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Timothy D Veenstra

Publications and source records attributed to Timothy D Veenstra.

At least 73 records · Page 4Linked to original sources
PubMed2004

Sheathless electrospray ionization interfaces for capillary electrophoresis-mass spectrometric detection advantages and limitations.

A review of sheathless interfaces for capillary electrophoresis (CE)-mass spectrometry (MS) is presented. The review discusses the on-line CE-MS system requirements, advantages and weaknesses of current sheathless interface designs for CE-electrospray ionization MS, and comparison between sheath flow and sheathless interfaces. The advantages and limitations of three sheathless designs are discussed and commented upon, these include single-capillary, two-capillary and three-piece designs.

Electrophoresis, Capillary↗
PubMed2004

Proteomic patterns for early cancer detection.

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 on 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, breast, and prostate cancer. This technology is capable of analyzing hundreds of clinical samples per day and has the potential to be a novel, highly sensitive diagnostic tool for the early detection of diseases, or as a predictor of response to therapy.

Biomarkers, Tumor↗
PubMed2004

Measuring seven endogenous ketolic estrogens simultaneously in human urine by high-performance liquid chromatography-mass spectrometry.

A rapid, sensitive, and specific high-performance liquid chromatography-electrospray ionization-multistage mass spectrometry (MS) method for measuring endogenous ketolic estrogen metabolites in human urine has been developed. The method requires a single hydrolysis/extraction/derivatization step and only 2.5 mL of urine, yet is able to simultaneously quantify estrone and its 2-methoxy and 2-, 4-, and 16alpha-hydroxy derivatives, 16-ketoestradiol, and 2-hydroxyestrone-3-methyl ether metabolites. The combination of a simple hydrazone derivatization step with multistage MS greatly enhances the sensitivity and specificity of the analysis of endogenous estrogen within human urine. Standard curves are linear over a 100-fold concentration range with linear regression correlation coefficients typically greater than 0.99. The lower limit of quantitation for each ketolic estrogen is 0.2 ng/2.5-mL urine sample (10 pg on column), with an accuracy of 93-103% and an overall precision, including the hydrolysis, extraction, and derivatization steps, of 1-13% relative standard derivation (RSD) for samples prepared concurrently and 8-16% RSD for samples prepared in separate batches. This method also allows for the identification of 2-hydroxyestrone-3-methyl ether in urine obtained from both pre- and postmenopausal women. This potentially protective estrogen metabolite has been previously reported only in the urine of pregnant women. Since individual patterns of estrogen metabolism may influence the risk of breast cancer, accurate and specific measurement of estrogen metabolites in biological matrixes will facilitate future research on breast cancer prevention, screening, and treatment.

Adult↗
PubMed2004

An antiproliferative factor from interstitial cystitis patients is a frizzled 8 protein-related sialoglycopeptide.

Approximately 1 million people in the United States suffer from interstitial cystitis, a chronic painful urinary bladder disorder characterized by thinning or ulceration of the bladder epithelial lining; its etiology is unknown. We have identified a glycosylated frizzled-related peptide inhibitor of cell proliferation that is secreted specifically by bladder epithelial cells from patients with this disorder. This antiproliferative factor (APF) profoundly inhibits bladder cell proliferation by means of regulation of cell adhesion protein and growth factor production. The structure of APF was deduced by using ion trap mass spectrometry (MS), enzymatic digestion, lectin affinity chromatography, and total synthesis, and confirmed by coelution of native and synthetic APF derivatives on microcapillary reversed-phase liquid chromatography (microRPLC)/MS. APF was determined to be an acidic, heat-stable sialoglycopeptide whose peptide chain has 100% homology to the putative sixth transmembrane domain of frizzled 8. Both synthetic and native APF had identical biological activity in normal bladder epithelial cells and T24 bladder cancer cells. Northern blot analysis indicated binding of a probe containing the sequence for the frizzled 8 segment with mRNA extracted from cells of patients with interstitial cystitis but not controls. APF is therefore a frizzled-related peptide growth inhibitor shown to contain exclusively a transmembrane segment of a frizzled protein and is a potential biomarker for interstitial cystitis.

