ATP5PO levels regulate enteric nervous system development in zebrafish, linking Hirschsprung disease to Down Syndrome.

Hirschsprung disease (HSCR) is a complex genetic disorder characterized by the absence of enteric nervous system (ENS) in the distal region of the intestine. Down Syndrome (DS) patients have a >50-fold higher risk of developing HSCR than the general population, suggesting that overexpression of human chromosome 21 (Hsa21) genes contribute to HSCR etiology. However, identification of responsible genes remains challenging. Here, we describe a genetic screening of potential candidate genes located on Hsa21, using the zebrafish. Candidate genes were located in the DS-HSCR susceptibility region, expressed in the human intestine, were known potential biomarkers for DS prenatal diagnosis, and were present in the zebrafish genome. With this approach, four genes were selected: RCAN1, ITSN1, ATP5PO and SUMO3. However, only overexpression of ATP5PO, coding for a component of the mitochondrial ATPase, led to significant reduction of ENS cells. Paradoxically, in vitro studies showed that overexpression of ATP5PO led to a reduction of ATP5PO protein levels. Impaired neuronal differentiation and reduced mitochondrial ATP production, were also detected in vitro, after overexpression of ATP5PO in a neuroblastoma cell line. Finally, epistasis was observed between ATP5PO and ret, the most important HSCR gene. Taken together, our results identify ATP5PO as the gene responsible for the increased risk of HSCR in DS patients in particular if RET variants are also present, and show that a balanced expression of ATP5PO is required for normal ENS development.

Taccalonolide binding to tubulin imparts microtubule stability and potent in vivo activity.

The taccalonolides are highly acetylated steroids that stabilize cellular microtubules and overcome multiple mechanisms of taxane resistance. Recently, two potent taccalonolides, AF and AJ, were identified that bind to tubulin directly and enhance microtubule polymerization. Extensive studies were conducted to characterize these new taccalonolides. AF and AJ caused aberrant mitotic spindles and bundling of interphase microtubules that differed from the effects of either paclitaxel or laulimalide. AJ also distinctly affected microtubule polymerization in that it enhanced the rate and extent of polymerization in the absence of any noticeable effect on microtubule nucleation. In addition, the resulting microtubules were found to be profoundly cold stable. These data, along with studies showing synergistic antiproliferative effects between AJ and either paclitaxel or laulimalide, suggest a distinct binding site. Direct binding studies demonstrated that AJ could not be displaced from microtubules by paclitaxel, laulimalide, or denaturing conditions, suggesting irreversible binding of AJ to microtubules. Mass spectrometry confirmed a covalent interaction of AJ with a peptide of ß-tubulin containing the cyclostreptin-binding sites. Importantly, AJ imparts strong inter-protofilament stability in a manner different from other microtubule stabilizers that covalently bind to tubulin, consistent with the distinct effects of the taccalonolides as compared with other stabilizers. AF was found to be a potent and effective antitumor agent that caused tumor regression in the MDA-MB-231 breast cancer xenograft model. The antitumor efficacy of some taccalonolides, which stabilize microtubules in a manner different from other microtubule stabilizers, provides the impetus to explore the therapeutic potential of this site.

Frontal affinity chromatography for the screening of mixtures.

A protein stationary phase for frontal affinity chromatography was prepared, containing biotinylated beta-galactosidase immobilized to controlled pore glass beads via covalently bonded streptavidin. Single microaffinity columns of approximately 30 pmol of active beta-galactosidase were prepared from this material and characterized with a known ligand by frontal analysis. These columns were used to measure the specific interactions between the bound beta-galactosidase and a library of modified beta-galactopyranosides using electrospray mass spectrometry as the means of detection. The library contained 89 entries, each representing 4 diastereomers for a total of 356 library members. A single entry was analysed revealing differential activity among the 4 isomers. The library was grouped into 10 mixtures of 24-40 members each with each mixture infused under frontal chromatographic conditions. This deconvolution procedure led to the identification of 34 entries containing isomers with K(d) values better than 10 microM. A method based on a displacement principle was implemented as a rapid prescreen which served as the basis for a parallel column high throughput screening assay.

Frontal affinity chromatography coupled to mass spectrometry for screening mixtures of enzyme inhibitors.

Frontal affinity chromatography coupled online to mass spectrometry (FAC/MS) has previously been used to estimate binding constants for individual protein ligands present in mixtures of compounds. In this study FAC/MS is used to determine enzyme substrate kinetic parameters and binding constants for enzyme inhibitors. Recombinant human N-acetylglucosaminyltransferase V was biotinylated and adsorbed onto immobilized streptavidin in a microcolumn (20 microL). The enzyme was shown to be catalytically competent transferring GlcNAc from the donor UDP-GlcNAc to beta-d-GlcpNAc-(1-->2)-alpha-d-Manp-(1-->6)-beta-d-Glcp-OR acceptor giving beta-d-GlcpNAc-(1-->2)-[beta-d-GlcpNAc-(1-->6)]-alpha-d-Manp-(1-->6)-beta-d-Glcp-OR as the reaction product. The kinetic parameters K(m) and V(max) for the immobilized enzyme could be determined by FAC/MS and were comparable to those measured in solution. Analysis of a mixture of eight trisaccharide analogs in a single run yielded K(d) values for each of the eight compounds ranging from 0.3 to 36 microM. These K(d) values were 2 to 10 times lower than the inhibition constants, K(I)'s, determined in solution using a standard radiochemical assay. However, the ranking order of K(d)'s was the same as the ranking of K(I) values. FAC/MS assays can therefore be employed for the rapid estimation of inhibitor K(d) values making it a valuable tool for enzyme inhibitor evaluations.

