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Biomedical subjects

R Aebersold

Publications and source records attributed to R Aebersold.

At least 55 records · Page 3Linked to original sources
PubMed2000

Proteins of rat serum V: adjuvant arthritis and its modulation by nonsteroidal anti-inflammatory drugs.

The effect of adjuvant arthritis (AA) on the pattern of rat serum proteins includes the upregulation of haptoglobin, orosomucoid, alpha2-macroglobulin, serine protease inhibitor-3, thiostatin, alpha1-antitrypsin, C-reactive protein, and the downregulation of kallikrein-binding protein, alpha1-inhibitor III, apolipoprotein A-I, alpha2-HS-glycoprotein, albumin, apolipoprotein A-IV, transthyretin and transferrin. Minor changes (+/- 20%) are observed for Gc-globulin, ceruloplasmin, and alpha1-macroglobulin. AA thus grossly resembles the acute inflammatory response elicited by the injection of turpentine, although the changes in the levels of negative acute-phase proteins (APP) are smaller in acute inflammation. Indomethacine and ibuprofen inhibit the effects of arthritis on the synthesis of rat serum proteins in different ways: The former is, on average, three times as effective as the latter. Each drug interferes differently with different proteins. In animals without AA, both nonsteroidal anti-inflammatory drugs (NSAID) mimic the inflammatory pattern to a certain extent, with more effect on the negative than on the positive APPs. Overall, the shifts in serum protein levels parallel changes in inflammatory parameters such as joint swelling and serum interleukin-6 (IL-6) activity. Protein quantitation after two-dimensional electrophoresis (2-DE) reveals some effects of the drugs per se which escape detection by other routine tests.

Animals↗
PubMed2000

A comprehensive characterization of the T-cell antigen receptor complex composition by microcapillary liquid chromatography-tandem mass spectrometry.

It has become apparent that many intracellular signaling processes involve the dynamic reorganization of cellular proteins into complex signaling assemblies that have a specific subunit composition, function, and subcellular location. Since the elements of such assemblies interact physically, multiprotein signaling complexes can be isolated and analyzed. Recent technical advances in highly sensitive protein identification by electrospray-tandem mass spectrometry have dramatically increased the sensitivity with which such analyses can be performed. The T-cell antigen receptor (TCR) is an oligomeric transmembrane protein complex that is essential to T-cell recognition and function. The extracellular protein domains are responsible for ligand binding while intracellular domains generate and transduce signals in response to specific receptor-ligand interactions. We used microbore capillary chromatography-tandem mass spectrometry to investigate the composition of the TCR protein complex isolated from resting and activated cells of the murine T-cell line CD11.3. We identified all the previously known subunits of the TCR/CD3 complex as well as proteins previously not known to associate with the TCR. The catalytic activities of some of these proteins could potentially be used to interfere pharmacologically with TCR signaling.

Amino Acid Sequence↗
PubMed2000

Measuring gene expression by quantitative proteome analysis.

Proteome analysis is most commonly accomplished by the combination of two-dimensional gel electrophoresis for protein separation, visualization, and quantification and mass spectrometry for protein identification. Over the past year, exceptional progress has been made towards developing a new technology base for the precise quantification and identification of proteins in complex mixtures, that is, quantitative proteomics.

Biochemistry↗
PubMed2000

Mass spectrometry and proteomics.

Proteomics is the systematic analysis of the proteins expressed by a cell or tissue, and mass spectrometry is its essential analytical tool. In the past two years, incremental advances in standard proteome technology have increased the speed of protein identification with higher levels of automation and sensitivity. Furthermore, new approaches have provided landmark advances in determining functionally relevant properties of proteins, including their quantity and involvement within protein complexes.

Mass Spectrometry↗
PubMed2000

Quantitative proteome analysis: methods and applications.

