Biomedical subjects

T D Veenstra

Publications and source records attributed to T D Veenstra.

At least 19 recordsLinked to original sources

PubMed2006

Cytokinesis regulator ECT2 changes its conformation through phosphorylation at Thr-341 in G2/M phase.

The Rho activator ECT2 functions as a key regulator in cytokinesis. ECT2 is phosphorylated during G2/M phase, but the physiological significance of this event is not well known. In this study, we show that phosphorylation of ECT2 at threonine-341 (T341) affects the autoregulatory mechanism of ECT2. In G2/M phase, ECT2 was phosphorylated at T341 most likely by Cyclin B/Cyclin-dependent kinase 1 (Cdk1), and then dephosphorylated before cytokinesis. Depletion of ECT2 by RNA interference (RNAi) efficiently induced multinucleate cells. Expression of the phospho-deficient mutant of ECT2 at T341 suppressed the multinucleation induced by RNAi to ECT2, indicating that ECT2 is biologically active even when it is not phosphorylated at T341. However, the phospho-mimic mutation at T341 weakly stimulates the catalytic activity of ECT2 as detected by serum response element reporter gene assays. As T341 is located at the hinge region of the N-terminal regulatory domain and C-terminal catalytic domain, phosphorylation of T341 may help accessing downstream signaling molecules to further activate ECT2. We found that the phospho-mimic mutation T341D increases binding with itself or the N-terminal half of ECT2. These results suggest a conformational change of ECT2 upon phosphorylation at T341. Therefore, ECT2 activity might be regulated by the phosphorylation status of T341. We propose that T341 phosphorylation by Cyclin B/Cdk1 could be a trigger for further activation of ECT2.

Amino Acid Sequence↗

PubMed2005

Low molecular weight proteomic information distinguishes metastatic from benign pheochromocytoma.

Metastatic lesions occur in up to 36% of patients with pheochromocytoma. Currently there is no way to reliably detect or predict which patients are at risk for metastatic pheochromocytoma. Thus, the discovery of biomarkers that could distinguish patients with benign disease from those with metastatic disease would be of great clinical value. Using surface-enhanced laser desorption ionization protein chips combined with high-resolution mass spectrometry, we tested the hypothesis that pheochromocytoma pathologic states can be reflected as biomarker information within the low molecular weight (LMW) region of the serum proteome. LMW protein profiles were generated from the serum of 67 pheochromocytoma patients from four institutions and analyzed by two different bioinformatics approaches employing pattern recognition algorithms to determine if the LMW component of the circulatory proteome contains potentially useful discriminatory information. Both approaches were able to identify combinations of LMW molecules which could distinguish all metastatic from all benign pheochromocytomas in a separate blinded validation set. In conclusion, for this study set low molecular mass biomarker information correlated with pheochromocytoma pathologic state using blinded validation. If confirmed in larger validation studies, efforts to identify the underlying diagnostic molecules by sequencing would be warranted. In the future, measurement of these biomarkers could be potentially used to improve the ability to identify patients with metastatic disease.

Adolescent↗

PubMed2004

High-resolution serum proteomic features for ovarian cancer detection.

