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Timothy D Veenstra

Publications and source records attributed to Timothy D Veenstra.

At least 91 records · Page 5Linked to original sources
PubMed2004

Organic solvent extraction of proteins and peptides from serum as an effective sample preparation for detection and identification of biomarkers by mass spectrometry.

A method to extract peptides and low molecular weight proteins from serum under denaturing conditions using acetonitrile containing 0.1% trifluoroacetic acid has been developed. The extraction procedure precipitates large, abundant proteins to simplify subsequent mass spectral analysis. This sample preparation method provides an efficient way to extract serum peptides, enabling them to be compared and identified using different mass spectrometry approaches. Surface-enhanced laser desorption/ionization-time of flight mass spectrometry analysis of mouse blood serum samples prepared by this method allowed detection of two markers which were significantly reduced in mice with B cell lymphoma tumor. One of these markers has been identified as apolipoprotein A-II.

Acetonitriles↗
PubMed2004

Lessons from Kitty Hawk: from feasibility to routine clinical use for the field of proteomic pattern diagnostics.

Proteomic pattern diagnostics is a rapidly evolving field of science. Despite the increasingly large number of laboratories reporting exciting success with this concept, recent speculation concerning reproducibility and the nature and identities of the information content of the pattern constituents have served to defocus and polarize the community. These controversies will be rendered obsolete as the field accelerates into a new realm of clinical diagnostics. This new era will see the currently dry biomarker pipeline flooded with new candidate molecules, and the mass spectrometer will continue its maturation into a dominant clinical platform. We reflect on the important lessons gleaned from the Wright brothers' attempts at controlled heavier-than-air flight as a model for perseverance and a view to the very near future for proteomic pattern diagnostics.

Biomarkers↗
PubMed2004

Identification of oxidized mitochondrial proteins in alcohol-exposed human hepatoma cells and mouse liver.

Heavy alcohol consumption can damage various cells and organs partly through production of reactive oxygen species (ROS) and mitochondrial dysfunction. Treatment with antioxidants can significantly reduce the degree of damage. Despite well established roles of ROS in alcohol-induced cell injury, the proteins that are selectively oxidized by ROS are poorly characterized. We hypothesized that certain cysteinyl residues of target proteins are oxidized by ROS upon alcohol exposure, and these modified proteins may play roles in mitochondrial dysfunction. A targeted proteomics approach utilizing biotin-N-maleimide (biotin-NM) as a specific probe to label oxidized cysteinyl residues was employed to investigate which mitochondrial proteins are modified during and after alcohol exposure. Human hepatoma HepG2 cells with transduced CYP2E1 (E47 cells) were used as a model to generate ROS through CYP2E1-mediated ethanol metabolism. Following exposure to 100 mM ethanol for 4 and 8 h, the biotin-NM-labeled oxidized proteins were purified with agarose coupled to either streptavidin or monoclonal antibody against biotin. The purified proteins were resolved by two-dimensional gel electrophoresis and protein spots that displayed differential abundances were excised from the gel, in-gel digested with trypsin and analyzed for identity utilizing either matrix-assisted laser desorption-time of flight mass spectrometry or microcapillary reversed-phase liquid chromatography-tandem mass spectrometry. The results demonstrate that heat shock protein 60, protein disulfide isomerase, mitochondrial aldehyde dehydrogenases, prohibitin, and other proteins were oxidized after alcohol exposure. The identity of some of the proteins purified with streptavidin-agarose was also confirmed by immunoblot analyses using the specific antibody to each target protein. This method was also used to identify oxidized mitochondrial proteins in the alcohol-fed mouse liver. These results suggest that exposure to ethanol causes oxidation of various mitochondrial proteins that may negatively affect their function and contribute to alcohol-induced mitochondrial dysfunction and cellular injury.

Alcohol Drinking↗
PubMed2004

Proteomic patterns as a diagnostic tool for early-stage cancer: a review of its progress to a clinically relevant tool.

