Evy Vierstraete

Gel-Based Versus Gel-Free Proteomics: A Review

With the sequencing of the genome of over 150 organisms, the field of biology has been revolutionised. Instead of studying one gene or protein at the time, it is now possible to study the effect of physiological or pathological changes on the expression of all genes or proteins in the organism. Proteomics aims at the simultaneous analysis of all proteins expressed by a cell, tissue or organism in a specific physiological condition. Because proteins are the effector molecules in all organisms, it is evident that changes in the physiological condition of an organism will be reflected by changes in protein expression and/or processing. Since the formulation of the concept of proteomics in the mid 90s proteomics has relied heavily on 2 dimensional gel electrophoresis (2DGE) for the separation and visualization of proteins. 2DGE, however, has a number of inherent drawbacks. 2DGE is costly, fairly insensitive to low copy proteins and cannot be used for the entire proteome. Therefore, over the years, several gel-free proteomics techniques have been developed to either fill the gaps left by 2DGE or to entirely abolish the gel based techniques. This review summarizes the most important gel-free and gel-based proteomics techniques and compares their advantages and drawbacks.

Proteomics in Drosophila melanogaster: first 2D database of larval hemolymph proteins

A proteomic approach was used for the identification of larval hemolymph proteins of Drosophila melanogaster. We report the initial establishment of a two-dimensional gel electrophoresis reference map for hemolymph proteins of third instar larvae of D. melanogaster. We used immobilized pH gradients of pH 4-7 (linear) and a 12-14% linear gradient polyacrylamide gel. The protein spots were silver-stained and analyzed by nanoLC-Q-Tof MS/MS (on-line nanoscale liquid chromatography quadrupole time of flight tandem mass spectrometry) or by Matrix assisted laser desorption time of flight MS (MALDI-TOF MS). Querying the SWISSPROT database with the mass spectrometric data yielded the identity of the proteins in the spots. The presented proteome map lists those protein spots identified to date. This map will be updated continuously and will serve as a reference database for investigators, studying changes at the protein level in different physiological conditions.

Phenotypic and Genome-Wide Analysis of an Antibiotic-Resistant Small Colony Variant (SCV) of Pseudomonas aeruginosa

Background Small colony variants (SCVs) are slow-growing bacteria, which often show increased resistance to antibiotics and cause latent or recurrent infections. It is therefore important to understand the mechanisms at the basis of this phenotypic switch. Methodology/Principal Findings One SCV (termed PAO-SCV) was isolated, showing high resistance to gentamicin and to the cephalosporine cefotaxime. PAO-SCV was prone to reversion as evidenced by emergence of large colonies with a frequency of 10−5 on media without antibiotics while it was stably maintained in presence of gentamicin. PAO-SCV showed a delayed growth, defective motility, and strongly reduced levels of the quorum sensing Pseudomonas quinolone signal (PQS). Whole genome expression analysis further suggested a multi-layered antibiotic resistance mechanism, including simultaneous over-expression of two drug efflux pumps (MexAB-OprM, MexXY-OprM), the LPS modification operon arnBCADTEF, and the PhoP-PhoQ two-component system. Conversely, the genes for the synthesis of PQS were strongly down-regulated in PAO-SCV. Finally, genomic analysis revealed the presence of mutations in phoP and phoQ genes as well as in the mexZ gene encoding a repressor of the mexXY and mexAB-oprM genes. Only one mutation occurred only in REV, at nucleotide 1020 of the tufA gene, a paralog of tufB, both encoding the elongation factor Tu, causing a change of the rarely used aspartic acid codon GAU to the more common GAC, possibly causing an increase of tufA mRNA translation. High expression of phoP and phoQ was confirmed for the SCV variant while the revertant showed expression levels reduced to wild-type levels. Conclusions By combining data coming from phenotypic, gene expression and proteome analysis, we could demonstrate that resistance to aminoglycosides in one SCV mutant is multifactorial including overexpression of efflux mechanisms, LPS modification and is accompanied by a drastic down-regulation of the Pseudomonas quinolone signal quorum sensing system.

Antimicrobial Compounds of Low Molecular Mass are Constitutively Present in Insects: Characterisation of β-Alanyl-Tyrosine

The number of bacterial and fungal strains that have developed resistance against the classical antibiotics continues to grow. The intensified search for new antibiotic lead compounds has resulted in the discovery of numerous endogenous peptides with antimicrobial properties in plants, bacteria and animals. Their possible applications as anti-infective agents are often limited by their size, in reference to production costs and susceptibility to proteases. In this article, we report recent isolations of antimicrobial compounds from insects, with molecular masses less than 1 kDa. Experimental approaches are discussed and the first data on the antimicrobial properties of beta-alanyl-tyrosine (252 Da), one of such low molecular mass compounds isolated from the fleshfly Neobellieria bullata, are presented. We also offer evidence for the constitutive presence of antimicrobial compounds in insects of different orders, in addition to the previously identified inducible antimicrobial peptides.

The hemolymph proteome of the honeybee: Gel‐based or gel‐free?

The honeybee has an invaluable economic impact and is a model for studying immunity, development and social behavior. The recent sequencing and annotation of the honeybee genome facilitates the study of its hemolymph, which reflects the physiological condition and mediates immune responses. We aimed at making a proteomic reference map of honeybee hemolymph and compared gel‐free and gel‐based techniques. One hundered and four 2‐DE spots corresponding to 62 different proteins were identified. Eight identical 2‐DLC experiments resulted in the identification of 32 unique proteins. One repeat was clearly not representative for the potential of the given 2‐DLC setup. Only 27% of the identified hemolymph proteins were found by both techniques. In addition, we found proteins of three different viruses which creates possibilities for biomarker design. Future hemolymph studies will benefit from this work.

Differential proteomics for studying Drosophila immunity

Abstract: Here, we report the identification of proteins associated with the immune response of Drosophila by analyzing the hemolymph profiles after infection. Two‐dimensional difference gel electrophoresis was used to study the secretome in the hemolymph of Drosophila larvae. Shortly after induction with lipopolysaccharides, we identified 10 proteins, which we designated ”Drosophila instantly released immune proteins”. Infection with Micrococcus luteus or Saccharomyces cerevisiae induced 20 and 19 differential protein spots, respectively. Next to known immune proteins, new candidates that require further investigation were identified.