Serum proteomic pattern diagnostics is an emerging paradigm employing low-resolution mass spectrometry (MS) to generate a set of biomarker classifiers. In the present study, we utilized a well-controlled ovarian cancer serum study set to compare the sensitivity and specificity of serum proteomic diagnostic patterns acquired using a high-resolution versus a low-resolution MS platform. In blinded testing sets, the high-resolution mass spectral data contained multiple diagnostic signatures that were superior to the low-resolution spectra in terms of sensitivity and specificity (P<0.00001) throughout the range of modeling conditions. Four mass spectral feature set patterns acquired from data obtained exclusively with the high-resolution mass spectrometer were 100% specific and sensitive in their diagnosis of serum samples as being acquired from either unaffected patients or those suffering from ovarian cancer. Important to the future of proteomic pattern diagnostics is the ability to recognize inferior spectra statistically, so that those resulting from a specific process error are recognized prior to their potentially incorrect (and damaging) diagnosis. To meet this need, we have developed a series of quality-assurance and in-process control procedures to (a) globally evaluate sources of sample variability, (b) identify outlying mass spectra, and (c) develop quality-control release specifications. From these quality-assurance and control (QA/QC) specifications, we identified 32 mass spectra out of the total 248 that showed statistically significant differences from the norm. Hence, 216 of the initial 248 high-resolution mass spectra were determined to be of high quality and were remodeled by pattern-recognition analysis. Again, we obtained four mass spectral feature set patterns that also exhibited 100% sensitivity and specificity in blinded validation tests (68/68 cancer: including 18/18 stage I, and 43/43 healthy). We conclude that (a) the use of high-resolution MS yields superior classification patterns as compared with those obtained with lower resolution instrumentation; (b) although the process error that we discovered did not have a deleterious impact on the present results obtained from proteomic pattern analysis, the major source of spectral variability emanated from mass spectral acquisition, and not bias at the clinical collection site; (c) this variability can be reduced and monitored through the use of QA/QC statistical procedures; (d) multiple and distinct proteomic patterns, comprising low molecular weight biomarkers, detected by high-resolution MS achieve accuracies surpassing individual biomarkers, warranting validation in a large clinical study.

Biomarkers, Tumor↗

PubMed2003

A comparison of microLC/electrospray ionization-MS and GC/MS for the measurement of stable isotope enrichment from a [2H2]-glucose metabolic probe in T-cell genomic DNA.

Measurement of the proliferation of lymphocytes and other high-turnover cell populations in vivo can be accomplished through the incorporation of an isotopically labeled DNA precursor into actively dividing cells and the subsequent determination of the isotope enrichment in the isolated genomic DNA from selected cell populations. Two published gas chromatography/mass spectrometry (GC/MS) methods were successfully modified by our laboratory whereby a postinjection methylation reaction, rather than silylation or acetylation, was used to form a volatile derivative of deoxyadenosine (dA). We also developed a second robust microcapillary liquid chromatography-electrospray ionization (microLC-ESI)/MS method that is faster and more sensitive than the GC/MS method and does not require sample derivatization. Following administration of [6,6-(2)H(2)]-glucose to human immunodeficiency virus-infected patients, peripheral blood was drawn; cells were obtained by lymphapheresis and fractionated. DNA was isolated from the desired cell subtypes and enzymatically hydrolyzed to the free deoxyribonucleosides. The digest was analyzed using both capillary GC/MS and microLC/ESI-MS to measure the levels of the dA and [(2)H(2)]-dA or their reaction products. Sample enrichments were calculated by comparison to standard curves prepared from dA and [(2)H(2)]-dA. The microLC/ESI-MS method required fewer cells, less sample preparation, shorter analysis times, and a single calibration curve. Overall, the microLC/ESI-MS method is superior to the GC/MS method in terms of precision and accuracy, while providing a 4-fold increase in sensitivity (from 20 pmol at 0.2% [(2)H(2)]-dA enrichment to 5 pmol at 0.1% [(2)H(2)]-dA enrichment).

Chromatography, Liquid↗

PubMed2001

Phosphoprotein isotope-coded affinity tag approach for isolating and quantitating phosphopeptides in proteome-wide analyses.

A method has been developed that utilizes phosphoprotein isotope-coded affinity tags (PhIAT) that combines stable isotope and biotin labeling to enrich and quantitatively measure differences in the O-phosphorylation states of proteins. The PhIAT labeling approach involves hydroxide ion-mediated beta-elimination of the O-phosphate moiety and the addition of 1,2-ethanedithiol containing either four alkyl hydrogens (EDT-D0) or four alkyl deuteriums (EDT-D4) followed by biotinylation of the EDT-D0/D4 moiety using (+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine. The PhIAT reagent, which contains the nucleophilic sulfhydryl and isotopic label covalently linked to a biotin moiety, was synthesized and has the potential utility to reduce the O-phosphorylation derivatization into a one-step process. The PhIAT labeling approach was initially demonstrated using the model phosphoprotein beta-casein. After proteolytic digestion, the PhIAT-labeled peptides were affinity isolated using immobilized avidin and analyzed using capillary reversed-phase liquid chromatography-mass spectrometry. PhIAT-labeled beta-casein peptides corresponding to peptides containing known sites of O-phosphorylation were isolated and identified. The PhIAT labeling method was also applied to a yeast protein extract. The PhIAT labeling technique provides a reliable method for making quantitative measurements of differences in the O-phosphorylation state of proteins.