The pace of development in novel technologies that promise improvements in the early diagnosis of disease is truly impressive. One such technology at the forefront of this revolution is mass spectrometry. New capabilities in mass spectrometry have provided the means for the development of proteomics, and the race is on to find innovative ways to apply this powerful technology to solving the problems faced in clinical medicine. One area that has garnered much attention over the past few years is the use of mass spectral patterns for cancer diagnostics. The use of these so-called 'proteomic patterns' for disease diagnosis relies fundamentally on the pattern of signals observed within a mass spectrum rather than the more conventional identification and quantitation of a biomarker such as in the case of cancer antigen-125- or prostate-specific antigen. The inherent throughput of proteomic pattern technology enables the analysis of hundreds of clinical samples per day. Currently, there are two primary means by which proteomic patterns can be acquired, surface-enhanced laser desorption/ionization (SELDI) and an electrospray ionization (ESI) method that has been popularized under the name, OvaCheck. In this review, an historical perspective on the development of proteomic patterns for the diagnosis of early-stage cancers is described. In addition, a critical assessment of the overall technology is presented with an emphasis on the steps required to enable proteomic pattern analysis to become a viable clinical tool for diagnosing early-stage cancers.

Early Diagnosis↗
PubMed2004

Serum proteomic profiling can discriminate prostate cancer from benign prostates in men with total prostate specific antigen levels between 2.5 and 15.0 ng/ml.

PURPOSE: Artificial intelligence based pattern recognition algorithms have been developed and successfully used to analyze complex serum proteomic data streams generated by surface enhanced, laser desorption ionization time-of-flight mass spectroscopy. In the current study we used a high performance, hybrid quadrupole time-of-flight mass spectrometer to generate discriminatory serum proteomic profiles to determine if this technology could be used to determine the need for prostate biopsy in men with elevated prostate specific antigen (PSA). MATERIALS AND METHODS: Serum samples were collected from 154 men with serum PSA 2.5 to 15.0 ng/ml and/or abnormal digital rectal examination prior to transrectal ultrasound guided biopsy. Serum samples were applied to WCX2 (weak cation exchange protein chip) Protein Arrays (Ciphergen Biosystems, Fremont, California) by a Biomek 2000 robotic liquid handler (Beckman-Coulter, Chaska, Minnesota) and low molecular weight (less than 20 kDa) proteomic patterns were generated with an API QSTAR Pulsar i LC/MS/MS System (Applied Biosystems, Framingham, Massachusetts). High resolution mass spectra were analyzed with a pattern recognition bioinformatics tool, that is Proteome Quest beta version 1.0 (Correlogic Systems, Inc., Bethesda, Maryland), in an attempt to identify and discover key discriminating ion signatures. Serum samples from 63 men (2 or more negative prostate biopsies in 23, 1 negative biopsy in 10 and biopsy detected prostate cancer [CaP] in 30) were used to train the diagnostic algorithm. The remaining 91 samples, including 28 of prostate cancer and 63 of 1 or more negative biopsies, were analyzed in blinded fashion. RESULTS: The most discriminatory model was found using the WCX2 chip. Testing the remaining 91 men with this model yielded 100% sensitivity and 67% specificity. In other words, if the proteomic pattern had been used to determine the need for prostate biopsy in this cohort of men with PSA between 2.5 and 15.0 ng/ml, 67% (42 of 63) with negative biopsies would have avoided unnecessary biopsy, while no cancers would have been missed. CONCLUSIONS: Our data demonstrate that high resolution mass spectroscopy can generate serum proteomic patterns that discriminate men with elevated PSA due to benign processes from men with CaP even when PSA is within the diagnostic gray zone. We are currently expanding the testing set to determine the reliability of this new technology to decrease unnecessary prostate biopsies without compromising the detection of curable CaP.