Antineoplastic Agents↗
PubMed2004

Global analysis of the cortical neuron proteome.

In this study, a multidimensional fractionation approach was combined with MS/MS to increase the capability of characterizing complex protein profiles of mammalian neuronal cells. Proteins extracted from primary cultures of cortical neurons were digested with trypsin followed by fractionation using strong cation exchange chromatography. Each of these fractions was analyzed by microcapillary reversed-phase LC-MS/MS. The analysis of the MS/MS data resulted in the identification of over 15,000 unique peptides from which 3,590 unique proteins were identified based on protein-specific peptide tags that are unique to a single protein in the searched database. In addition, 952 protein clusters were identified using cluster analysis of the proteins identified by the peptides not unique to a single protein. This identification revealed that a minimum of 4,542 proteins could be identified from this experiment, representing approximately 16% of all known mouse proteins. An evaluation of the number of false-positive identifications was undertaken by searching the entire MS/MS dataset against a database containing the sequences of over 12,000 proteins from archaea. This analysis allowed a systematic determination of the level of confidence in the identification of peptides as a function of SEQUEST cross correlation (Xcorr) and delta correlation (DeltaCn) scores. Correlation charts were also constructed to show the number of unique peptides identified for proteins from specific classes. The results show that low-abundance proteins involved in signal transduction and transcription are generally identified by fewer peptides than high-abundance proteins that play a role in maintaining mammalian cellular structure and motility. The results presented here provide the broadest proteome coverage for a mammalian cell to date and show that MS-based proteomics has the potential to provide high coverage of the proteins expressed within a cell.

Amino Acid Sequence↗
PubMed2004

Serum proteomic profiles suggest celecoxib-modulated targets and response predictors.

Cyclooxygenase-2 is a valid target for cancer prevention and treatment. This has been shown in preclinical and clinical cancer prevention studies by using a cyclooxygenase-2 inhibitor, celecoxib. When used in a randomized cancer prevention clinical trial on patients with the inherited autosomal dominant condition, familial adenomatous polyposis, celecoxib proved efficacious. However, a remarkable heterogeneity in patients' responses to the chemopreventive effects of celecoxib was observed. Proteomic profiling of sera from these patients identified several markers, the expression of which was specifically modulated after treatment with celecoxib. A decision tree algorithm identified classifiers for response to celecoxib with relatively high sensitivity but moderate to low specificity. In particular, a spectral feature at m/z 16,961.4 was identified as a strong discriminator between response and nonresponse to celecoxib at the highest dose.

Adenomatous Polyposis Coli↗
PubMed2004

Proteomic investigation of natural killer cell microsomes using gas-phase fractionation by mass spectrometry.

We have explored the utility of gas-phase fractionation by mass spectrometry (MS) in the mass-to-charge (m/z) dimension (GPF(m/z)) for increasing the effective number of protein identifications in cases where sample quantity limits the use of multi-dimensional chromatographic fractionation. A peptide digestate from proteins isolated from the membrane fraction of natural killer (NK) cells was analyzed by microcapillary reversed-phase liquid chromatography coupled online to an ion-trap (IT) mass spectrometer. Performing GPF(m/z) using eight narrow precursor ion scan m/z ranges enabled the identification of 340 NK cell proteins from 12 microg of digestate, representing more than a fivefold increase in the number of proteins identified as compared to the same experiment employing a standard precursor ion survey scan m/z range (i.e., m/z 400-2000). The results show that GPF(m/z) represents an effective technique for increasing protein identifications in global proteomic investigations especially when sample quantity is limited.

Amino Acid Sequence↗
PubMed2004

Proteome analysis of DNA damage-induced neuronal death using high throughput mass spectrometry.

