeptides. A denatured solution of beta-casein was labeled using the PhIAT method, and after proteolytic digestion, the labeled peptides were isolated using immobilized avidin. The recovered peptides were separated by capillary reversed-phase liquid chromatography and identified by tandem mass spectrometry. PhIAT-labeled peptides corresponding to known O-phosphorylated peptides from beta-casein were identified along with the phosphorylated peptides from alphas1-casein and alphas2-casein, known low-level (<5%) contaminants of commercially available beta-casein. All of the casein-phosphorylated residues identified by the present PhIAT approach correspond to previously documented sites of phosphorylation. The results illustrate the efficacy of the PhIAT-labeling strategy to not only enrich mixtures for phosphopeptides but also, more importantly, permit the detection and identification of low-level phosphopeptides. In addition, the differences in the phosphorylation state could be determined between phosphopeptides in comparative samples by stoichiometric conversion using the light and heavy isotopic versions of the PhIAT reagents. Overall, our results exemplify the application of the PhIAT approach and demonstrate its utility for proteome-wide phosphoprotein identification and quantitation.

Affinity Labels↗
PubMed2002

New tools for quantitative phosphoproteome analysis.

Recent advances in analytical methods, particularly in the area of mass spectrometry, have brought the field of proteomics to the forefront in biological science. The ultimate goal of proteomics--to characterize proteins expressed within a cell under a specific set of conditions--is daunting due to the complexity and dynamic nature the of protein population within the cell. While much of the effort has focused on developing methods to identify expressed proteins, the identification of posttranslational modifications is equally important for comprehensive proteome characterization. Of all the known posttranslational modifications, phosphorylation arguably plays the largest role in the context of cellular homeostasis. This review discusses some of the recent progress made in the development of techniques not only to identify, but also to quantitatively determine sites of phosphorylation.

Animals↗
PubMed2002

Proteome analysis of camptothecin-treated cortical neurons using isotope-coded affinity tags.

Isotope-coded affinity tags (ICATs) were employed to identify and quantitate changes in protein expression between control and camptothecin-treated mouse cortical neurons. Proteins extracted from control cortical neurons and those treated with camptothecin were labeled with the light and heavy isotopic versions of the ICAT reagents, respectively. ICAT-labeled samples were combined, proteolytically digested, and the derivatized peptides isolated using immobilized avidin chromatography. The peptides thus isolated were analyzed by reversed-phase liquid chromatography coupled directly to either a conventional ion-trap mass spectrometer (IT-MS) or a Fourier transform ion cyclotron resonance mass spectrometer (FTICR). While a majority of the peptide identifications were accomplished using IT-MS, FTICR was used to quantitate the relative abundances of the ICAT-labeled peptides taking advantage of its high resolution, sensitivity, and duty cycle. By using this combination of MS technologies we have thus far identified and quantified the expression of greater than 125 proteins from control and camptothecin-treated mouse cortical neurons. While proteins from most functional classes of proteins were identified, a particularly large percentage of the enzymes involved in glycolysis and the tricarboxylic acid cycle were observed.

Animals↗
PubMed2002

Methods for fractionation, separation and profiling of proteins and peptides.

In the last few years there has been an increased effort to develop technologies capable of identifying and quantifying large numbers of proteins expressed within a cell system (i.e., the proteome). The complexity of the mixtures being analyzed has made the development of effective fractionation and separation methods a critical component of this effort. This review highlights many of the protein and peptide fractionation and separation methods, such as electrophoresis and high-performance liquid chromatography (HPLC), which have experienced significant development over the past forty years. Modern instrumental strategies for the resolution of cell proteins, based on separations employing a single high-resolution or multidimensional approach, and the relative merits of each, will be discussed. The focus of this manuscript will be on the development of multidimensional separations such as two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), HPLC/HPLC, and HPLC-capillary electrophoresis and their application to the characterization of complex proteome mixtures.

Animals↗
PubMed2002

An accurate mass tag strategy for quantitative and high-throughput proteome measurements.

We describe and demonstrate a global strategy that extends the sensitivity, dynamic range, comprehensiveness, and throughput of proteomic measurements based upon the use of peptide "accurate mass tags" (AMTs) produced by global protein enzymatic digestion. The two-stage strategy exploits Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometry to validate peptide AMTs for a specific organism, tissue or cell type from "potential mass tags" identified using conventional tandem mass spectrometry (MS/MS) methods, providing greater confidence in identifications as well as the basis for subsequent measurements without the need for MS/MS, and thus with greater sensitivity and increased throughput. A single high resolution capillary liquid chromatography separation combined with high sensitivity, high resolution and accurate FT-ICR measurements has been shown capable of characterizing peptide mixtures of significantly more than 10(5) components with mass accuracies of < 1 ppm, sufficient for broad protein identification using AMTs. Other attractions of the approach include the broad and relatively unbiased proteome coverage, the capability for exploiting stable isotope labeling methods to realize high precision for relative protein abundance measurements, and the projected potential for study of mammalian proteomes when combined with additional sample fractionation. Using this strategy, in our first application we have been able to identify AMTs for >60% of the potentially expressed proteins in the organism Deinococcus radiodurans.

Amino Acid Sequence↗
PubMed2002

Selective incorporation of isotopically labeled amino acids for identification of intact proteins on a proteome-wide level.