Algorithms↗
PubMed2004

A proteomic characterization of the plasma membrane of human epidermis by high-throughput mass spectrometry.

Membrane proteins are responsible for many critical cellular functions and identifying cell surface proteins on different keratinocyte populations by proteomic approaches would improve our understanding of their biological function. The ability to characterize membrane proteins, however, has lagged behind that of soluble proteins both in terms of throughput and protein coverage. In this study, a membrane proteomic investigation of keratinocytes using a two-dimensional liquid chromatography (LC) tandem-mass spectrometry (MS/MS) approach that relies on a buffered methanol-based solubilization, and tryptic digestion of purified plasma membrane is described. A highly enriched plasma membrane fraction was prepared from newborn foreskins using sucrose gradient centrifugation, followed by a single-tube solubilization and tryptic digestion of membrane proteins. This digestate was fractionated by strong cation-exchange chromatography and analyzed using microcapillary reversed-phase LC-MS/MS. In a set of 1306 identified proteins, 866 had a gene ontology (GO) annotation for cellular component, and 496 of these annotated proteins (57.3%) were assigned as known integral membrane proteins or membrane-associated proteins. Included in the identification of a large number of aqueous insoluble integral membrane proteins were many known intercellular adhesion proteins and gap junction proteins. Furthermore, 121 proteins from cholesterol-rich plasma membrane domains (caveolar and lipid rafts) were identified.

Caveolae↗
PubMed2004

Clinical proteomics and biomarker discovery.

Early detection of disease generally provides much-improved outcomes by a definitive medical procedure or through lifestyle modification along with specific medical management strategies. For serum biomarkers, which are central to the diagnosis of many diseases, to become truly useful sentinels of pathogenesis, their sensitivity and specificity in both early detection and recurrence monitoring must be improved. Currently, the detection and monitoring of disease markers is based on solitary proteins, and this approach is not always reliable. New classes of biomarkers derived from mass spectroscopy analysis of the low molecular weight proteome have shown improved abilities in the early detection of disease and hence in patient risk stratification and outcome. The development of a modular platform technology with sufficient flexibility and design abstractions allowing for concurrent experimentation, test, and refinement will help speed the progress of mass spectroscopy-derived proteomic pattern-based diagnostics from the scientific laboratory to the medical clinic. For acceptance by scientists, physicians, and regulatory personnel, new bioinformatic tools are essential system components for data management, analysis, and intuitive display of these new and complex data. Clinically engineered mass spectroscopy systems are essential for the further development and validation of multiplexed biomarkers that have shown tremendous promise for the early detection of disease.

Biomarkers↗
PubMed2004

Characterization and quantitation of membrane proteomes using multidimensional MS-based proteomic technologies.

A major goal of proteomics is to develop methods that enable the systematic characterization of every protein within the cell or particular subcellular proteome using a single analytical platform. Although the equivalent has already been achieved in genomics, reaching this goal in proteomics represents a much greater challenge due to the wide dynamic range of protein expression, numerous post-translational modifications and remarkable physicochemical heterogeneity of proteins. A major analytical challenge has involved developing more effective means for proteome-scale investigations of membrane proteins, whose solubility differs drastically from that of cytoplasmic proteins. Fortunately, rapid progress has increased the ability to characterize this critically important class of proteins on a scale analogous to that of aqueous soluble proteins.

Cell Adhesion↗
PubMed2004

The utility of proteomic patterns for the diagnosis of cancer.

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 termed proteomic pattern analysis has emerged as an effective method for the early diagnosis of diseases such as ovarian, breast, and prostate cancer. Proteomic pattern analysis relies on the pattern of proteins observed and does not rely on the identification of a traceable biomarker. Utilizing this technology, hundreds of clinical samples per day can be analyzed with the potential to be a novel, highly sensitive diagnostic tool for the early detection of diseases or as a predictor of response to therapy.