Isotope-coded affinity tag reagents and high throughput mass spectrometry were used to quantitate changes in the expression of 150 proteins in mouse wild-type (p53(+/+)) cortical neurons undergoing DNA damage-induced death. Immunological techniques confirmed several of the changes in protein expression, but microarray analysis indicated that many of these changes were not accompanied by altered mRNA expression. Proteome analysis revealed perturbations in mitochondrial function, free radical production, and neuritogenesis that were not observed in p53-deficient neurons. Changes in Tau, cofilin, and other proteins recapitulated abnormalities observed in neurodegenerative states in vivo. Additionally, DNA damage caused a p53-dependent decrease in expression of members of the protein kinase A (PKA) signaling pathway. PKA inhibition promoted death in the absence of DNA damage, revealing a novel mechanism by which endogenous down-regulation of PKA signaling may contribute to p53-dependent neuronal death. These data demonstrate the power of high throughput mass spectrometry for quantitative analysis of the neuronal proteome.

Animals↗
PubMed2004

Coupling morphogenesis to mitotic entry.

In eukaryotes, cyclin B-bound cyclin-dependent protein kinase 1 promotes mitotic entry but is held in check, in part, by Wee1 protein kinase. Timely mitotic entry in budding yeast requires inactivation of Swe1 (Wee1 ortholog). Perturbations of the septin collar at the bud neck lead to Swe1 stabilization, delaying the G(2)/M transition. Swe1 is recruited to the neck and hyperphosphorylated before ubiquitin-mediated degradation. Hsl1 kinase (Nim1 ortholog), a negative regulator of Wee1, is required for efficient Swe1 localization at the neck but seems not to phosphorylate Swe1. Here, we show that two other kinases targeted sequentially to the neck, Cla4/PAK and Cdc5/Polo, are responsible for stepwise phosphorylation and down-regulation of Swe1. This mechanism links assembly of a cellular structure to passage into mitosis.

Cell Cycle Proteins↗
PubMed2004

The human plasma proteome: a nonredundant list developed by combination of four separate sources.

We have merged four different views of the human plasma proteome, based on different methodologies, into a single nonredundant list of 1175 distinct gene products. The methodologies used were 1) literature search for proteins reported to occur in plasma or serum; 2) multidimensional chromatography of proteins followed by two-dimensional electrophoresis and mass spectroscopy (MS) identification of resolved proteins; 3) tryptic digestion and multidimensional chromatography of peptides followed by MS identification; and 4) tryptic digestion and multidimensional chromatography of peptides from low-molecular-mass plasma components followed by MS identification. Of 1,175 nonredundant gene products, 195 were included in more than one of the four input datasets. Only 46 appeared in all four. Predictions of signal sequence and transmembrane domain occurrence, as well as Genome Ontology annotation assignments, allowed characterization of the nonredundant list and comparison of the data sources. The "nonproteomic" literature (468 input proteins) is strongly biased toward signal sequence-containing extracellular proteins, while the three proteomics methods showed a much higher representation of cellular proteins, including nuclear, cytoplasmic, and kinesin complex proteins. Cytokines and protein hormones were almost completely absent from the proteomics data (presumably due to low abundance), while categories like DNA-binding proteins were almost entirely absent from the literature data (perhaps unexpected and therefore not sought). Most major categories of proteins in the human proteome are represented in plasma, with the distribution at successively deeper layers shifting from mostly extracellular to a distribution more like the whole (primarily cellular) proteome. The resulting nonredundant list confirms the presence of a number of interesting candidate marker proteins in plasma and serum.

Biomarkers, Tumor↗
PubMed2004

Direct ampholyte-free liquid-phase isoelectric peptide focusing: application to the human serum proteome.

In this study, we utilized a multidimensional peptide separation strategy combined with tandem mass spectrometry (MS/MS) for the identification of proteins in human serum. After enzymatically digesting serum with trypsin, the peptides were fractionated using liquid-phase isoelectric focusing (IEF) in a novel ampholyte-free format. Twenty IEF fractions were collected and analyzed by reversed-phase microcapillary liquid chromatography (microLC)-MS/MS. Bioinformatic analysis of the raw MS/MS spectra resulted in the identification of 844 unique peptides, corresponding to 437 proteins. This study demonstrates the efficacy of ampholyte-free peptide autofocusing, which alleviates peptide losses in ampholyte removal strategies. The results show that the separation strategy is effective for high-throughput characterization of proteins from complex proteomic mixtures.