Affinity Labels↗

PubMed2001

Quantitative analysis of bacterial and mammalian proteomes using a combination of cysteine affinity tags and 15N-metabolic labeling.

We describe the combined use of 15N-metabolic labeling and a cysteine-reactive biotin affinity tag to isolate and quantitate cysteine-containing polypeptides (Cys-polypeptides) from Deinococcus radiodurans as well as from mouse B16 melanoma cells. D. radiodurans were cultured in both natural isotopic abundance and 15N-enriched media. Equal numbers of cells from both cultures were combined and the soluble proteins extracted. This mixture of isotopically distinct proteins was derivatized using a commercially available cysteine-reactive reagent that contains a biotin group. Following trypsin digestion, the resulting modified peptides were isolated using immobilized avidin. The mixture was analyzed by capillary reversed-phase liquid chromatography (LC) online with ion trap mass spectrometry (MS) as well as Fourier transform ion cyclotron resonance (FTICR) MS. The resulting spectra contain numerous pairs of Cyspolypeptides whose mass difference corresponds to the number of nitrogen atoms present in each of the peptides. Designation of Cys-polypeptide pairs is also facilitated by the distinctive isotopic distribution of the 15N-labeled peptides versus their 14N-labeled counterparts. Studies with mouse B16 cells maintained in culture allowed the observation of hundreds of isotopically distinct pairs of peptides by LC-FTICR analysis. The ratios of the areas of the pairs of isotopically distinct peptides showed the expected 1:1 labeling of the 14N and 15N versions of each peptide. An additional benefit from the present strategy is that the 15N-labeled peptides do not display significant isotope-dependent chromatographic shifts from their 14N-labeled counterparts, therefore improving the precision for quantitating peptide abundances. The methodology presented offers an alternate, cost-effective strategy for conducting global, quantitative proteomic measurements.

Animals↗

PubMed2001

Packed capillary reversed-phase liquid chromatography with high-performance electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry for proteomics.

In this study, high-efficiency packed capillary reversed-phase liquid chromatography (RPLC) coupled on-line with high-performance Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has been investigated for the characterization of complex cellular proteolytic digests. Long capillary columns (80-cm) packed with small (3-micron) C18 bonded particles provided a total peak capacity of approximately 1000 for cellular proteolytic polypeptides when interfaced with an ESI-FTICR mass spectrometer under composition gradient conditions at a pressure of 10,000 psi. Large quantities of cellular proteolytic digests (e.g., 500 micrograms) could be loaded onto packed capillaries of 150-micron inner diameter without a significant loss of separation efficiency. Precolumns with suitable inner diameters were found useful for improving the elution reproducibility without a significant loss of separation quality. Porous particle packed capillaries were found to provide better results than those containing nonporous particles because of their higher sample capacity. Two-dimensional analyses from the combination of packed capillary RPLC with high-resolution FTICR yield a combined capacity for separations of > 1 million polypeptide components and simultaneously provided information for the identification of the separated components based upon the accurate mass tag concept previously described.

Chromatography, Liquid↗

PubMed2001

Design and performance of an ESI interface for selective external ion accumulation coupled to a Fourier transform ion cyclotron mass spectrometer.

