Biomedical subjects

Timothy D Veenstra

Publications and source records attributed to Timothy D Veenstra.

At least 37 records · Page 2Linked to original sources

PubMed2006

Identification of the SELDI ProteinChip human serum retentate by microcapillary liquid chromatography-tandem mass spectrometry.

Surface-enhanced laser desorption/ionization (SELDI) time-of-flight (TOF) mass spectrometry (MS) has been widely applied for conducting biomarker research with the goal of discovering patterns of proteins and/or peptides from biological samples that reflect disease status. Many diseases, ranging from cancers of the colon, breast, and prostate to Alzheimer's disease, have been studied through serum protein profiling using SELDI-based methods. Although the results from SELDI-based diagnostic studies have generated a great deal of excitement and skepticism alike, the basis of the molecular identities of the features that underpin the diagnostic potential of the mass spectra is still largely unexplored. A detailed investigation has been undertaken to identify the compliment of serum proteins that bind to the commonly used weak cation exchange (WCX-2) SELDI protein chip. Following incubation and washing of a standard serum sample on the WCX-2 sorbent, proteins were harvested, digested with trypsin, fractionated by strong cation exchange liquid chromatography (LC), and subsequently analyzed by microcapillary reversed-phase LC coupled online with an ion-trap mass spectrometer. This analysis resulted in the identification of 383 unique proteins in the WCX-2 serum retentate. Among the proteins identified, 50 (13%) are documented clinical biomarkers with 36 of these (72%) identified from multiple peptides.

Biomarkers↗

PubMed2006

Proteomic analysis of plasma membrane from hypoxia-adapted malignant melanoma.

Hypoxic conditions often persist within poorly vascularized tumors. At the cellular level constitutive activation of transcriptional regulators of the hypoxic response leads to the emergence of clones with aggressive phenotypes. The primary interface between the cell and the hypoxic environment is the plasma membrane. A detailed investigation of this organelle is expected to yield further targets for therapeutic perturbation of the response to hypoxia. In the present study, quantitative proteomic analysis of plasma membrane from hypoxia-adapted murine B16F10 melanoma was performed using differential 16O/18O stable isotopic labeling and multidimensional liquid chromatography-tandem mass spectrometry. The analysis resulted in the identification of 24,853 tryptic peptides, providing quantitative information for 2,433 proteins. For a subset of plasma membrane and secreted proteins, quantitative RT-PCR was used to gain further insight into the genomic regulatory events underlying the response to hypoxia. Consistent increases at the proteomic and transcriptomic levels were observed for aminopeptidase N (CD13), carbonic anhydrase IX, potassium-transporting ATPase, matrix metalloproteinase 9, and stromal cell derived factor I (SDF-1). Antibody-based analysis of a panel of human melanoma cell lines confirmed that CD13 and SDF-1 were consistently upregulated during hypoxia. This study provides the basis for the discovery of novel hypoxia-induced membrane proteins.

Amino Acid Sequence↗

PubMed2006

Mass spectrometry reveals specific and global molecular transformations during viral infection.

Mass spectrometry analysis was used to target three different aspects of the viral infection process: the expression kinetics of viral proteins, changes in the expression levels of cellular proteins, and the changes in cellular metabolites in response to viral infection. The combination of these methods represents a new, more comprehensive approach to the study of viral infection revealing the complexity of these events within the infected cell. The proteins associated with measles virus (MV) infection of human HeLa cells were measured using a label-free approach. On the other hand, the regulation of cellular and Flock House Virus (FHV) proteins in response to FHV infection of Drosophila cells was monitored using stable isotope labeling. Three complementary techniques were used to monitor changes in viral protein expression in the cell and host protein expression. A total of 1500 host proteins was identified and quantified, of which over 200 proteins were either up- or down-regulated in response to viral infection, such as the up-regulation of the Drosophila apoptotic croquemort protein, and the down-regulation of proteins that inhibited cell death. These analyses also demonstrated the up-regulation of viral proteins functioning in replication, inhibition of RNA interference, viral assembly, and RNA encapsidation. Over 1000 unique metabolites were also observed with significant changes in over 30, such as the down-regulated cellular phospholipids possibly reflecting the initial events in cell death and viral release. Overall, the cellular transformation that occurs upon viral infection is a process involving hundreds of proteins and metabolites, many of which are structurally and functionally uncharacterized.