The post-genomic era and increased demands for broad proteome measurements have greatly increased the needs for protein identification. We describe a strategy that uses accurate mass measurements and partial amino acid content information to unambiguously identify intact proteins, and show its initial application to the proteomes of Escherichia coli and Saccharomyces cerevisiae. Proteins were extracted from the organisms grown in minimal medium or minimal medium to which isotopically labeled leucine (Leu-D(10)) had been added. The two protein extracts were mixed and analyzed by capillary isoelectric focusing (CIEF) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FTICR). The incorporation of the isotopically labeled residue has no effect on the CIEF separation of proteins, and both isotopically labeled and unlabeled versions of specific proteins are observed within the same mass spectrum. The difference in the mass of the unlabeled and labeled proteins is used to determine the number of Leu residues present in a particular protein. Proteins can then often be unambiguously identified based on their accurately determined molecular mass and the additional constraint provided by number of Leu residues. The identities of proteins were further confirmed by repeating CIEF/FTICR measurements with samples that contain other isotopically labeled amino acid residues (e.g. His, Arg, Ile, Phe, Lys). A theoretical study of the amino acid composition (for a difference in the amino acid sequence) showed the constraints needed in order to identify the protein unambiguously. Additionally, the mass differences between the predicted and the experimental accurate mass measurement provide insights into the nature of simple post-translational modifications.

Amino Acids↗
PubMed2002

The postgenomic age: characterization of proteomes.

Global analysis of biological systems is becoming increasingly feasible as technologies that facilitate genome-wide analyses of gene expression are developed. Proteomics is the global analysis of expressed proteins (including posttranslational modifications) and seeks to establish the relationship between genome sequence, expressed proteins, protein-protein interactions, and cell and tissue phenotype. While the relative abundance of transcripts can be quantified using gene expression microarrays, the identification and quantitation of expressed proteins is more challenging. Nevertheless, the potential payoff for global protein analyses is immense because identification of distinctive protein signatures associated with cell function may provide novel therapeutic targets, molecular markers of disease, and increased understanding of determinants of cell phenotype. The challenges and promises of applications of established and emerging proteome strategies to detect and quantify differentially expressed proteins in culture cells are discussed.

Animals↗
PubMed2002

Proteomic analysis in the neurosciences.

Proteomics is a field of study directed toward providing a comprehensive view of the characteristics and activity of every cellular protein. Rapid innovations in the core technologies required to characterize proteins on a global scale are poised to bring about a comprehensive understanding of how dynamic changes in protein expression, post-translational modification, and function affect complex signaling and regulatory networks. These advances have significant implications for understanding the multitude of pathways that govern behavior and cognition and the response of the nervous system to injury and disease.

Animals↗
PubMedDate not supplied

Evaluation of the acid-cleavable isotope-coded affinity tag reagents: application to camptothecin-treated cortical neurons.

The new generation of isotope-coded affinity tag (ICAT) reagents have been evaluated by labeling an equimolar amount of bovine serum albumin (BSA) with ICAT-12C9 and ICAT-13C9, combining the mixtures, digesting them with trypsin and analyzing the digestate both by muRPLC-tandem MS and by matrix-assisted laser desorption ionization (MALDI) TOF/TOF MS. The use of 13C in place of 2H resulted in both of the labeled peptides having identical elution characteristics in a reversed-phase separation. This similarity in elution allows ICAT-labeled peptides to be effectively analyzed using a muRPLC-MALDI-MS strategy as well. All of the cysteinyl-containing tryptic peptides from BSA were identified with only a 10% variation in the relative abundance measurements between the light and heavy versions of each peptide. A facile method for the removal of contaminants that arise from the cleaved biotin moiety that otherwise interfere with downstream separations and MS analysis has also been developed. The new ICAT reagents were then applied to the analysis of a cortical neuron proteome sample to identify proteins regulated by the antitumor drug, camptothecin.

Amino Acid Sequence↗
PubMedDate not supplied

Quantitative profiling of the detergent-resistant membrane proteome of iota-b toxin induced vero cells.

Enzyme-mediated 18O/16O differential labeling of proteome samples often suffers from incomplete exchange of the carboxy-terminus oxygen atoms, resulting in ambiguity in the measurable abundance differences. In this study, an 18O/16O labeling strategy was optimized for and applied to the solution-based comparative analysis of the detergent-resistant membrane proteome (DRMP) of untreated and Iota-b (Ib)-induced Vero cells. Solubilization and tryptic digestion of the DRMP was conducted in a buffer containing 60% methanol. Unfortunately, the activity of trypsin is attenuated at this methanol concentration hampering the ability to obtain complete oxygen atom turnover. Therefore, the incorporation of the 18O atoms was decoupled from the protein digestion step by carrying out the trypsin-mediated heavy atom incorporation in a buffer containing 20% methanol; a concentration at which trypsin activity is enhanced compared to purely aqueous conditions. After isotopic labeling, the samples were combined, fractionated by strong cation exchange and analyzed by microcapillary reversed-phase liquid chromatography coupled on-line with electrospray ionization tandem mass spectrometry. In total, over 1400 unique peptides, corresponding to almost 600 proteins, were identified and quantitated, including all known caveolar and lipid raft marker proteins. The quantitative profiling of Ib-induced DRMP from Vero cells revealed several proteins with altered expression levels suggesting their possible role in Ib binding/uptake.

ADP Ribose Transferases↗