The coupling of Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) with electrospray ionization has advanced the analysis of large biopolymers and provided the basis for high-throughput protein characterization (e.g., for rapid "proteome" analyses). In this work, the combination of high-performance capillary liquid chromatography with FTICR mass spectrometry and external ion accumulation has been shown to increase both sensitivity and analysis duty cycle. Instrument versatility is further improved by ion preselection followed by ion accumulation in an external linear quadrupole ion trap. The interface was tested with a 3.5-T FTICR mass spectrometer and evaluated with a number of peptides and proteins whose molecular weights ranged from 500 to 66000. A significant increase in the sensitivity, duty cycle, and dynamic range over that of the previously used accumulated trapping was achieved, exhibiting a detection limit of approximately 10 zmol (approximately 6000 molecules) for smaller proteins such as cytochrome c. Capillary LC external accumulation interface with FTICR was successfully applied for the study of whole-proteome mouse tryptic digests.

Animals↗

PubMed2001

Rapid quantitative measurements of proteomes by Fourier transform ion cyclotron resonance mass spectrometry.

The patterns of gene expression, post-translational modifications, protein/biomolecular interactions, and how these may be affected by changes in the environment, cannot be accurately predicted from DNA sequences. Approaches for proteome characterization are generally based upon mass spectrometric analysis of in-gel digested two dimensional polyacrylamide gel electrophoresis (2-D PAGE) separated proteins, allowing relatively rapid protein identification compared to conventional approaches. This technique, however, is constrained by the speed of the 2-D PAGE separations, the sensitivity limits intrinsic to staining necessary for protein visualization, the speed and sensitivity of subsequent mass spectrometric analyses for identification, and the limited ability for accurate quantitative measurements based on differences in spot intensity. We are presently developing alternative approaches for proteomics based upon the combination of fast capillary electrophoresis, or other suitable chromatographic separations, and the high mass accuracy and sensitivity obtainable with unique Fourier transform ion cyclotron resonance (FTICR) mass spectrometers available at our laboratory. Several approaches are presently being pursued; one based upon the analysis of intact proteins and the second upon approaches for global protein digestion and accurate peptide mass analysis. Quantitation of protein/peptide levels are based on using two or more stable-isotope labeled versions of proteomes which are combined to obtain precise quantitation of relative protein abundances. We describe the status of our efforts towards the development of a high-throughput proteomics capability and present initial results for application to several microorganisms and discuss our efforts for extending the developed capability to mammalian proteomes.

Bacterial Proteins↗

PubMed2001

RNA-RNA noncovalent interactions investigated by microspray ionization mass spectrometry.

Electrospray ionization mass spectrometry is playing an increasing role in the study of noncovalent interactions involving biomolecules. RNA-RNA complexes are important in many areas of biology, including RNA catalysis, RNA splicing, ribosome function, and gene regulation. Here, microelectrospray mass spectrometry (microESI-MS) is used to study noncovalent base-pairing interactions between RNA oligonucleotides, an area not previously explored by this technique. Using a set of complementary RNA oligonucleotides, we demonstrate the formation of the expected double-helical RNA complexes composed of three distinct oligonucleotides. The ability to study specific RNA noncovalent interactions by microESI-MS has the potential to provide a unique method by which to analyze and assign precise molecular masses to RNA-RNA complexes.

Base Sequence↗

PubMed2000

Utility of accurate mass tags for proteome-wide protein identification.

An enabling capability for proteomics would be the ability to study protein expression on a global scale. While several different separation and analysis options are being investigated to advance the practice of proteomics, mass spectrometry (MS) is rapidly becoming the core instrumental technology used to characterize the large number of proteins that constitute a proteome. To be most effective, proteomic measurements must be high-throughput, ideally allowing thousands of proteins to be identified on a time scale of hours. Most strategies of identification by MS rely on the analysis of enzymatically produced peptides originating from an isolated protein followed by either peptide mapping or tandem MS (MS/MS) to obtain sequence information for a single peptide. In the case of peptide mapping, several peptide masses are needed to unambiguously identify a protein with the typically achieved mass measurement accuracies (MMA). The ability to identify proteins based on the mass of a single peptide (i.e., an accurate mass tag; AMT) is proposed and is largely dependent on the MMA that can be achieved. To determine the MMA necessary to enable the use of AMTs for proteome-wide protein identification, we analyzed the predicted proteins and their tryptic fragments from Saccharomyces cerevisiae and Caenorhabditis elegans. The results show that low ppm (i.e., approximately 1 ppm) level measurements have practical utility for analysis of small proteomes. Additionally, up to 85% of the peptides predicted from these organisms can function as AMTs at sub-ppm MMA levels attainable using Fourier transform ion cyclotron resonance MS. Additional information, such as sequence constraints, should enable even more complex proteomes to be studied at more modest mass measurement accuracies. Once AMTs are established, subsequent high-throughput measurements of proteomes (e.g., after perturbations) will be greatly facilitated.