Animals↗

PubMed2006

Increased serum levels of complement C3a anaphylatoxin indicate the presence of colorectal tumors.

BACKGROUND & AIMS: Late diagnosis of colorectal carcinoma results in a significant reduction of average survival times. Yet despite screening programs, about 70% of tumors are detected at advanced stages (International Union Against Cancer stages III/IV). We explored whether detection of malignant disease would be possible through identification of tumor-specific protein biomarkers in serum samples. METHODS: A discovery set of sera from patients with colorectal malignancy (n = 58) and healthy control individuals (n = 32) were screened for potential differences using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. Candidate proteins were identified and their expression levels were validated in independent sample sets using a specific immunoassay (enzyme-linked immunosorbent assay). RESULTS: By using class comparison and custom-developed algorithms we identified several m/z values that were expressed differentially between the malignant samples and the healthy controls of the discovery set. Characterization of the most prominent m/z values revealed a member of the complement system, the stable form of C3a anaphylatoxin (ie, C3a-desArg). Based on a specific enzyme-linked immunosorbent assay, serum levels of complement C3a-desArg predicted the presence of colorectal malignancy in a blinded validation set (n = 59) with a sensitivity of 96.8% and a specificity of 96.2%. Increased serum levels were also detected in 86.1% of independently collected sera from patients with colorectal adenomas (n = 36), whereas only 5.6% were classified as normal. CONCLUSIONS: Complement C3a-desArg is present at significantly higher levels in serum from patients with colorectal adenomas (P < .0001) and carcinomas (P < .0001) than in healthy individuals. This suggests that quantification of C3a-desArg levels could ameliorate existing screening tests for colorectal cancer.

Adenoma↗

PubMed2006

Proteomic and biochemical analysis of purified human immunodeficiency virus type 1 produced from infected monocyte-derived macrophages.

Human immunodeficiency virus type 1 (HIV-1) infects CD4(+) T lymphocytes and monocytes/macrophages, incorporating host proteins in the process of assembly and budding. Analysis of the host cell proteins incorporated into virions can provide insights into viral biology. We characterized proteins in highly purified HIV-1 virions produced from human monocyte-derived macrophages (MDM), within which virus buds predominantly into intracytoplasmic vesicles, in contrast to the plasmalemmal budding of HIV-1 typically seen with infected T cells. Liquid chromatography-linked tandem mass spectrometry of highly purified virions identified many cellular proteins, including 33 previously described proteins in HIV-1 preparations from other cell types. Proteins involved in many different cellular structures and functions were present, including those from the cytoskeleton, adhesion, signaling, intracellular trafficking, chaperone, metabolic, ubiquitin/proteasomal, and immune response systems. We also identified annexins, annexin-binding proteins, Rab proteins, and other proteins involved in membrane organization, vesicular trafficking, and late endosomal function, as well as apolipoprotein E, which participates in cholesterol transport, immunoregulation, and modulation of cell growth and differentiation. Several tetraspanins, markers of the late endosomal compartment, were also identified. MDM-derived HIV contained 26 of 37 proteins previously found in exosomes, consistent with the idea that HIV uses the late endosome/multivesicular body pathway during virion budding from macrophages.

Cell Line↗

PubMed2006

Proteomic profiling of differentiating osteoblasts.

