Jan van Oostrum

GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE, THE PUTATIVE TARGET OF THE ANTIAPOPTOTIC COMPOUNDS CGP 3466 AND R-(-)-DEPRENYL

R-(−)-Deprenyl (Selegiline) represents one of the drugs currently used for the treatment of Parkinson’s disease. This compound was shown to protect neurons or glias from programmed cell death in a variety of models. The mechanism of action of neuroprotection as well as inhibition of apoptosis remains elusive. CGP 3466 is a structurally related analog ofR-(−)-deprenyl that exhibits virtually no monoamine oxidase type B inhibiting activity but is neuroprotective in the picomolar concentration range. We showed specific binding of CGP 3466 to glyceraldehyde-3-phosphate dehydrogenase by affinity binding, by affinity labeling, and by means of BIAcore® technology. Apoptosis assays based on the human neuroblastoma cell line PAJU established the importance of this interaction for mediating drug-induced inhibition of programmed cell death.

Towards high performance two‐dimensional gel electrophoresis using ultrazoom gels

Proteomics is the analysis of protein expression in cells or tissues, e.g., to study cellular processes at the molecular level. Ultimately, a proteome analysis should encompass most if not all protein species in a biological sample, including those present in low copy numbers. We are developing two‐dimensional gel electrophoresis technology by applying narrow pH range ultrazoom gels to enhance resolution and to improve the detection of low abundance proteins. Ultrazoom gels in the acidic pH range allow the detection of proteins down to 300 copies per cell of a B‐lymphoma cell line. Protein separation in the alkaline pH range, however, still requires optimization, especially in conjunction with high sample loads.

Preparative two‐dimensional gel electrophoresis at alkaline pH using narrow range immobilized pH gradients

A reproducible high‐resolution protein separation method is the basis for a successful differential proteome analysis. Of the techniques currently available, two‐dimensional gel electrophoresis is most widely used, because of its robustness under various experimental conditions. With the introduction of narrow range immobilized pH gradient (IPG) strips (also referred to as ultra‐zoom gels) in the first dimension, the depth of analysis, i.e. the number of proteins that can be resolved, has increased substantially. However, for poorly understood reasons isoelectric focusing on ultra‐zoom gels in the alkaline region above pH 7 has suffered from problems with resolution and reproducibility. To tackle these difficulties we have optimized the separation of semipreparative amounts of proteins on alkaline IPG strips by focusing on two important phenomena: counteracting water transport during isoelectric focusing and migration of dithiothreitol (DTT) in alkaline pH gradients. The first problem was alleviated by the addition of glycerol and isopropanol to the focusing medium, leading to a significant improvement in the resolution above pH 7. Even better results were obtained by the introduction of excess of the reducing agent DTT at the cathode. With these adaptations together with an optimized composition of the IPG strip, separation efficiency in the pH 6.2–8.2 range is now comparable to the widely used acidic ultra‐zoom gels. We further demonstrated the usefulness of these modifications up to pH 9.5, although further improvements are still needed in that range. Thus, by extending the range covered by conventional ultra‐zoom gels, the depth of analysis of two‐dimensional gel electrophoresis can be significantly increased, underlining the importance of this method in differential proteomics.

Sialylated carbohydrate chains of recombinant human glycoproteins expressed in Chinese hamster ovary cells contain traces of N-glycolylneuraminic acid

HPLC analysis of sialic acid released from recombinant variants of human tissue plasminogen activator, human chimeric plasminogen activator, human erythropoietin, and human follitropin, expressed in Chinese hamster ovary cells, demonstrates for each glycoprotein the presence of N‐acetylneuraminic and N‐glycolylneuraminic acid in a ratio of 97:3. Structural analysis by 500 MHz1H‐NMR spectroscopy, of the enzymatically released N‐linked carbohydrate chains of chimeric plasminogen activator and of erythropoietin, showed that α2‐3 linked N‐glycolylneuraminic acid can occur in different N‐acetyllactosamine type antennary structures.

Characterization of Histone H2A and H2B Variants and Their Post-translational Modifications by Mass Spectrometry

The nucleosome, the fundamental structural unit of chromatin, contains an octamer of core histones H3, H4, H2A, and H2B. Incorporation of histone variants alters the functional properties of chromatin. To understand the global dynamics of chromatin structure and function, analysis of histone variants incorporated into the nucleosome and their covalent modifications is required. Here we report the first global mass spectrometric analysis of histone H2A and H2B variants derived from Jurkat cells. A combination of mass spectrometric techniques, HPLC separations, and enzymatic digestions using endoproteinase Glu-C, endoproteinase Arg-C, and trypsin were used to identify histone H2A and H2B subtypes and their modifications. We identified nine histone H2A and 11 histone H2B subtypes, among them proteins that only had been postulated at the gene level. The two main H2A variants, H2AO and H2AC, as well as H2AL were either acetylated at Lys-5 or phosphorylated at Ser-1. For the replacement histone H2AZ, acetylation at Lys-4 and Lys-7 was found. The main histone H2B variant, H2BA, was acetylated at Lys-12, -15, and -20. The analysis of core histone subtypes with their modifications provides a first step toward an understanding of the functional significance of the diversity of histone structures.