Blood Proteins↗
PubMed2004

An investigation into the human serum "interactome".

The protein content of human serum is composed of a millieu of proteins from almost every type of cell and tissue within the body. The serum proteome has been shown to contain information that directly reflects pathophysiological states and represents an invaluable source of diagnostic information for a variety of different diseases. Unfortunately, the dynamic range of protein abundance, ranging from >> mg/mL level to << pg/mL level, renders complete characterization of this proteome nearly impossible with current analytical methods. To study low-abundance proteins, which have potential value for clinical diagnosis, the high-abundant species, such as immunoglobulins and albumin, are generally eliminated as the first step in many analytical protocols. This step, however, is hypothesized to concomitantly remove proteins/peptides associated with the high-abundant proteins targeted for depletion. In this study, immunoprecipitation was combined with microcapillary reversed-phase liquid chromatography (microRPLC) coupled on-line with tandem mass spectrometry (MS/MS) to investigate the low-molecular-weight proteins/peptides that associate with the most abundant species in serum. By this targeted isolation of select highly abundant serum proteins, the associated proteins/peptides can be enriched and effectively identified by microRPLC-MS/MS. Among the 210 proteins identified, 73% and 67% were not found in previous studies of the low-molecular-weight or whole-serum proteome, respectively.

Albumins↗
PubMed2004

Proteomic analysis of detergent-resistant membrane rafts.

A combined, detergent- and organic solvent-based proteomic method for the analysis of detergent-resistant membrane rafts (DRMR) is described. These specialized domains of the plasma membrane contain a distinctive and dynamic protein and/or lipid complement, which can be isolated from most mammalian cells. Lipid rafts are predominantly involved in signal transduction and adapted to mediate and produce different cellular responses. To facilitate a better understanding of their biology and role, DRMR were isolated from Vero cells as a Triton X-100 insoluble fraction. After detergent removal, sonication in 60% buffered methanol was used to extract, solubilize and tryptically digest the resulting protein complement. The peptide digestate was analyzed by microcapillary reversed-phase liquid chromatography-tandem mass spectrometry. Gas-phase fractionation in the mass-to-charge range was employed to broaden the selection of precursor ions and increase the number of identifications in an effort to detect less abundant proteins. A total of 380 proteins were identified including all known lipid raft markers. A total of 91 (24%) proteins were classified as integral alpha-helical membrane proteins, of which 51 (56%) were predicted to have multiple transmembrane domains.

Amino Acid Sequence↗
PubMed2004

Quantitative proteomic analysis of inorganic phosphate-induced murine MC3T3-E1 osteoblast cells.

Cleavable isotope-coded affinity tag (cICAT) reagents were utilized to identify and quantitate protein expression differences in control and inorganic phosphate-treated murine MC3T3-E1 osteoblast cells. Proteins extracted from control and treated cells were labeled with the light and heavy isotopic versions of cICAT reagents, respectively. The cICAT-labeled samples were combined, proteolytically digested, and the cICAT-derivatized peptides isolated using immobilized avidin chromatography. The cICAT-labeled peptides were resolved into 96 fractions by strong cation-exchange (SCX) liquid chromatography (LC). Analysis of the SCX-LC cICAT peptide fractions by microcapillary reversed-phase LC-tandem mass spectrometry resulted in the identification and quantitation of 7227 unique peptides corresponding to 2501 proteins, or roughly 9% of the proteins currently predicted to be encoded by the mouse genome. A false positive analysis indicated a 98% confidence in the peptide identifications. To corroborate changes in abundance measured by cICAT with those detectable in traditionally prepared cell lysate, we chose to analyze cyclin D1. Cyclin D1 has been previously identified as a phosphate-responsive gene and was likewise identified as a phosphate-responsive protein in the current analysis. The 1.76-fold increase in abundance in cyclin D1 determined from cICAT corresponds well with the 2.41-fold increase as determined by Western blotting. These results demonstrate that quantitative proteomics is capable of providing a quantitative view of thousands of proteins in mammalian cells within a defined set of experiments.