The major event that triggers osteogenesis is the transition of mesenchymal stem cells into bone-forming, differentiating osteoblast cells. Osteoblast differentiation is the primary event of bone formation, exemplified by the synthesis, deposition and mineralization of extracellular matrix. Osteoblast differentiation is controlled tightly by sequential activation of diverse transcription factors that regulate the expression of specific genes. The spatial and temporal regulation of the differentiation process is not completely understood at the cellular or molecular level. Recent advances in mass spectrometry-based proteomics have allowed for the systematic qualitative and quantitative profiling of differentiating osteoblasts, enabling a better understanding of the multiple factors and signaling events that control the differentiation process at a molecular level. This review focuses on recent developments in the proteomic analysis of differentiating osteoblasts, including advances, challenges and future prospects of using mass spectrometry to investigate the local and systemic factors regulating bone formation and its homeostasis.

Animals↗

PubMed2006

Multiplexed immunofluorescence microscopy for the interrogation of cellular protein complexes.

Knowing that a specific protein is present within a cell provides little insight into its function. In a study by Schubert and colleagues, the investigators present a multidimensional method that utilizes fluorescence microscopy and automated antibody introduction and detection, which is potentially capable of localizing hundreds of proteins within individual cells. The method, referred to as multiepitope-ligand cartography, is validated in the analysis of cell-surface receptors in peripheral mononuclear blood cells, and then used to map protein complexes in a series of disease models, including psoriasis and chronic constriction injury. Within each experiment, the locales of each protein are presented in a binary format and the data are interpreted to recognize specific proteins that control the topology of the protein network. The hope is that by identifying partnerships between proteins and those proteins that are most responsible for these interactions, novel diagnostic features and therapeutic targets can be established.

Comment↗

PubMed2006

Sampling and analytical strategies for biomarker discovery using mass spectrometry.

There is an often unspoken truth behind the course of scientific investigation that involves not what is necessarily academically worthy of study, but rather what is scientifically worthy in the eyes of funding agencies. The perception of worthy research is, as cost is driven in the simplest sense in economics, often driven by demand. Presently, the demand for novel diagnostic and therapeutic protein biomarkers that possess high sensitivity and specificity is placing major impact on the field of proteomics. The focal discovery technology that is being relied on is mass spectrometry (MS), whereas the challenge of biomarker discovery often lies not in the application of MS but in the underlying proteome sampling and bioinformatic processing strategies. Although biomarker discovery research has been historically technology-driven, it is clear from the meager success in generating validated biomarkers that increasing attention must be placed at the pre-analytic stage, such as sample retrieval and preparation. As diseases vary, so do the combinations of sampling and sample analyses necessary to discover novel biomarkers. In this review, we highlight different strategies used toward biomarker discovery and discuss them in terms of their reliance on technology and methodology.

Biomarkers↗

PubMed2006

Plasma proteomic profiling: search for lung cancer diagnostic and early detection markers.

Environmental and occupational exposure to asbestos is among the established risk factors for lung cancer, the leading cause of cancer-related deaths in the United States. This link between exposure to asbestos and the excessive death rate from lung cancer was evident in a study of former workers of an asbestos pipe insulation manufacturing plant in Tyler, TX. We performed comparative proteomic profiling of plasma samples that were collected from nine patients within 12 months before death and their age-, race- and exposure-matched disease-free controls on strong anion exchange chips using surface-enhanced laser desorption ionization time-of-flight mass spectrometry. A distance-dependent K-nearest neighbor (KNN) classification algorithm identified spectral features of m/z values 7558.9 and 15103.0 that were able to distinguish lung cancer patients from disease-free individuals with high sensitivity and specificity. The high correlation between the intensities of these two peaks (r=0.987) strongly suggests that they are the doubly and singly charged ions of the same protein product. Examination of these proteomic markers in the plasma samples of subjects from >5 years before death from lung cancer suggested that they are related to the early development of lung cancer. Validation of these biomarkers would have significant implications for the early detection of lung cancer and better management of high-risk patients.

Adult↗

PubMed2005

A transcription inhibitor, actinomycin D, enhances HIV-1 replication through an interleukin-6-dependent pathway.

