Proteomics application exercise of the Swiss Proteomics Society: report of the SPS'02 session

After the success of the mass spectrometry (MS) round table that was held at the first Swiss Proteomics Society congress (SPS'01) in Geneva, the SPS has organized a proteomics application exercise and allocated a full session at the SPS'02 congress. The main objective was to encourage the exchange of expertise in protein identification, with a focus on the use of mass spectrometry, and to create a bridge between the users' questions and the instrument providers' solutions. Two samples were sent to fifteen interested labs, including academic groups and MS hardware providers. Participants were asked to identify and partially characterize the samples. They consisted of a complex mixture of peptide/proteins (sample A) and an almost pure recombinant peptide carrying post-translational modifications (sample B). Sample A was an extract of snake venom from the species Bothrops jararaca. Sample B was a recombinant and modified peptide derived from the shrimp Penaeus vannamei penaeidin 3a. The eight labs that returned results reported the use of a wide range of MS instrumentation and techniques. They mentioned a variety of time and manpower allocations. The origin of sample A was generally identified together with a number of database protein entries. The difficulty of the sample identification lay in the incomplete knowledge of the Bothrops species genome sequence and is discussed. Sample B was generally and correctly identified as penaeidin. However, only one group reported the full primary structure. Interestingly, the approaches were again varied and are discussed in the text.     

Proteomic analysis of Lactococcus lactis,a lactic acid bacterium

Lactococcus lactis is a Gram-positive bacteria, which belongs to the group of lactic acid bacteria among which several genera play an essential role in the manufacture of food products. Cytosolic proteins of L. lactis IL1403 cultivated in M17 broth have been resolved by two-dimensional gel electrophoresis using two pH gradients (pH 4-7, 4.5-5.5). More than 230 spots were identified by peptide mass fingerprints, corresponding to 25% of the predicted acid proteome. The present study made it possible to describe at the proteome level a significant number of cellular pathways (glycolysis, fermentation, nucleotide metabolism, proteolysis, fatty acid and peptidoglycan synthesis) related to important physiological processes and technological properties. It also indicated that the fermentative metabolism, which characterizes L. lactis is associated with a high expression of glycolytic enzymes. Thirty-four proteins were matched to open reading frames for which there is no assigned function. The comparison at the proteome level of two strains of L. lactis showed an important protein polymorphism. The comparison of the proteomes of glucose- and lactose-grown cells revealed an unexpected link between the nature of the carbon source and the metabolism of pyrimidine nucleotides.     

Frog alien species: a way for genetic invasion?

European water frogs are characterized by anthropic introductions and Rana ridibunda may be considered as an invasive species. As such translocations may result in introgression of exotic genes in native populations, i.e. genetic pollution, we studied genetic characteristics (on 11 allozymic loci) of natural versus introduced water frogs. Our study contributed to (1) disclose 3 genetic markers allowing the identification of exotic frogs; (2) quantify the proportion of exotic frogs found in natural populations; and (3) suggest how genetic pollution may arise in these frogs.     

The future of bacteriophage biology

After an illustrious history as one of the primary tools that established the foundations of molecular biology, bacteriophage research is now undergoing a renaissance in which the primary focus is on the phages themselves rather than the molecular mechanisms that they explain. Studies of the evolution of phages and their role in natural ecosystems are flourishing. Practical questions, such as how to use phages to combat human diseases that are caused by bacteria, how to eradicate phage pests in the food industry and what role they have in the causation of human diseases, are receiving increased attention. Phages are also useful in the deeper exploration of basic molecular and biophysical questions.     

Real-time PCR based on SYBR-Green I fluorescence: An alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions

Background

Real-time PCR is increasingly being adopted for RNA quantification and genetic analysis. At present the most popular real-time PCR assay is based on the hybridisation of a dual-labelled probe to the PCR product, and the development of a signal by loss of fluorescence quenching as PCR degrades the probe. Though this so-called 'TaqMan' approach has proved easy to optimise in practice, the dual-labelled probes are relatively expensive.

Results

We have designed a new assay based on SYBR-Green I binding that is quick, reliable, easily optimised and compares well with the published assay. Here we demonstrate its general applicability by measuring copy number in three different genetic contexts; the quantification of a gene rearrangement (T-cell receptor excision circles (TREC) in peripheral blood mononuclear cells); the detection and quantification of GLI, MYC-C and MYC-N gene amplification in cell lines and cancer biopsies; and detection of deletions in the OPA1 gene in dominant optic atrophy.

Conclusion

Our assay has important clinical applications, providing accurate diagnostic results in less time, from less biopsy material and at less cost than assays currently employed such as FISH or Southern blotting.     

Light scattering studies of the solution properties of chitosans of varying degrees of acetylation

The use of two techniques, differential interferometry and quasi-elastic light scattering (QELS), allowed us to study solutions of chitosan varying in degree of acetylation (DA), degree of dissociation (alpha), and concentration (C(p)). With the first technique, we demonstrated the modification of the electric polarizability of the polymer chains, through a law of behavior of the variation of the refractive index increment dn/dC with DA and alpha. This brought us information on the various kinds of interactions (H-bonds, electrostatic, and hydrophobic) involved in the evolution of the solution properties. QELS experiments performed in dilute regime showed the presence of supramolecular structures depending on DA and alpha. The topology and the nature of these objects are discussed. The typical presence of aggregates and their evolution with concentration was also demonstrated in semidilute regime.     

Optimization of chitin extraction from shrimp shells

The aim of this paper is to define optimal conditions for the extraction of chitin from shrimp shells. The kinetics of both demineralization and deproteinization with, in the latter case, the role of temperature are studied. The characterization of the residual calcium and protein contents, the molecular weights, and degrees of acetylation (DA) allows us to propose the optimal conditions as follows. The demineralization is completely achieved within 15 min at ambient temperature in an excess of HCl 0.25 M (with a solid-to-liquid ratio of about 1/40 (w/v)). The deproteinization is conveniently obtained in NaOH 1 M within 24 h at a temperature close to 70 degrees C with no incidence on the molecular weight or the DA. In these conditions, the residual content of calcium in chitin is below 0.01%, and the DA is almost 95%.     

Crab shell chitin whisker reinforced natural rubber nanocomposites. 2. Mechanical behavior

In a previous work (part 1), nanocomposite materials were obtained using a latex of either unvulcanized or prevulcanized natural rubber as the matrix and a colloidal suspension of crab chitin whiskers as the reinforcing phase. The mechanical behavior of the resulting nanocomposite films was analyzed in both the linear and the nonlinear range in the present study. The effects of the filler and processing technique were evaluated, and the results are discussed based on the knowledge of the structural morphology and swelling behavior reported in our previous work. The reinforcing effect of chitin whiskers strongly depended on their ability to form a rigid three-dimensional network, resulting from strong interactions such as hydrogen bonds between the whiskers. The results emanating from the successive tensile test experiments give clear evidence for the presence of a three-dimensional chitin network within the evaporated samples. Cross-linking of the matrix was found to interfere with the formation of this network.