Cyclotrons↗

PubMed2000

Characterization of microorganisms and biomarker development from global ESI-MS/MS analyses of cell lysates.

The capability for sensitive and accurate identification of microorganisms has potential applications that include the monitoring of industrial bioprocessing operations, food safety analyses, disease diagnosis, and detection of potential biological hazards. Efforts based upon matrix-assisted laser desorption/ionization mass spectrometry to detect and identify specific microorganisms have been actively pursued for several years. We report a new method being developed to select useful biomarkers for the identification of microorganisms based upon electrospray ionization (ESI)-ion trap mass spectrometry. Crude cell lysates are processed using a recently developed dualmicrodialysis device and then directly infused into an ion trap MS. The low ESI flow rate and precursor ion accumulation capability of the ion trap MS enables high-sensitivity MS/MS analyses. Precursor ions are automatically selected and analyzed using tandem MS (MS/MS) to produce "global" MS/MS surveys and processed to yield two-dimensional MS/MS spectral displays. Such global MS/MS surveys are demonstrated for Escherichia coli lysates. The distinctive MS/MS spectral patterns can be used to identify mass spectrometric-detected species useful as biomarkers, which then provide a basis for confident microorganism identification. The results presented demonstrate the application of this method for the identification of microorganisms, as well as for detection of bacteriophage MS2 in the presence of a large excess of E. coli.

Biomarkers↗

PubMed2000

Stepwise mobilization of focused proteins in capillary isoelectric focusing mass spectrometry.

A stepwise mobilization strategy has been developed for the elution of complex protein mixtures, separated by capillary isoelectric focusing (CIEF) for detection using on-line electrospray ionization mass spectrometry (ESI-MS). Carrier polyampholytes are used to establish a pH gradient as well as to control the electroosmotic flow arising from the use of uncoated fused-silica capillaries. Elution of focused protein zones is achieved by controlling the mobilization pressure and voltage, leaving the remaining protein zones focused inside the capillary. Protein zones are stepwise eluted from the capillary by changing the mobilization conditions. Stepwise mobilization improves separation resolution and simplifies coupling with multistage MS (i.e., MSn) analysis since it allows more effective temporal control of protein elution from the CIEF capillary. We also describe a modified configuration for coupling CIEF with ESI-MS using a coaxial sheath flow interface that facilitate the automation of on-line CIEF-ESI-MS analyses. The stepwise mobilization strategy is demonstrated for the analysis of standard protein mixtures and soluble E. coli lysate proteins using CIEF-ESI-MS. These results indicate that inlet pressure or voltage programming to control the elution of the protein zones from the capillary (i.e., gradient mobilization) may allow for the optimization of the mobilization conditions and provide higher resolution for CIEF separation of complex mixtures with on-line MS.

Electrophoresis, Capillary↗

PubMed2000

Proteome analysis using selective incorporation of isotopically labeled amino acids.

A method is described for identifying intact proteins from genomic databases using a combination of accurate molecular mass measurements and partial amino acid content. An initial demonstration was conducted for proteins isolated from Escherichia coli (E. coli) using a multiple auxotrophic strain of K12. Proteins extracted from the organism grown in natural isotopic abundance minimal medium and also minimal medium containing isotopically labeled leucine (Leu-D10), 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 Leu residue has no effect on the CIEF separation of the protein, therefore both versions of the protein are observed within the same FTICR spectrum. The difference in the molecular mass of the natural isotopic abundance and Leu-D10 isotopically labeled proteins is used to determine the number of Leu residues present in that particular protein. Knowledge of the molecular mass and number of Leu residues present can be used to unambiguously identify the intact protein. Preliminary results show the efficacy of this method for unambiguously identifying proteins isolated from E. coli.