We previously demonstrated that Actinomycin D (ActD) enhanced HIV-1 replication in the MT-2 cell, a human T-cell leukemia virus type-1-infected cell line. The MT-2 cell is known to produce multiple cytokines spontaneously. In this study, we investigated the impact of ActD on the cytokine production from MT-2 cells and HIV-1 replication in a latently infected cell line, U1. MT-2 cells were pulse-treated with 0 or 200 nM of ActD, and culture supernatants were collected 3 days after incubation. Supernatants from untreated cells (Sup0) induced HIV-1 replication by 150-fold in U1 cells. Culture supernatants from ActD-treated cells (Sup200) enhanced HIV-1 replication by 1200-fold. A combination of a sequential chromatographic approach and mass spectrometric analysis identified that the HIV-inducing factors in Sup200 were interleukin (IL)-6 and tumor necrosis factor (TNF)-beta. Quantitative analysis revealed that ActD treatment increased the concentration of IL-6 in Sup200 by 600% compared with that in Sup0 but decreased the amount of TNFbeta in Sup200 by 85%. Northern blot analysis showed that ActD treatment increased IL-6 transcripts; however, no change was seen in TNFbeta transcripts. These results suggest that ActD induces replication of HIV-1 through modulation of cytokine production.

Blotting, Northern↗

PubMed2005

Activation-induced deaminase cloning, localization, and protein extraction from young VH-mutant rabbit appendix.

Studies in mouse, human, and chicken suggest that activation-induced deaminase (AID) is involved in three known processes leading to antibody diversification: somatic hypermutation, gene conversion, and class-switch recombination. Developing rabbit appendix provides a particularly good site for studying all three of these B cell maturation events. We report here successful cloning of rabbit AID and isolation of AID protein from rabbit appendix-cell nuclear and cytoplasmic extracts. We succeeded in identifying and locating AID protein in cells by immunohistochemical and immunofluorescent staining techniques and examined colocalization of AID and other molecules important for Ab diversification. This report extends our knowledge about AID to a mammalian species that uses gene conversion to diversify rearranged Ig genes. Although much work remains to understand fully the mechanism of action of AID and its association with other cellular components, the rabbit system now offers a particularly useful model for future studies of these dynamics.

Amino Acid Sequence↗

PubMed2005

Deamidation of peptides in aerobic nitric oxide solution by a nitrosative pathway.

Hydrolytic deamidation of asparagine (Asn) and glutamine (Gln) residues to aspartate (Asp) and glutamate (Glu), respectively, can have significant biological consequences. We hypothesize that a wholly different mechanism of deamidation might occur in the presence of aerobic nitric oxide (NO). Accordingly, we examined the deamidating ability of aerobic NO toward three model peptides, 2,4-dinitrophenyl (DNP)-Pro-Gln-Gly, Lys-Trp-Asp-Asn-Gln, and Ser-Glu-Asn-Tyr-Pro-Ile-Val, incubating them with the NO-generating compound, Et(2)N[N(O)NO]Na (DEA/NO, 30-48 mM), in aerobic, pH 7.4, buffer at 37 degrees C. DNP-Pro-Glu-Gly was detected after 2 h, while Lys-Trp-Asp-Asp-Gln, Lys-Trp-Asp-Asn-Glu, and Ser-Glu-Asp-Tyr-Pro-Ile-Val were detected within 10 min, accumulating in apparent yields of up to approximately 10%. In the latter case, tyrosine nitration was also observed, producing the expected nitrotyrosine residue. DEA/NO solutions preincubated to exhaust the NO before the peptides were added did not induce detectable deamidation. The data demonstrate that aerobic NO exposures can lead to nitrosative deamidation of peptides, a pathway that differs from the established hydrolytic deamidation mechanism in several key respects: the by-products of the former are molecular nitrogen and an acid (HONO) while that of the latter is a base (NH(3)); the nitrosative path can in principle proceed in the absence of water molecules; Asn is much more easily deamidated than Gln in the hydrolytic pathway, while the two amino acid residues were deamidated to a similar extent by exposure to NO in the presence of oxygen.