With the completion of a rapidly increasing number of complete genomic sequences, much attention is currently focused on how the information contained in sequence databases might be interpreted in terms of the structure, function, and control of biological systems. Quantitative proteome analysis, the global analysis of protein expression, has been proposed as a method to study steady-state gene expression and perturbation-induced changes. Here, we discuss the rationale for quantitative proteome analysis, highlight the limitations in the current standard technology, and introduce a new experimental approach to quantitative proteome analysis.

Amino Acid Sequence↗
PubMed1999

Feedback phosphorylation of an RGS protein by MAP kinase in yeast.

Regulators of G protein signaling (RGS proteins) are well known to accelerate G protein GTPase activity in vitro and to promote G protein desensitization in vivo. Less is known about how RGS proteins are themselves regulated. To address this question we purified the RGS in yeast, Sst2, and used electrospray ionization mass spectrometry to identify post-translational modifications. This analysis revealed that Sst2 is phosphorylated at Ser-539 and that phosphorylation occurs in response to pheromone stimulation. Ser-539 lies within a consensus mitogen-activated protein (MAP) kinase phosphorylation site, Pro-X-Ser-Pro. Phosphorylation is blocked by mutations in the MAP kinase genes (FUS3, KSS1), as well as by mutations in components needed for MAP kinase activation (STE11, STE7, STE4, STE18). Phosphorylation is also blocked by replacing Ser-539 with Ala, Asp, or Glu (but not Thr). These point mutations do not alter pheromone sensitivity, as determined by growth arrest and reporter transcription assays. However, phosphorylation appears to slow the rate of Sst2 degradation. These findings indicate that the G protein-regulated MAP kinase in yeast can act as a feedback regulator of Sst2, itself a regulator of G protein signaling.

Amino Acid Sequence↗
PubMed1999

Characterization of an activated ribosomal S6 kinase variant from maturing sea star oocytes: association with phosphatase 2A and substrate specificity.

Two ribosomal protein S6 kinases (i.e., pp52(S6K) and pp70(S6K)) of the p70 S6 kinase family were markedly activated during meiotic maturation of Pisaster ochraceus sea star oocytes. A rapid protocol was developed for the purification from the oocyte cytosol of pp52(S6K) by approximately 50,000-fold with a specific enzyme activity of 1.6 micromol per min per mg. The purified enzyme apparently featured the N- and C-terminal regions of pp70(S6K) as it immunoreacted with antibodies directed to peptides patterned after these amino acid sequences in mammalian pp70(S6K). pp52(S6K) was inhibited by fluoride (IC(50) approximately 60 mM), but was relatively insensitive to beta-glycerolphosphate, EGTA, dithiothreitol, spermine, heparin, NaCl, and metal ions such as Mn(2+), Zn(2+), and Ca(2+). The consensus sequence for substrate phosphorylation was determined to be RXXSXR, which was partially distinct from mammalian p70(S6K) in its requirement for an amino-terminal arginine. Phosphorylation of ribosomal protein S6 by p52(S6K) occurred exclusively on serine on at least five tryptic peptides. Inhibition of sea star p52(S6K) phosphotransferase activity after treatment with protein serine/threonine phosphatases confirmed that p52(S6K) was still regulated by phosphorylation. The sea star S6 kinase was purified to near homogeneity with the regulatory and catalytic subunits of protein-serine phosphatase 2A and the heat shock protein 60. The association of an S6 kinase with phosphatase 2A was confirmed by coimmunoprecipitation of S6 kinase activity with phosphatase 2A-specific antibodies. The purified S6 kinase and the sea star oocyte system will be useful for analysis of upstream and downstream signaling events that lead to phosphorylation of the S6 protein and other targets.

Amino Acid Sequence↗
PubMed1999

Microfabricated polymer devices for automated sample delivery of peptides for analysis by electrospray ionization tandem mass spectrometry.