MALDI mass spectrometry combined with avidin-biotin chemistry for analysis of protein modifications.

A general mass spectrometric method that combines purification and analysis in one step is described for the rapid and sensitive determination of protein modification that involves covalent attachment of a modifying group. In this method, the modifying group is first labeled with a biotin moiety, and the covalent interaction of this group with the targeted protein results in a biotinylated product. The modified protein can then be subjected to enzymatic digestion, followed by the isolation of the biotinylated peptide based on a previously described MALDI method incorporating the avidin-biotin interaction (Schriemer, D. C.; Li, L. Anal. Chem. 1996, 68, 3382-3387). To illustrate the validity of the method, a study of a model system was undertaken, involving the interaction between avian skeletal muscle troponin C and a sulfhydryl-specific biotinylation reagent. It is shown that isolation of a modified peptide with an immobilized avidin product could be achieved, even in the presence of an excess of contaminating protein. Exoproteases could be added to the crude tryptic digest to generate peptide ladders, each containing biotin, which could be analyzed by the avidin-biotin/MALDI method for sequence information. Complementary sequence information could be obtained from the application of this technique in a tandem sector/time-of-flight mass spectrometer for MALDI MS/MS analysis, which allowed for the identification of the modification site.

Deconvolution approaches in screening compound mixtures.

Combinatorial chemistry initiatives can be used to generate compound mixtures, and it has become a challenge to effectively screen these mixtures for activity against a specified target. Overall mixture activity can be readily measured, however it is not a straightforward matter to identify all those components giving rise to the total activity (a process referred to as deconvolution). This review discusses the attraction in preparing and screening mixtures, and also addresses the difficulties inherent in their screening. Attention is paid to the current approaches of achieving mixture deconvolution, including synthetic pooling, fractionation, affinity-based isolation, and techniques requiring no physical separation. Recent successes in mixture screening suggest a rekindled interest in this area.

Time-lag focusing MALDI time-of-flight mass spectrometry for polymer characterization: oligomer resolution, mass accuracy, and average weight information.

We report a polymer characterization study by matrix-assisted laser description/ionization (MALDI) on a linear time-of-flight instrument equipped with pulsed ion extraction for time-lag focusing. It is demonstrated that time-lag focusing MALDI provides improved mass resolution and mass accuracy over continuous extraction instruments. Oligomer resolution is extended to a much higher mass range than that observed even by continuous extraction reflectron systems. This allows new opportunities to study the chemical composition and determine the molecular weight of individual components in a mixture of higher molecular weight polymers. It is shown that oligomer resolution can be obtained for poly(ethylene glycol) (repeat unit mass of 44) of mass up to 25,000 u and poly(styrene) (repeat unit mass of 104) up to 55,000 u. Mass measurement accuracy of 80 ppm or better is demonstrated, and the relevance to end-group analysis is shown for two derivative of poly(ethylene glycol) used as slow-release drugs. The analysis of the molecular weight distribution was investigated at several extraction pulse potentials to determine if there was an effect on the relative peak area. We found that the values of the number-average molecular weight (Mn and the weight-average molecular weight (Mw) do not change significantly for a poly(styrene) blend with oligomer masses between 2,000 and 15,000 u and a polydispersity of 1.155. The values are within the 1.6% standard deviation observed for repeat analyses at the same extraction pulse.

Combining avidin-biotin chemistry with matrix-assisted laser desorption/ionization mass spectrometry.

Molecular recognition based on the high affinity of avidin for biotin has been combined with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The rich chemistry of biotinylation reagents underlies the development of a mass tracer method for the rapid and sensitive analysis of biotinylated analyte in complex mixtures. In this method, the biotinylated analyte is captured with immobilized avidin agarose beads. The beads with the bound complex are deposited on the MALDI sample probe, followed by elution of the retained compounds and removal of the beads. The use of MALDI matrix solutions in conjunction with drying of the beads is shown to be sufficient to overcome the avidin-biotin interaction. The studies of several different avidin-biotin interaction schemes tailored to the MALDI analysis are presented. It is demonstrated that, with an optimized scheme, the extremely high selectivity of the avidin-biotin interaction is preserved, generating species represented in the MALDI spectra that arise only from the avidin-biotin interaction. Biotinylated species in the nanomolar range can be isolated and analyzed with this technique.

Detection of High Molecular Weight Narrow Polydisperse Polymers up to 1.5 Million Daltons by MALDI Mass Spectrometry.

The detection of very high molecular weight narrow polydisperse poly(styrene) samples by MALDI time-of-flight mass spectrometry is reported. It is shown that accurate molecular weight determinations of samples up to 1 million can be achieved very rapidly from the singly charged polymeric species. For a poly(styrene) with a molecular weight of approximately 1.5 million, signals corresponding to the multiply charged ions of the principal distribution are observed. The molecular weights obtained by MALDI are in good agreement with classical molecular weight determination techniques. all-trans-Retinoic acid was used as the organic matrix for the laser desorption procedure, and the samples were analyzed as their silver cation adducts. This work demonstrates that, with proper matrix selection and sample preparation, MALDI can be a very useful tool for high molecular weight polymer sample analysis.