Amino Acids↗

PubMed2005

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

A sensitive, specific, accurate, and precise high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry method for measuring the absolute quantities of 15 endogenous estrogens and their metabolites in human urine has been developed and validated. The method requires a single hydrolysis/extraction/derivatization step and only 0.5 mL of urine, yet is capable of simultaneously quantifying estrone and its 2-, 4-methoxy and 2-, 4-, and 16alpha-hydroxy derivatives, and 2-hydroxyestrone-3-methyl ether; estradiol and its 2-, 4-methoxy and 2-, 16alpha-hydroxy derivatives, 16-epiestriol, 17-epiestriol, and 16-ketoestradiol in pre- and postmenopausal women as well as men. Standard curves are linear over a 10(3)-fold concentration range with the standard error of the estimate (SEE) and the relative standard error of the estimate (RSEE) for the linear regression line ranging from 0.0131 to 0.1760 and 1.2 to 7.3%, respectively. The lower limit of quantitation for each estrogen is 0.02 ng/0.5 mL urine sample (2 pg on column), with the percent recovery of a known added amount of compound (accuracy) of 96-107% and an overall precision, including the hydrolysis, extraction, and derivatization steps, of 1-5% relative standard deviation (RSD) for samples prepared concurrently and 1-12% RSD for samples prepared in separate batches. Since individual patterns of estrogen metabolism may influence the risk of breast cancer, accurate, precise, and specific measurement of endogenous estrogen metabolites in biological matrixes will facilitate future research on breast cancer prevention, screening, and treatment.

Adult↗

PubMed2005

Analysis of mass spectral serum profiles for biomarker selection.

MOTIVATION: Mass spectrometric profiles of peptides and proteins obtained by current technologies are characterized by complex spectra, high dimensionality and substantial noise. These characteristics generate challenges in the discovery of proteins and protein-profiles that distinguish disease states, e.g. cancer patients from healthy individuals. We present low-level methods for the processing of mass spectral data and a machine learning method that combines support vector machines, with particle swarm optimization for biomarker selection. RESULTS: The proposed method identified mass points that achieved high prediction accuracy in distinguishing liver cancer patients from healthy individuals in SELDI-QqTOF profiles of serum. AVAILABILITY: MATLAB scripts to implement the methods described in this paper are available from the HWR's lab website http://lombardi.georgetown.edu/labpage

Algorithms↗

PubMed2005

Pooled ORF expression technology (POET): using proteomics to screen pools of open reading frames for protein expression.

We have developed a pooled ORF expression technology, POET, that uses recombinational cloning and proteomic methods (two-dimensional gel electrophoresis and mass spectrometry) to identify ORFs that when expressed are likely to yield high levels of soluble, purified protein. Because the method works on pools of ORFs, the procedures needed to subclone, express, purify, and assay protein expression for hundreds of clones are greatly simplified. Small scale expression and purification of 12 positive clones identified by POET from a pool of 688 Caenorhabditis elegans ORFs expressed in Escherichia coli yielded on average 6 times as much protein as 12 negative clones. Larger scale expression and purification of six of the positive clones yielded 47-374 mg of purified protein/liter. Using POET, pools of ORFs can be constructed, and the pools of the resulting proteins can be analyzed and manipulated to rapidly acquire information about the attributes of hundreds of proteins simultaneously.

Animals↗

PubMed2005

Analysis of albumin-associated peptides and proteins from ovarian cancer patients.

