␣-Latrotoxin (LTX) stimulates massive exocytosis of synaptic vesicles and may help to elucidate the mechanism of regulation of neurosecretion. We have recently isolated latrophilin, the synaptic Ca 2؉ -independent LTX receptor. Now we demonstrate that latrophilin is a novel member of the secretin family of G protein-coupled receptors that are involved in secretion. Northern blot analysis shows that latrophilin message is present only in neuronal tissue. Upon expression in COS cells, the cloned protein is indistinguishable from brain latrophilin and binds LTX with high affinity. Latrophilin physically interacts with a G␣ o subunit of heterotrimeric G proteins, because the two proteins co-purify in a twostep affinity chromatography. Interestingly, extracellular domain of latrophilin is homologous to olfactomedin, a soluble neuronal protein thought to participate in odorant binding. Our findings suggest that latrophilin may bind unidentified endogenous ligands and transduce signals into nerve terminals, thus implicating G proteins in the control of synaptic vesicle exocytosis.

Motivation: Metabolite fingerprinting is a technology for providing information from spectra of total compositions of metabolites. Here, spectra acquisitions by microchip-based nanoflow-direct-infusion QTOF mass spectrometry, a simple and high throughput technique, is tested for its informative power. As a simple test case we are using Arabidopsis thaliana crosses. The question is how metabolite fingerprinting reflects the biological background. In many applications the classical principal component analysis (PCA) is used for detecting relevant information. Here a modern alternative is introduced-the independent component analysis (ICA). Due to its independence condition, ICA is more suitable for our questions than PCA. However, ICA has not been developed for a small number of high-dimensional samples, therefore a strategy is needed to overcome this limitation. Results: To apply ICA successfully it is essential first to reduce the high dimension of the dataset, by using PCA. The number of principal components determines the quality of ICA significantly, therefore we propose a criterion for estimating the optimal dimension automatically. The kurtosis measure is used to order the extracted components to our interest. Applied to our A. thaliana data, ICA detects three relevant factors, two biological and one technical, and clearly outperforms the PCA.

The TTGA-binding factor is a transcriptional regulator activated by DIF, the chlorinated hexaphenone that induces prestalk cell differentiation in Dictyostelium. The same activity also functions as a repressor, controlling stalk cell differentiation. We show that the TTGA-binding factor is a STAT protein. Like the metazoan STATs, it functions via the reciprocal interaction of a phosphotyrosine residue on one molecule with an SH2 domain on a dimerizing partner. Furthermore, it will bind specifically to a mammalian interferon-stimulated response element. In Saccharomyces cerevisiae, where the entire genomic sequence is known, SH2 domains have not been identified. It would seem, therefore, that SH2 signaling pathways arose very early in the evolution of multicellular organisms, perhaps to facilitate intercellular comunication.

Carbohydrates represent a major class of biopolymers, which occur in nature either as oligosaccharides or glycoconjugates, in which the sugar moiety is linked to proteins or lipids. The significance of mass spectrometry for highly sensitive analysis of complex carbohydrates increased after the introduction of the electrospray ionization and matrix assisted laser desorption/ionization methods and the possibility of tandem MS for sequencing of single molecular species in complex mixtures. Rapid and sensitive characterization of carbohydrates in biological systems by automated nanoscale liquid delivery and chip-based electrospray interface techniques have not been developed so far. In this contribution, the implementation and optimization of a fully automated chip-based nanoelectrospray assembly (NanoMate system), operating in the negative ion mode, in combination with QTOF-tandem MS for mapping/sequencing and computer-assisted structure assignment for carbohydrate components in complex mixtures is presented.