Animals↗
PubMed2003

On-column sample enrichment for capillary electrophoresis sheathless electrospray ionization mass spectrometry: evaluation for peptide analysis and protein identification.

Although several designs have been advanced for coupling sample enrichment devices to a sheathless electrospray ionization-mass spectrometry (MS) interface on a capillary electrophoresis (CE) column, most of these approaches suffer from difficulties in fabrication, and the CE separation efficiency is degraded as a result of the presence of coupling sleeves. We have developed a design that offers significant improvements in terms of ease of fabrication, durability, and maintenance of the integrity of the CE-separated analyte zones. Capillaries with different inside and outside diameters were evaluated to optimize the performance of the CE-MS system, resulting in a mass limit of detection of 500 amol for tandem MS analysis of a standard peptide using a 20-microm-i.d. capillary. The improved design incorporates an efficient method to preconcentrate a sample directly within the CE capillary followed by its electrophoretic separation and detection using a true zero dead-volume sheathless CE-MS interface. Testing of this novel CE-MS system showed its ability to characterize proteomic samples such as protein digests, in-gel-digested proteins, and hydrophobic peptides as well as to quantitate ICAT-labeled peptides.

Amino Acid Sequence↗
PubMed2003

Protein phosphatase 2A positively regulates Ras signaling by dephosphorylating KSR1 and Raf-1 on critical 14-3-3 binding sites.

BACKGROUND: Kinase Suppressor of Ras (KSR) is a conserved component of the Ras pathway that acts as a molecular scaffold to facilitate signal transmission through the MAPK cascade. Although recruitment of KSR1 from the cytosol to the plasma membrane is required for its scaffolding function, the precise mechanism(s) regulating the translocation of KSR1 have not been fully elucidated. RESULTS: Using mass spectrometry to analyze the KSR1-scaffolding complex, we identify the serine/threonine protein phosphatase PP2A as a KSR1-associated protein and show that PP2A is a critical regulator of KSR1 activity. We find that the enzymatic core subunits of PP2A (PR65A and catalytic C) constitutively associate with the N-terminal domain of KSR1, whereas binding of the regulatory PR55B subunit is induced by growth factor treatment. Specific inhibition of PP2A activity prevents the growth factor-induced dephosphorylation event involved in the membrane recruitment of KSR1 and blocks the activation of KSR1-associated MEK and ERK. Moreover, we find that PP2A activity is required for activation of the Raf-1 kinase and that both Raf and KSR1 must be dephosphorylated by PP2A on critical regulatory 14-3-3 binding sites for KSR1 to promote MAPK pathway activation. CONCLUSIONS: These findings identify KSR1 as novel substrate of PP2A and demonstrate the inducible dephosphorylation of KSR1 in response to Ras pathway activation. Further, these results elucidate a common regulatory mechanism for KSR1 and Raf-1 whereby their localization and activity are modulated by the PP2A-mediated dephosphorylation of critical 14-3-3 binding sites.

14-3-3 Proteins↗
PubMed2003

Characterization of the low molecular weight human serum proteome.

Serum potentially carries an archive of important histological information whose determination could serve to improve early disease detection. The analysis of serum, however, is analytically challenging due to the high dynamic concentration range of constituent protein/peptide species, necessitating extensive fractionation prior to mass spectrometric analyses. The low molecular weight (LMW) serum proteome is that protein/peptide fraction from which high molecular weight proteins, such as albumin, immunoglobulins, transferrin, and lipoproteins, have been removed. This LMW fraction is made up of several classes of physiologically important proteins such as cytokines, chemokines, peptide hormones, as well as proteolytic fragments of larger proteins. Centrifugal ultrafiltration of serum was used to remove the large constituent proteins resulting in the enrichment of the LMW proteins/peptides. Because albumin is known to bind and transport small molecules and peptides within the circulatory system, the centrifugal ultrafiltration was conducted under solvent conditions effecting the disruption of protein-protein interactions. The LMW serum proteome sample was digested with trypsin, fractionated by strong cation exchange chromatography, and analyzed by microcapillary reversed-phase liquid chromatography coupled on-line with electrospray ionization tandem mass spectrometry. Analysis of the tandem mass spectra resulted in the identification of over 340 human serum proteins; however, not a single peptide from serum albumin was observed. The large number of proteins identified demonstrates the efficacy of this method for the removal of large abundant proteins and the enrichment of the LMW serum proteome.

