Synthesis of modified nucleoside 5'-triphosphates for in vitro selection of catalytic nucleic acids

2'-Modified pyrimidine nucleoside 5'-triphosphates comprising amino, imidazole and carboxylate functionality attached to the 5-position of the base were synthesized. Two different phosphorylation methods were used to optimize the yields of these highly modified triphosphates.     

Cerebrin prohormone processing, distribution and action in Aplysia californica

The isolation, characterization, and bioactivity in the feeding circuitry of a novel neuropeptide in the Aplysia californica central nervous system are reported. The 17-residue amidated peptide, NGGTADALYNLPDLEKIamide, has been termed cerebrin due to its primary location in the cerebral ganglion. Liquid chromatographic purification guided by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry allowed the isolation of the peptide with purity adequate for Edman sequencing. The cerebrin cDNA has been characterized and encodes an 86 amino acid prohormone that predicts cerebrin and one additional peptide. Mapping using in situ hybridization and immunocytochemistry showed that cerebrin containing neuronal somata are localized almost exclusively in the cerebral ganglion, mostly in the F- and C-clusters. Both immunostaining and mass spectrometry demonstrated the presence of cerebrin in the neurohemal region of the upper labial nerve. In addition, immunoreactive processes were detected in the neuropil of all of the ganglia, including the buccal ganglia, and in some interganglionic connectives, including the cerebral-buccal connective. This suggests that cerebrin may also function as a local signaling molecule. Cerebrin has a profound effect on the feeding motor pattern elicited by the command-like neuron CBI-2, dramatically shortening the duration of the radula protraction in a concentration-dependent manner, mimicking the motor-pattern alterations observed in food induced arousal states. These findings suggest that cerebrin may contribute to food-induced arousal in the animal. Cerebrin-like immunoreactivity is also present in Lymnaea stagnalis suggesting that cerebrin-like peptides may be widespread throughout gastropoda.     

d-Alanine in the islets of Langerhans of rat pancreas

Relatively high levels of d-alanine (d-Ala), an endogenous d-amino acid, have been found in the endocrine systems of several animals, especially in the anterior pituitary; however, its functional importance remains largely unknown. We observed d-Ala in islets of Langerhans isolated from rat pancreas in significantly higher levels than in the anterior/intermediate pituitary; specifically, 180 ± 60 fmol d-Ala per islet (300 ± 100 nmol/g islet), and 10 ± 2.5 nmol/g of wet tissue in pituitary. Additionally, 12 ± 5% of the free Ala in the islets was in the d form, almost an order of magnitude higher than the percentage of d-Ala found in the pituitary. Surprisingly, glucose stimulation of the islets resulted in d-Ala release of 0.6 ± 0.5 fmol per islet. As d-Ala is stored in islets and released in response to changes in extracellular glucose, d-Ala may have a hormonal role.     

Effects of Piper hispidinervum on spermatogenesis and histochemistry of ovarioles of Spodoptera frugiperda

The fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), not only damages crops, but controlling its population also requires synthetic insecticides, which leads to selection of resistant populations and environmental contamination. Essential oils are an alternative for controlling this insect. There are few studies of the effects of these oils on the insect's reproductive system. We evaluated the effects of the long pepper, Piper hispidinervum, essential oil on the gonads of the armyworm and tested its possible influence on the fertility of this insect. Dosages of 30 and 50 mg/ml were tested in 3^rd instar caterpillars using the leaf immersion method. Testes and ovarioles were collected, fixed with 10% formalin and embedded in Historesin. The sections were stained with toluidine blue and Mallory trichrome to detect connective tissue, periodic acid-Schiff to detect neutral carbohydrates, and bromophenol blue to detect proteins. We found that the long pepper essential oil affected negatively the spermatogenesis and altered the histochemistry of the ovarioles of S. frugiperda. The effects of long pepper oil suggest that it is a promising tool for controlling the armyworm pest.     

Accurate Assignment of Significance to Neuropeptide Identifications Using Monte Carlo K-Permuted Decoy Databases

In support of accurate neuropeptide identification in mass spectrometry experiments, novel Monte Carlo permutation testing was used to compute significance values. Testing was based on k-permuted decoy databases, where k denotes the number of permutations. These databases were integrated with a range of peptide identification indicators from three popular open-source database search software (OMSSA, Crux, and X! Tandem) to assess the statistical significance of neuropeptide spectra matches. Significance p-values were computed as the fraction of the sequences in the database with match indicator value better than or equal to the true target spectra. When applied to a test-bed of all known manually annotated mouse neuropeptides, permutation tests with k-permuted decoy databases identified up to 100% of the neuropeptides at p-value < 10⁻⁵. The permutation test p-values using hyperscore (X! Tandem), E-value (OMSSA) and Sp score (Crux) match indicators outperformed all other match indicators. The robust performance to detect peptides of the intuitive indicator “number of matched ions between the experimental and theoretical spectra” highlights the importance of considering this indicator when the p-value was borderline significant. Our findings suggest permutation decoy databases of size 1×10⁵ are adequate to accurately detect neuropeptides and this can be exploited to increase the speed of the search. The straightforward Monte Carlo permutation testing (comparable to a zero order Markov model) can be easily combined with existing peptide identification software to enable accurate and effective neuropeptide detection. The source code is available at http://stagbeetle.animal.uiuc.edu/pepshop/MSMSpermutationtesting.     