Amino Acid Sequence↗
PubMed2004

Two-dimensional liquid chromatography-capillary zone electrophoresis-sheathless electrospray ionization-mass spectrometry: evaluation for peptide analysis and protein identification.

A peptide separation strategy that combines two-dimensional (2-D) liquid chromatography (LC)-capillary zone electrophoresis (CZE) with tandem mass spectrometry (MS/MS) is described for the identification of proteins in complex mixtures. To test the effectiveness of this strategy, a serum sample was depleted of the high-abundance proteins by methanol precipitation, digested with trypsin to generate a complex peptide mixture, and separated into 96 fractions by reversed-phase (RP)-LC. Compared to ion-exchange LC separations, RPLC provides much higher resolution and peak capacity. Fractions were collected off-line from the RPLC separation, and subjected to short 20 min CZE separations. The separated zones were introduced to the mass spectrometer through a sheathless electrospray ionization interface that is integrated on the separation capillary. The ease of fabrication of the interface and its durability allowed for the analysis of all fractions on a single capillary in a relatively short analysis time. A stable electrospray was produced at nanoliter flowrates by augmenting analyte electrophoretic and electroosmotic mobilities with pressure-assisted flow. Unlike first-dimensional ion-exchange LC fractionation, where there is a large degree of overlap, the CZE-MS results show less than 15% overlap between neighboring RPLC fractions.

Blood Proteins↗
PubMed2004

A detergent- and cyanogen bromide-free method for integral membrane proteomics: application to Halobacterium purple membranes and the human epidermal membrane proteome.

A simple and rapid method for characterizing hydrophobic integral membrane proteins and its utility for membrane proteomics using microcapillary liquid chromatography coupled on-line with tandem mass spectrometry (microLC-MS/MS) is described. The present technique does not rely on the use of detergents, strong organic acids or cyanogen bromide-mediated proteolysis. A buffered solution of 60% methanol was used to extract, solubilize, and tryptically digest proteins within a preparation of Halobacterium (H.) halobium purple membranes. Analysis of the digested purple membrane proteins by microLC-MS/MS resulted in the identification of all the predicted tryptic peptides of bacteriorhodopsin, including those that are known to be post-translationally modified. In addition, 40 proteins from the purple membrane preparation were also identified, of which 80% are predicted to contain between 1 and 16 transmembrane domains. To evaluate the general applicability of the method, the same extraction, solubilization, and digestion conditions were applied to a plasma membrane fraction prepared from human epidermal sheets. A total of 117 proteins was identified in a single microLC-MS/MS analysis, of which 55% are known to be integral or associated with the plasma membrane. Due to its simplicity, efficiency, and absence of MS interfering compounds, this technique can be used for the characterization of other integral membrane proteins and may be concomitantly applied for the analysis of membrane protein complexes or large-scale proteomic studies of different membrane samples.

Bacteriorhodopsins↗
PubMed2004

Profiling the activity of G proteins in patient-derived tissues by rapid affinity-capture of signal transduction proteins (GRASP).

The next phase in molecular medicine will require the ability to identify signal transduction events inside a cell, in the biologic context of the disease-host interface and at a given point in time. New technologies are needed to profile the activity of these signaling pathways in patient tissue rather than cultured cell lines since the tumor-host microenvironment influences the cellular proteome. We introduce such a technology, rapid affinity capture of signaling proteins (GRASP), to investigate the activity of signaling pathways from patient-derived carcinomas and benign epithelial surfaces and apply it to studying important signaling events in ovarian carcinoma. During the progression from benign ovarian epithelium to invasive carcinoma, there is loss of repression of Rho A as evidenced by its dissociation from its inhibitor, Rho Guanine Nucleotide Dissociation Inhibitor (RhoGDI). GRASP is more informative than simply profiling transcript or protein levels. Furthermore, GRASP coupled with mass spectrometry allowed us to identify a protein-binding partner of RhoGDI, demonstrating the power of this technology in the discovery of potentially novel protein-protein interactions. GRASP represents an advance in the field of proteomics as it detects protein interactions present in cells as they exist in their native tissue microenvironment.

Carcinoma↗