PepC: proteomics software for identifying differentially expressed proteins based on spectral counting.

UNLABELLED: Identifying biologically significant changes in protein abundance between two conditions is a key issue when analyzing proteomic data. One widely used approach centers on spectral counting, a label-free method that sums all the tandem mass spectra for a protein observed in an analysis. To assess the significance of the results, we recently combined the t-test and G-test, with random permutation analysis, and we validated this approach biochemically. To automate the statistical method, we developed PepC, a software program that balances the trade-off between the number of differentially expressed proteins identified and the false discovery rate. This tool can be applied to a wide range of proteomic datasets, making data analysis rapid, reproducible and easily interpretable by proteomics specialists and non-specialists alike. AVAILABILITY AND IMPLEMENTATION: The software is implemented in Java. It has been added to the Trans Proteomic Pipeline project's 'Petunia' web interface, but can also be run as a command line program. The source code is GNU Lesser General Public License and the program is freely available on the web. http://sashimi.svn.sourceforge.net/viewvc/sashimi/trunk/trans_proteomic_pipeline/src/Quantitation/Pepc.

Sequence information, distinction and quantitation of C-terminal leucine and isoleucine in ternary complexes of tripeptides with Cu(II) and 2,2'-bipyridine.

Tripeptides form ternary complexes with Cu(2+) and 2,2'-bipyridine (bpy) that self-assemble upon mixing the components in aqueous methanol solution. Electrospray ionization (ESI) of the complex solutions provides abundant singly charged [Cu(peptide -- H)bpy](+) and doubly charged [Cu(peptide)bpy](2+) ions. Collision-induced dissociation (CID) at low ion kinetic energies of several tripeptides, AGG, GGA, LGG, GGL, GGI, FGG, GGF, LGF, GLF, GFL, GYA and GAY, showed fragments that were indicative of the amino acid sequence in the peptide. In addition, CID of single and doubly charged complexes of isomeric tripeptides GGL and GGI provided unambiguous distinction of the isomeric leucine and isoleucine residues. Leucine peptides eliminated C(3)H(7) radicals from the amino acid side-chain whereas isoleucine eliminated C(2)H(5) radicals. CID of gas-phase doubly charged peptide complexes in a quadrupole ion trap produced a series of singly charged sequence fragments that following isolation and further CID furnished distinct fragments that allowed quantitation of leucine and isoleucine-containing peptides in mixtures.

Saponins from Mussaenda pubescens.

Two new triterpenoid saponins, named mussaendosides G and K, were isolated from aerial parts of Mussaenda pubescens by normal and reverse phase chromatography. On the basis of chemical and spectroscopic methods, their structures have been elucidated as heinsiagenin A 3-O-{alpha-L-rhamnopyranosyl(1-->2)-[beta-D-glucopyranosyl (1-->6)]-beta-D-glucopyranosyl(1-->2)}-alpha-L -rhamnopyranosyl(1-->4)-O-beta-D-glucopyranoside and 3 beta,19 alpha-dihydroxyl-olean-12-en-24, 28-dioic acid-24,28-di-O-beta-D-glucopyranoside, respectively.

Lability of N-alkylated peptides towards TFA cleavage.

Trifluoroacetic acid (TFA) is a common reagent in both solid-phase and solution peptide synthesis. It is used for the deprotection and/or cleavage of the synthesized peptide from the resin. The use of TFA under these standardized conditions is thought to be sufficiently mild, thereby preventing degradation of the desired product. However, peptides of the general structure R1-(N-alkyl X1)-X2-R2 are hydrolyzed by standard TFA solid-phase peptide synthesis (SPPS) cleavage/deprotection conditions providing fragments R1-(N-alkyl X1)-OH and H-X2-R2. The fragmentation is observed during a TFA cleavage both from the resin and in solution. The hydrolysis is proposed to proceed via an oxazolone-like intermediate in which equilibration of the chiral center of the N-alkylated residue occurs. This mechanism is supported by H/D exchange as observed MS and NMR in conjunction with HPLC.

Carboxylate and amine terminus directed fragmentations in gaseous dipeptide complexes with copper (II) and diimine ligands formed by electrospray.

Singly and doubly charged peptide complexes with copper(II) and 2,2'-bipyridyl (bpy) are formed in the gas phase by electrospraying water-methanol solutions containing the components. Collisionally activated dissociations at low kinetic energies of singly charged complexes of the [CuII(peptide-H)(bpy)].+ type provide information about the amino acid sequence for L-Phe-Leu, L-Leu-Phe, L-Phe-Pro, L-Pro-Phe, L-Phe-Met, L-Met-Phe, L-Ser-Phe, L-Asp-Phe, and L-His-Phe. Dissociations of doubly charged complexes of the [CuII(peptide)(bpy).2+ type also allow identification of the N- and C-terminal amino acid residues. Leucine and isoleucine residues are readily distinguished in L-Ala-Leu and L-Ala-Ile through dissociations of their Cu complexes. Ion dissociation mechanisms, as elucidated by deuterium labeling, are discussed.

Synthesis of O-[2-acetamido-2-deoxy-6-O-stearoyl- and -6-O-(2-tetradecylhexadecanoyl)-beta-D-glucopyranosyl]-(1-->4)-N- acetylnormuramoyl-L-alpha-aminobutanoyl-D-isoglutamine, lipophilic disaccharide analogues of MDP.

Silver triflate-promoted condensation of 3,4,6-tri-O-acetyl-2-deoxy-2- phthalimido-beta-D-glucopyranosyl bromide (1) with benzyl 2-acetamido-6-O-benzyl-2-deoxy-3-O- (methoxycarbonyl)methyl-alpha-D-glucopyranoside (4) afforded the key compound, benzyl 2-acetamido-6-O-benzyl-2-deoxy-3-O- (methoxy-carbonyl)methyl-4-O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b eta-D- glucopyranosyl)-alpha-D-glucopyranoside (5), which after deprotection was transformed into acid 10. Condensation of 10 with the benzyl ester of L-alpha-aminobutanoyl-D-isoglutamine and deisopropylidenation of the product 11 afforded the benzyl ester of N-(2-O-[benzyl 2-acetamido-4-O-(2-acetamido-3-O-benzyloxymethyl-2- deoxy-beta-D-glucopyranosyl)-6-O-benzyl-2,3-dideoxy-alpha-D-glucopyra nosid-3- yl]glycoloyl)-L-alpha-aminobutanoyl-D-isoglutamine (12). Partial O-acylation of 12 and hydrogenolysis of protecting groups gave the 6-O-stearoyl- and 6-O-(2-tetradecylhexadecanoyl)-disaccharide-dipeptides 17 and 18, respectively. Pyrogenicity and adjuvant activity in cell-mediated immunity are reported.

Contents of poison apparatus and their relation to trail-following in the antDaceton armigerum.

The poison gland, which is the source of the trail pheromone inDaceton armigerum, contains a mixture of dimethylpyrazine, trimethylpyrazine and 3-ethyl-2,5-dimethylpyrazine in approximately equal proportions, similar to that of the antManica rubida. The cross-activity in trail-following tests with several other species can thus be explained. The Dufour gland ofD. armigerum contains a mixture of 26 identified hydrocarbons, chiefly 9-tricosene, tricosane, and 9-pentacosene. It also contains very small amounts of two sesquiterpene aldehydes, tetramorine-2 and tetramorine-3, already known in other myrmicine Dufour glands. No volatile substances were detected in the pygidial gland.