Analysis of Dynamic Changes in Post-translational Modifications of Human Histones during Cell Cycle by Mass Spectrometry

The N-terminal tails of the four core histones are subject to several types of covalent post-translational modifications that have specific roles in regulating chromatin structure and function. Here we present an extensive analysis of the core histone modifications occurring through the cell cycle. Our MS experiments characterized the modification patterns of histones from HeLa cells arrested in phase G1, S, and G2/M. For all core histones, the modifications in the G1 and S phases were largely identical but drastically different during mitosis. Modification changes between S and G2/M phases were quantified using the SILAC (stable isotope labeling by amino acids in cell culture) approach. Most striking was the mitotic phosphorylation on histone H3 and H4, whereas phosphorylation on H2A was constant during the cell cycle. A loss of acetylation was observed on all histones in G2/M-arrested cells. The pattern of cycle-dependent methylation was more complex: during G2/M, H3 Lys27 and Lys36 were decreased, whereas H4 Lys20 was increased. Our results show that mitosis was the period of the cell cycle during which many modifications exhibit dynamic changes.

Protein purification by Off-Gel electrophoresis

A novel free‐flow protein purification technique based on isoelectric electrophoresis is presented, where the proteins are purified in solution without the need of carrier ampholytes. The gist of the method is to flow protein solutions under an immobilised pH gradient gel (IPG) through which an electric field is applied perpendicular to the direction of the flow. Due to the buffering capacity of the IPG gel, proteins with an isoelectric point (pI) close to pH of the gel in contact with the flow chamber stay in solution because they are neutral and therefore not extracted by the electric field. Other proteins will be charged when approaching the IPG gel and are extracted into the gel by the electric field. Both a demonstration experiment with pI markers and a simulation of the electric field distribution are presented to highlight the principle of the system. In addition, an isoelectric fractionation of an Escherichia coli extract is shown to illustrate the possible applications.

Mode of action of the lanthionine-containing peptide antibiotics duramycin, duramycin B and C, and cinnamycin as indirect inhibitors of phospholipase A2

Effects of the lanthionine-containing peptide antibiotics duramycin, duramycin B, duramycin C and cinnamycin on the activity of phospholipase A2 from six different sources were studied, and their mode of action was investigated. The four antibiotics inhibited potently all tested phospholipases A2, with IC50 values of around 1 microM, using phosphatidylethanolamine or [1-14C]oleate-labelled Escherichia coli, whose phospholipids are rich in phosphatidylethanolamine, as substrates. No inhibition was observed when the substrate was phosphatidylcholine. Binding of the antibiotics to the lipid fraction of E. coli could be demonstrated by co-sedimentation with whole, but not with lipid-depleted E. coli. In addition, preincubation of duramycin B with vesicles of phosphatidylethanolamine, but not those of phosphatidylcholine, prevented the inhibition of phospholipase A2 activity. The interaction of duramycin B and C, but not that of the biologically inactive compounds actagardine and the duramycin B trisulphoxide, with phosphatidylethanolamine was demonstrated using circular dichroism studies. On the other hand, no interaction of duramycin B with phosphatidylcholine could be demonstrated. A strict correlation between the physico-chemical interaction of the studied lantibiotics, demonstrated by circular dichroism spectroscopy, and their inhibition of phospholipase A2 was observed. These results suggest that lanthionine-containing peptide antibiotics inhibit phospholipase A2 indirectly by specifically sequestering the substrate phosphatidylethanolamine. This mode of action is analogous to the one described for the protein lipocortin.

Neuroproteomics: expression profiling of the brain's proteomes in health and disease.

The term “proteome” describes the protein complement of a genome. Proteomes of cells are dynamic and are directly affected by environmental factors, such as stress and/or drug treatment, or as a result of aging and disease. One of the distinct advantages of proteomic analysis, not attainable with RNA expression data, is the ability to fractionate the cells proteins into various subpopulations. In neuroscience, “neuromics” (proteomics in the central nervous system) is in its infancy, with a paucity of studies in the context of the brain. One of the objectives of this review is to illustrate the potential of neuromics to profile differences in the distribution of thousands of proteins as a function of disease markers. We have previously used this approach to determine the effects of varied postmortem interval in examining human brain tissue and to identify biomarkers. Here we review proteomic studies of schizophrenia, Alzheimers disease, and Parkinsons disease. Experimental results regarding Parkinsons disease are presented to illustrate the potential of neuromics to identify pathways of pathogenesis and novel therapeutic targets.

Microfabricated polymer injector for direct mass spectrometry coupling

This paper demonstrates the coupling of a plasma etched polymer microfluidic system with an electrospray mass spectrometer by generation of a nanospray. Taking advantage of the microtechnology processes and polymer properties, high volume production with good reproducibility of hydrophobic interfaces could be obtained. The nanospray was directly produced from the outlet of the plastic microfabricated chip positioned in front of the capillary entrance of the mass spectrometer. No chemical background due to the polymer has been observed under standard nanospray conditions. The performances of the spray as well as its efficiency have been demonstrated by flow measurements, stability establishment and tandem mass spectrometry experiment on angiotensin II. The spray was actuated without additional flow in methanol: water:acetic acid (50:49:1%) solution. A 40 fmol/νL detection limit could be reached.