Amino Acids↗

PubMed1999

High-resolution capillary isoelectric focusing of complex protein mixtures from lysates of microorganisms.

High-resolution capillary isoelectric focusing separations of complex protein mixtures have been obtained for cellular lysates of Saccharomyces cerevisiae, Eschericia coli, and Deinococcus radiodurans. High quality separations are shown to be achievable for total protein concentrations of < 0.1 mg/mL. The separation reproducibility was examined, and the influence of the capillary inner wall coating on resolution investigated using fusedsilica capillaries coated with various hydrophilic polymers including hydroxypropyl cellulose, poly(vinyl alcohol), and linear polyacrylamide. Proteins having an isoelectric point (pI) difference of 0.004 are shown to be separated using a linear carrier ampholyte (linear pH gradient between two electrodes) of 3-10. Approximately 45 discrete peaks in the pI range of 5-7 were obtained for S. cerevisiae, approximately 80 peaks in the pI range of 4.5-8.5 for E. coli, and approximately 210 peaks in the pI range of 3-8.8 for D. radiodurans.

Bacterial Proteins↗

PubMed1999

Electrospray ionization mass spectrometry in the study of biomolecular non-covalent interactions.

In the past mass spectrometry has been limited to the study of small, stable molecules, however, with the emergence of electrospray ionization mass spectrometry (ESI-MS) large biomolecules as well as non-covalent biomolecular complexes can be studied. ESI-MS has been used to study non-covalent interactions involving proteins with metals, ligands, peptides, oligonucleotides, as well as other proteins. Although complementary to other well-established techniques such as circular dichroism and fluorescence spectroscopy, ESI-MS offers some advantages in speed, sensitivity, and directness particularly in the determination of the stoichiometry of the complex. One major advantage is the ability of ESI-MS to provide multiple signals each arising from a distinct population within the sample. In this review I will discuss some of the different types of non-covalent biomolecular interactions that have been studied using ESI-MS, highlighting examples which show the efficacy of using ESI-MS to probe the structure of biomolecular complexes.

Journal Article↗

PubMed1999

Electrospray ionization mass spectrometry: a promising new technique in the study of protein/DNA noncovalent complexes.

With the emergence of electrospray ionization mass spectrometry (ESI-MS), mass spectrometry is no longer restricted to the study of small, stable molecules, but has become a viable technique to study large biomolecules as well as noncovalent biomolecular complexes. ESI-MS has been used to study noncovalent interactions involving proteins with metals, ligands, peptides, oligonucleotides, and other proteins. An area where ESI-MS holds significant promise is in the study of protein/DNA interactions. The most common technique employed to study protein/DNA interactions is the electrophoretic gel mobility shift assay (EMSA). Although this technique has and will continue to provide excellent results, ESI-MS has shown the ability to provide detailed results not easily obtainable by EMSA. In this review I will discuss some of the protein/DNA noncovalent interactions that have been measured using ESI-MS, and contrast the results obtained by ESI-MS to those obtained by EMSA.

DNA↗

PubMed1999

Determination of the metal-binding cooperativity of wild-type and mutant calbindin D9K by electrospray ionization mass spectrometry.

Since the initial reports showing the ability of electrospray ionization mass spectrometry (ESI-MS) to study intact noncovalent biomolecular complexes, an increasing number of uses for this technique in studying biochemical systems is emerging. We have investigated the ability of ESI-MS to characterize the metal-binding properties of calcium (Ca2+) binding proteins by studying the incorporation of Ca2+ and cadmium (Cd2+) into wild-type and mutant calbindin D9K. ESI-MS showed that wild-type calbindin D9K binds two Ca2+ ions with similar affinities while the binding of two Cd2+ ions is sequential, as is the binding of the two Ca2+ or Cd2+ ions to the N56A mutant of calbindin. The binding of Ca2+ to the wild-type protein was clearly seen to be cooperative. These results demonstrate the potential efficacy of ESI-MS to discriminate between cooperative and independent site metal binding to metalloproteins.

Cadmium↗