Blood Proteins↗
PubMed2003

Development of a two-dimensional protein-peptide separation protocol for comprehensive proteome measurements.

We have developed an effective two-dimensional fractionation protocol of complex proteome mixtures that extends the ability to conduct more comprehensive proteome measurements. A sample containing intact proteins extracted from Saccharomyces cerevisiae was fractionated by liquid phase isoelectric focusing, followed by tryptic digestion and solid-phase extraction (SPE) clean-up and reversed-phase liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS-MS) of the resultant peptides. The clean-up step is designed to desalt the fractions and rid them of urea and ampholytes prior to analysis by LC-MS-MS. Fifty milligrams of protein were separated into 20 fractions by liquid-phase isoelectric focusing, spanning a pH range of 3-10. The effectiveness of the removal of ampholytes was monitored by capillary zone electrophoresis and LC-MS-MS. The ability to analyze all of the 20 fractions without any noticeable decrease in the separation efficiency demonstrates the overall effectiveness of the SPE clean-up step. The results show that the separation strategy is effective for high throughput characterization of proteins from complex proteomic mixtures.

Electrophoresis, Polyacrylamide Gel↗
PubMed2003

A sheathless nanoflow electrospray interface for on-line capillary electrophoresis mass spectrometry.

A novel, rugged capillary electrophoresis-electrospray ionization (CE-ESI) interface where the separation column, an electrical porous junction, and the spray tip are integrated on a single piece of a fused-silica capillary is described. ESI is accomplished by applying an electrical potential through an easily prepared porous junction across a 3-4-mm length of fused silica. A stable electrospray is produced at nanoflow rates generated in the capillary by electrophoretic and electroosmotic forces. The interface is particularly well suited for the detection of low-femtomole levels of proteins and peptides. The ruggedness of this interface was evident by the continuous operation of the same column for over a 2-week period with no detectable deterioration in separation or electrospray performance. The new interface was used for the LC-ESI-MS separation and analysis of peptides and proteins. Injection of 25 fmol of [Glu1]-fibrinopeptide B using the new device produced a CE-ESI-MS electropherogram with a signal-to-noise ratio of over 100 for this peptide.

Amino Acid Sequence↗
PubMed2003

The depletion of protein signals in metabonomics analysis with the WET-CPMG pulse sequence.

Nuclear magnetic resonance (NMR) spectroscopy is a powerful analytical tool capable of providing a comprehensive metabolic profile of biofluids such as urine, plasma, and serum. Unfortunately, when measuring serum and plasma, the high protein concentration can obscure the signals originating from low molecular weight metabolites. We evaluated the use of different parameters within the Carr-Purcell-Meiboom-Gill (CPMG) pulse train of fast spin-echoes to remove the macromolecular signal contribution in one-dimensional proton (1H) NMR spectra. Experimental parameters such as the refocusing delay in the CPMG pulse train, pulse miscalibration, and recycle time were examined to assess the ability to remove the protein signals from the spectrum without causing a deleterious effect on the signals originating from free, low molecular weight metabolites. The 1H-NMR spectra of a variety of serum samples spiked with 2'-deoxyadenosine were acquired using various acquisition parameters. Our results show that the delay used in the CPMG spin-echo and the combination of the acquisition pulse flip angle and recycle time are the two major factors affecting the observed metabolite signal amplitudes in the resulting 1H-NMR spectrum.

Body Fluids↗