Delivery of proteins and peptides to electrospray ionization mass spectrometers (ESI-MS) has been demonstrated using glass and quartz microfabricated devices. This paper reports the construction and use of poly(dimethylsiloxane) (PDMS) microfabricated soft polymer devices with mass spectrometry for protein analysis. The PDMS devices were fabricated using replica molding against a patterned photoresist generated by photolithographic techniques. The PDMS devices were connected to the mass spectrometer via a derivatized transfer capillary and samples were transferred by electroosmotic pumping. The formulation of PDMS was optimized for compatibility with ESI, and the devices were tested for performance. The practical application of PDMS devices was demonstrated by the identification of rat serum albumin separated by 2-D gel electrophoresis. Extended contact of the sample with the surface of the PDMS device did not significantly affect the sample analysis, and the limit of detection for samples run on a PDMS device was comparable to the limit of detection achieved on glass devices. This study suggests that PDMS devices fabricated using replica molding are compatible with ESI-MS. This will potentially lead to the construction of inexpensive microfabricated devices with complex designs and advanced functionalities.

Amino Acid Sequence↗
PubMed1999

Identification of flow-dependent endothelial nitric-oxide synthase phosphorylation sites by mass spectrometry and regulation of phosphorylation and nitric oxide production by the phosphatidylinositol 3-kinase inhibitor LY294002.

Endothelial cells release nitric oxide (NO) acutely in response to increased laminar fluid shear stress, and the increase is correlated with enhanced phosphorylation of endothelial nitric-oxide synthase (eNOS). Phosphoamino acid analysis of eNOS from bovine aortic endothelial cells labeled with [(32)P]orthophosphate demonstrated that only phosphoserine was present in eNOS under both static and flow conditions. Fluid shear stress induced phosphate incorporation into two specific eNOS tryptic peptides as early as 30 s after initiation of flow. The flow-induced tryptic phosphopeptides were enriched, separated by capillary electrophoresis with intermittent voltage drops, also known as "peak parking," and analyzed by collision-induced dissociation in a tandem mass spectrometer. Two phosphopeptide sequences determined by tandem mass spectrometry, TQpSFSLQER and KLQTRPpSPGPPPAEQLLSQAR, were confirmed as the two flow-dependent phosphopeptides by co-migration with synthetic phosphopeptides. Because the sequence (RIR)TQpSFSLQER contains a consensus substrate site for protein kinase B (PKB or Akt), we demonstrated that LY294002, an inhibitor of the upstream activator of PKB, phosphatidylinositol 3-kinase, inhibited flow-induced eNOS phosphorylation by 97% and NO production by 68%. Finally, PKB phosphorylated eNOS in vitro at the same site phosphorylated in the cell and increased eNOS enzymatic activity by 15-20-fold.

Amino Acid Sequence↗
PubMed1999

Spin-stretching of DNA and protein molecules for detection by fluorescence and atomic force microscopy.

We have developed a rapid and efficient way of stretching DNA and denatured protein molecules for detection by fluorescence microscopy and atomic force microscopy (AFM). In the described method, a viscous drag created by transient rotational flow stretches randomly coiled DNA molecules or denatured proteins. Stretching is achieved by dispensing a droplet of sample solution containing DNA or denatured protein on a MgCl2-soaked mica surface. We present fluorescent images of straightened lambdaDNA molecules and AFM images of stress-shared, reduced von Willebrand factor as well as straightened lambdaDNA. The described quick and reliable spin-stretching technique will find wide applications in the analysis of single biopolymer molecules.

DNA↗
PubMed1999

p90(RSK) is a serum-stimulated Na+/H+ exchanger isoform-1 kinase. Regulatory phosphorylation of serine 703 of Na+/H+ exchanger isoform-1.