BACKGROUND: Albumin binds low-molecular-weight molecules, including proteins and peptides, which then acquire its longer half-life, thereby protecting the bound species from kidney clearance. We developed an experimental method to isolate albumin in its native state and to then identify [mass spectrometry (MS) sequencing] the corresponding bound low-molecular-weight molecules. We used this method to analyze pooled sera from a human disease study set (high-risk persons without cancer, n = 40; stage I ovarian cancer, n = 30; stage III ovarian cancer, n = 40) to demonstrate the feasibility of this approach as a discovery method. METHODS: Albumin was isolated by solid-phase affinity capture under native binding and washing conditions. Captured albumin-associated proteins and peptides were separated by gel electrophoresis and subjected to iterative MS sequencing by microcapillary reversed-phase tandem MS. Selected albumin-bound protein fragments were confirmed in human sera by Western blotting and immunocompetition. RESULTS: In total, 1208 individual protein sequences were predicted from all 3 pools. The predicted sequences were largely fragments derived from proteins with diverse biological functions. More than one third of these fragments were identified by multiple peptide sequences, and more than one half of the identified species were in vivo cleavage products of parent proteins. An estimated 700 serum peptides or proteins were predicted that had not been reported in previous serum databases. Several proteolytic fragments of larger molecules that may be cancer-related were confirmed immunologically in blood by Western blotting and peptide immunocompetition. BRCA2, a 390-kDa low-abundance nuclear protein linked to cancer susceptibility, was represented in sera as a series of specific fragments bound to albumin. CONCLUSION: Carrier-protein harvesting provides a rich source of candidate peptides and proteins with potential diverse tissue and cellular origins that may reflect important disease-related information.

Albumins↗

PubMed2005

Proteomic analysis of formalin-fixed prostate cancer tissue.

Proteomic analysis of formalin-fixed paraffin-embedded (FFPE) tissue would enable retrospective biomarker investigations of this vast archive of pathologically characterized clinical samples that exist worldwide. These FFPE tissues are, however, refractory to proteomic investigations utilizing many state of the art methodologies largely due to the high level of covalently cross-linked proteins arising from formalin fixation. A novel tissue microdissection technique has been developed and combined with a method to extract soluble peptides directly from FFPE tissue for mass spectral analysis of prostate cancer (PCa) and benign prostate hyperplasia (BPH). Hundreds of proteins from PCa and BPH tissue were identified, including several known PCa markers such as prostate-specific antigen, prostatic acid phosphatase, and macrophage inhibitory cytokine-1. Quantitative proteomic profiling utilizing stable isotope labeling confirmed similar expression levels of prostate-specific antigen and prostatic acid phosphatase in BPH and PCa cells, whereas the expression of macrophage inhibitory cytokine-1 was found to be greater in PCa as compared with BPH cells.

Amino Acid Sequence↗

PubMed2005

A combined proteome and microarray investigation of inorganic phosphate-induced pre-osteoblast cells.

Inorganic phosphate, which is generated during osteoblast differentiation and mineralization, has recently been identified as an important signaling molecule capable of altering signal transduction pathways and gene expression. A large scale quantitative proteomic investigation of pre-osteoblasts stimulated with inorganic phosphate for 24 h resulted in the identification of 2501 proteins, of which 410 (16%) had an altered abundance ratio of greater than or equal to 1.75-fold, either up or down, revealing both novel and previously defined osteoblast-regulated proteins. A pathway/function analysis of these proteins revealed an increase in cell cycle and proliferation that was subsequently verified by conventional biochemical means. To further analyze the mechanisms by which inorganic phosphate regulates cellular protein levels, we undertook a mRNA microarray analysis of pre-osteoblast cells at 18, 21, and 24 h after inorganic phosphate exposure. Comparison of the mRNA microarray data with the 24-hour quantitative proteomic data resulted in a generally weak overall correlation; the 21-hour RNA sample showed the highest correlation to the proteomic data. However, an analysis of osteoblast relevant proteins revealed a much higher correlation at all time points. A comparison of the microarray and proteomic datasets allowed for the identification of a number of candidate proteins that are post-transcriptionally regulated by elevated inorganic phosphate, including Fra-1, a member of the activator protein-1 family of transcription factors. The analysis of the data presented here not only sheds new light on the important roles of inorganic phosphate in osteoblast function but also begins to address the contribution of post-transcriptional and post-translational regulation to a cell's expressed proteome. The ability to accurately measure changes in both protein abundance and mRNA levels on a system-wide scale represents a novel means to extract data from previously one-dimensional datasets.

3T3 Cells↗