Microscale NMR

NMR spectroscopy is increasingly being used to characterize microliter and smaller-volume samples. Substances at picomole levels have been identified using NMR spectrometers equipped with microcoil-based probes. NMR probes that incorporate multiple sample chambers enable higher-throughput NMR experiments. Hyphenation of capillary-scale separations and microcoil NMR has also decreased analysis time of mixtures. For example, capillary isotachophoresis/NMR allows the highest mass sensitivity nanoliter-volume flow cells to be used with low microliter volume samples because isotachophoresis concentrates the microliter volume sample into the nanoliter volume NMR detection probe. In addition, the diagnostic capabilities of NMR spectroscopy allow the physico-chemical aspects of a capillary separation process to be characterized on-line. Because of such advances, the application of NMR to smaller samples continues to grow.     

An enhanced protein crosslink identification strategy using CID-cleavable chemical crosslinkers and LC/MS(n) analysis

We describe a novel two-step LC/MS(n) strategy to effectively and confidently identify numerous crosslinked peptides from complex mixtures. This method incorporates the use of our gas-phase cleavable crosslinking reagent, disuccinimidyl-succinamyl-aspartyl-proline (SuDP), and a new data-processing algorithm CXLinkS (Cleavable Crosslink Selection), which enables unequivocal crosslink peptide selection and identification on the basis of mass measurement accuracy, high resolving power, and the unique fragmentation pattern of each crosslinked peptide. We demonstrate our approach with well-characterized monomeric and multimeric protein systems with and without database searching restrictions where inter-peptide crosslink identification is increased 8-fold over our previously published data-dependent LC/MS3 method and discuss its applicability to other CID-cleavable crosslinkers and more complex protein systems.     

Adapting the stretched sample method from tissue profiling to imaging

The characterization and localization of peptides and proteins in tissues provides information that aids in understanding their function and in characterizing disease states. Over the past decades, the use of MS for the profiling and imaging of biological compounds from tissues has evolved into a powerful modality to accomplish these studies. One recently described sampling approach, the stretched sample method (Monroe, E. B. et al.., Anal. Chem. 2006, 78, 6826-6832), places a tissue section onto an array of glass beads embedded on a Parafilm M membrane. When the membrane is stretched, it separates the tissue section into thousands of cell-sized pieces for tissue profiling by MALDI-MS. The physical separation between beads eliminates analyte redistribution during matrix application and allows long analyte extraction periods without loss of spatial resolution. Here, we enhance this sampling approach by introducing algorithms that enable the reconstruction of ion images from these stretched samples. As the first step, a sample-tailored data acquisition method is devised to obtain mass spectra exclusively from the beads, thereby reducing the time, instrument resources, and data handling required for such MS imaging (MSI) experiments. Next, an image reconstruction algorithm matches data acquired from the stretched sample to the initial bead locations. The efficacy of this method is demonstrated using peptide-coated beads with known peptide distributions and appears well-suited to the MSI of heterogeneous tissue samples.     

Prediction of neuropeptide cleavage sites in insects

MOTIVATION: The production of neuropeptides from their precursor proteins is the result of a complex series of enzymatic processing steps. Often, the annotation of new neuropeptide genes from sequence information outstrips biochemical assays and so bioinformatics tools can provide rapid information on the most likely peptides produced by a gene. Predicting the final bioactive neuropeptides from precursor proteins requires accurate algorithms to determine which locations in the protein are cleaved. RESULTS: Predictive models were trained on Apis mellifera and Drosophila melanogaster precursors using binary logistic regression, multi-layer perceptron and k-nearest neighbor models. The final predictive models included specific amino acids at locations relative to the cleavage sites. Correct classification rates ranged from 78 to 100% indicating that the models adequately predicted cleaved and non-cleaved positions across a wide range of neuropeptide families and insect species. The model trained on D.melanogaster data had better generalization properties than the model trained on A. mellifera for the data sets considered. The reliable and consistent performance of the models in the test data sets suggests that the bioinformatics strategies proposed here can accurately predict neuropeptides in insects with sequence information based on neuropeptides with biochemical and sequence information in well-studied species.