The Na+/H+ exchanger isoform-1 (NHE-1) is the key member of a family of exchangers that regulates intracellular pH and cell volume. Activation of NHE-1 by growth factors is rapid, correlates with increased NHE-1 phosphorylation and cell alkalinization, and plays a role in cell cycle progression. By two-dimensional tryptic peptide mapping of immunoprecipitated NHE-1, we identify serine 703 as the major serum-stimulated amino acid. Mutation of serine 703 to alanine had no effect on acid-stimulated Na+/H+ exchange but completely prevented the growth factor-mediated increase in NHE-1 affinity for H+. In addition, we show that p90 ribosomal S6 kinase (p90(RSK)) is a key NHE-1 kinase since p90(RSK) phosphorylates NHE-1 serine 703 stoichiometrically in vitro, and transfection with kinase-inactive p90(RSK) inhibits serum-induced phosphorylation of NHE-1 serine 703 in transfected 293 cells. These findings establish p90(RSK) as a serum-stimulated NHE-1 kinase and a mediator of increased Na+/H+ exchange in vivo.

Amino Acid Sequence↗
PubMed1999

Data-dependent modulation of solid-phase extraction capillary electrophoresis for the analysis of complex peptide and phosphopeptide mixtures by tandem mass spectrometry: application to endothelial nitric oxide synthase.

Electrospray ionization (ESI) tandem mass spectrometry (MS/MS) of peptides in conjunction with automated sequence database searching of the resulting collision-induced dissociation (CID) spectra has become a powerful method for the identification of purified proteins or the components of protein mixtures. The success of the method is critically dependent on the manner by which the peptides are introduced into the mass spectrometer. In this report, we describe a capillary electrophoresis-based system for the automated, sensitive analysis of complex peptide mixtures. The system consists of an ESI-MS/MS instrument, a solid-phase extraction (SPE)-capillary zone electrophoresis (CZE) device for peptide concentration and separation, and an algorithm written in Instrument Control Language (ICL) which modulates the electrophoretic conditions in a data-dependent manner to optimize available time for the generation of high-quality CID spectra of peptides in complex samples. We demonstrate that the data-dependent modulation of the electric field significantly expands the analytical window for each peptide analyzed and that the sensitivity of the SPE-CZE technique is not noticeably altered by the procedure. By applying the technique to the analysis of in vivo phosphorylation sites of endothelial nitric oxide synthase (eNOS), we demonstrate the power of this system for the MS/MS analysis of minor peptide species in complex samples such as phosphopeptides generated by the proteolytic digestion of a large protein, eNOS, phosphorylated at low stoichiometry.

Amino Acid Sequence↗
PubMed1999

Regulation of 4E-BP1 phosphorylation: a novel two-step mechanism.

The multisubunit eukaryotic translation initiation factor (eIF) 4F recruits 40S ribosomal subunits to the 5' end of mRNA. The eIF4F subunit eIF4E interacts directly with the mRNA 5' cap structure. Assembly of the eIF4F complex is inhibited by a family of repressor polypeptides, the eIF4E-binding proteins (4E-BPs). Binding of the 4E-BPs to eIF4E is regulated by phosphorylation: Hypophosphorylated 4E-BP isoforms interact strongly with eIF4E, whereas hyperphosphorylated isoforms do not. 4E-BP1 is hypophosphorylated in quiescent cells, but is hyperphosphorylated on multiple sites following exposure to a variety of extracellular stimuli. The PI3-kinase/Akt pathway and the kinase FRAP/mTOR signal to 4E-BP1. FRAP/mTOR has been reported to phosphorylate 4E-BP1 directly in vitro. However, it is not known if FRAP/mTOR is responsible for the phosphorylation of all 4E-BP1 sites, nor which sites must be phosphorylated to release 4E-BP1 from eIF4E. To address these questions, a recombinant FRAP/mTOR protein and a FRAP/mTOR immunoprecipitate were utilized in in vitro kinase assays to phosphorylate 4E-BP1. Phosphopeptide mapping of the in vitro-labeled protein yielded two 4E-BP1 phosphopeptides that comigrated with phosphopeptides produced in vivo. Mass spectrometry analysis indicated that these peptides contain phosphorylated Thr-37 and Thr-46. Thr-37 and Thr-46 are efficiently phosphorylated in vitro by FRAP/mTOR when 4E-BP1 is bound to eIF4E. However, phosphorylation at these sites was not associated with a loss of eIF4E binding. Phosphorylated Thr-37 and Thr-46 are detected in all phosphorylated in vivo 4E-BP1 isoforms, including those that interact with eIF4E. Finally, mutational analysis demonstrated that phosphorylation of Thr-37/Thr-46 is required for subsequent phosphorylation of several carboxy-terminal serum-sensitive sites. Taken together, our results suggest that 4E-BP1 phosphorylation by FRAP/mTOR on Thr-37 and Thr-46 is a priming event for subsequent phosphorylation of the carboxy-terminal serum-sensitive sites.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Sharp DNA bends as landmarks of protein-binding sites on straightened DNA.