The major constitutive phosphatidylinositol-3,4,5-P 3 (PtdIns) 5-phosphatase activity was purified and subjected to peptide sequence analysis providing extensive amino acid sequence which was subsequently used for cloning the cDNA. Peptide and cDNA sequences revealed that the purified PtdIns(3,4,5)P 3 5-phosphatase was identical to a splice variant of a recently cloned inositol polyphosphate 5-phosphatase termed synaptojanin. Since synaptojanin is not known to possess PtdIns(3,4,5)P 3 5-phosphatase activity, we verified that the purified PtdIns(3,4,5)P 3 5-phosphatase activity and synaptojanin are identical by Western blot using specific antibodies raised against synaptojanin sequences. Immunoprecipitation from crude lysates of rat brain tissue showed that synaptojanin accounts for the major part of the active PtdIns(3,4,5)P 3 5-phosphatase activity. It is also shown that the protein is localized to the soluble fraction. Expression of a truncated recombinant protein demonstrates that the conserved 5-phosphatase region of the synaptojanin gene expresses PtdIns(3,4,5)P 3 5-phosphatase activity. However, immunological analysis demonstrates that the PtdIns(3,4,5)P 3 5-phosphatase activity expressed from the synaptojanin gene in brain is due to a particular splice variant which contains a 16-amino acid insert as shown by immunoprecipitation using a specific antibody raised against this particular splice variant.The regulation of inositol phospholipid metabolism has provided a cornerstone to at least two signal transduction pathways, one involving the phospholipase C-dependent phosphodiesterase cleavage of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) 1 (1) and the second the phosphorylation of this lipid by PtdIns 3-kinases yielding PtdIns(3,4,5)P 3 (2, 3). This latter lipid second messenger has been implicated in a number of signaling pathways (PKB/akt, p70S6k , PKC, PRK) as has its metabolite PtdIns(3,4)P 2 (4 -9). It has been suggested that the 5-phosphatase responsible for the transformation of PtdIns(3,4,5)P 3 to PtdIns(3,4)P 2 (10) is due to a recently cloned 5-phosphatase termed SIP (11) or SHIP (p145 SHIP ) (12-14) which has specificity for 3-phosphorylated inositol phosphates (11)(12)(13)(14). However, other PtdIns(3,4,5)P 3 5-phosphatases have been identified (15,16) indicating that this issue is still not resolved.We have previously identified and purified the major constitutive PtdIns(3,4,5)P 3 5-phosphatase from rodent brain tissue (16). Here we demonstrate that this protein is encoded by a particular splice variant of the recently described synaptojanin gene. It is further shown that a recombinant synaptojanin protein has intrinsic PtdIns(3,4,5)P 3 5-phosphatase activity. Thus, we are able to show that synaptojanin which to date has not been associated with PtdIns 3-kinase signaling could play a major role in PtdIns 3-kinase-dependent pathways. EXPERIMENTAL PROCEDURES Microsequencing of Purified Rat Brain PIP 3 5-Phosphatase-The 5-phosphatase was purified (16), further fractionated by SDS-poly...

The introduction of chip-based electrospray (ESI) ion sources into biological mass spectrometry (MS) addressed the fundamental issue of how to analyze minute amounts of complex biological systems. The automation of sample delivery into the MS combined with the chip-based ESI allows for high quality bioanalysis in a high-throughput fashion. These advantages have already been demonstrated in proteomics, direct screening of drugs and drug discovery. As part of our continuing effort to implement automated chip-based mass spectrometry into the field of complex carbohydrate analysis, we hereby report the development of a chipESI MS and MS/MS methodology for the screening of gangliosides. A strategy to characterize a complex ganglioside mixture from human cerebellar tissue, by automated ESIchip-quadrupole time-of-flight (QTOF) MS and MS/MS is presented here. The feasibility of this method, and the general experimental requirements for automated chipESI MS analysis of these carbohydrate species is

A method for ligand screening by automated nano-electrospray ionization mass spectrometry (nano-ESI/MS) is described. The core of the system consisted of a chip-based platform for automated sample delivery from a 96-well plate and subsequent analysis based on noncovalent interactions. Human fatty acid binding protein, H-FABP (heart) and A-FABP (adipose), with small potential ligands was analyzed. The technique has been compared with a previously reported method based on nuclear magnetic resonance (NMR), and excellent correlation with the found hits was obtained. In the current MS screening method, the cycle time per sample was 1.1 min, which is approximately 50 times faster than NMR for single compounds and approximately 5 times faster for compound mixtures. High reproducibility was achieved, and the protein consumption was in the range of 88 to 100 picomoles per sample. Furthermore, a novel protocol for preparation of A-FABP without the natural ligand is presented. The described screening approach is suitable for ligand screening very early in the drug discovery process before conventional high-throughput screens (HTS) are developed and/or used as a secondary screening for ligands identified by

Nanoflow electrospray mass spectrometry has been applied previously to investigate noncovalent protein-protein and protein-ligand interactions. Here we evaluate a commercial microchip device for this application. We show that the microchip can be used to obtain mass spectra of the noncovalent tetramer transthyretin. The device showed a 10-fold increase in signal stability compared with a nanoflow capillary and a high level of nozzle-to-nozzle reproducibility. Binding of the natural ligand thyroxine was clearly observed, and a range of small molecules proposed as inhibitors of transthyretin amyloidosis were shown to be effective in stabilizing the tetramer. We propose that measuring the ability of small molecules to stabilize protein complexes using this automated microchip technology will enable high-throughput screening of multi-protein complexes by mass spectrometry.