Bioinformatics pipeline for metaproteomics data analysis : investigation on microbial populations and their respective functional role in cheese

The investigating functional molecular pathways of an organism are at the basis of the understanding of complex microbial ecosystems. In the case of unicellular organisms, the description of protein pathways provides information on the functionality of a microbial consortium. We recently developed a semi-automated pipeline for the analysis of metaproteomics data to depict the newborn mouse gut phylotypes.1 Here we present a refined bioinformatics tool to extend the analysis to a more interesting description of the metabolic functions correlated with the characterized microbial taxa, using a typical PDO Italian cheese as model. Metaproteomics raw data are analyzed to obtain a representation of the bacterial population at different taxonomic levels on the bases of the taxon-specificity of a tryptic peptide list, comparing it with that coming from the already available metaproteomics and metagenomics applications such as Megan2. As a further development we managed for the automated association of taxa to metabolic pathways (KEGG database) and of proteins to groups of cluster orthologs (COG). We optimized parameters to have the maximum number of protein and minimum FDR, and then developed a series of python scripts to integrate and improve, the output of available application, and manipulate raw data. This investigation aims at providing a functional insight in metagenomic analysis, and at offer a direct evaluation of protein functional pathways which are actually controlling the consortia homeostasis

Determination of the absolute configurations of chiral organometallic complexes via density functional theory calculations of their vibrational circular dichroism spectra: The chiral chromium tricarbonyl complex of N-pivaloyl-tetrahydroquinoline

The racemate of the chiral tricarbonyl-η6-arene-chromium(0) complex, tricarbonyl-η6-N-pivaloyl-tetrahydroquinoline-chromium(0), 1, has been synthesized and resolved using chromatography on a (R,R)-Whelk-O1 column. The Absolute Configuration (AC) of 1 has been determined using vibrational circular dichroism (VCD) spectroscopy. The VCD spectrum of 1 has been predicted using the Stephens equation for vibrational rotational strengths, implemented using density functional theory (DFT) in the gaussian program. Using the B3PW91 functional and the 6-311++G (2d,2p) basis set, the predicted VCD spectrum of S-1 is in excellent agreement with the experimental VCD spectrum of (+)-1, leading unambiguously to the AC S-(+). It is concluded that VCD is a useful technique for determining the ACs of chiral organometallic complexes, given the use of optimum functionals and basis sets

pH-regulated formation of side products in the reductive amination approach for differential labeling of peptides in relative quantitative experiments

Among the most common stable-isotope labeling strategies, the reaction of formaldehyde with peptides in the presence of NaCNBH₃ features many attractive aspects that are conducive to its employment in quantitation experiments in proteomics. Reductive amination, with formaldehyde and d(2)-formaldehyde, is reported to be a fast, easy, and specific reaction, undoubtedly inexpensive if compared with commercially available kits for differential isotope coding. Acetaldehyde and d(4)-acetaldehyde could be employed as well without a substantial increase in terms of cost, and should provide a wider spacing between the differentially tagged peptides in the mass spectrum. Nevertheless, only a single paper reports about a diethylation approach for quantitation. We undertook a systematic analytical investigation on the reductive amination of some standard peptides pointing out the occasional occurrence of side reactions in dependence of pH or reagents order of addition, particularly observing the formation of cyclic adducts ascribable to rearrangements involving the generated Schiff-base and all the nucleophilic sites of its chemical environment. We also tried to evaluate how much this side-products amount may impair isotope coded relative quantitation

The "Bridge" Game: Role of the Fourth Player in Chiral Recognition

A new team player: The “three-point interaction” model, which is usually employed to rationalize chiral recognition, does not account for the amazing enantioselectivity measured for the receptors of many proteic acceptors. Gas-phase experiments have indicated that at least a fourth “player” must be considered: the rigidity that a receptor opposes to distortions of its cavity resulting from noncovalent interactions with a chiral molecule