We have developed a fluorescence-based method for mapping single or multiple protein-binding sites on straightened, large-size DNA molecules (> 5 kbp). In the described method, protein-DNA complexes were straightened and immobilized on a flat surface using surface tension. A fraction of the immobilized complexes displayed a sharp DNA bend with two DNA segments extending from the apex. The presence of DNA-binding proteins at the apex was verified by atomic force microscopy. The position of protein binding relative to the ends of the DNA molecule was determined by measuring the length of two DNA segments using fluorescence microscopy. We demonstrate the potential of the fluorescence-based method to localize protein-binding sites on the DNA template and to evaluate relative binding affinity. The proposed protein-binding-site mapping technique is simple and easy to perform. Practical applications include screening for DNA-binding proteins and the localization of protein-binding sites on large segments of DNA.

Bacteriophage lambda↗
PubMed1999

Molecular characterization of mitochondrial apoptosis-inducing factor.

Mitochondria play a key part in the regulation of apoptosis (cell death). Their intermembrane space contains several proteins that are liberated through the outer membrane in order to participate in the degradation phase of apoptosis. Here we report the identification and cloning of an apoptosis-inducing factor, AIF, which is sufficient to induce apoptosis of isolated nuclei. AIF is a flavoprotein of relative molecular mass 57,000 which shares homology with the bacterial oxidoreductases; it is normally confined to mitochondria but translocates to the nucleus when apoptosis is induced. Recombinant AIF causes chromatin condensation in isolated nuclei and large-scale fragmentation of DNA. It induces purified mitochondria to release the apoptogenic proteins cytochrome c and caspase-9. Microinjection of AIF into the cytoplasm of intact cells induces condensation of chromatin, dissipation of the mitochondrial transmembrane potential, and exposure of phosphatidylserine in the plasma membrane. None of these effects is prevented by the wide-ranging caspase inhibitor known as Z-VAD.fmk. Overexpression of Bcl-2, which controls the opening of mitochondrial permeability transition pores, prevents the release of AIF from the mitochondrion but does not affect its apoptogenic activity. These results indicate that AIF is a mitochondrial effector of apoptotic cell death.

Amino Acid Sequence↗
PubMed1999

Protein analysis by mass spectrometry and sequence database searching: tools for cancer research in the post-genomic era.

The post-genomic era is characterized by the deposition of sequence information for entire genomes in databases. Currently, besides the protein sequences for known human proteins, there are partial sequences from thousands more human proteins for which no biological function has been assigned. A powerful new tool for the unambiguous identification and characterization of gel-separated proteins is accomplished by the combination of mass spectrometry and sequence database searching. This combination provides the cancer biologist with the ability to (i) identify the potential protein:protein associations and (ii) fully characterize function-critical post-translational modifications, both directly from silver-stained polyacrylamide gels. In this report we describe the application of tandem mass spectrometry and database searching to two problems which are prototypical for cancer research and indeed for biomedical research in general. The first is the identification of gel-separated, low abundance proteins based on amino acid sequence composition following coimmunoprecipitation with the human apoptosis inhibitor protein BclX(L). The second is the determination of the precise sites of phosphorylation of the human regulatory protein 4E-BP1, which controls mRNA translation.

Amino Acid Sequence↗