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Pathogenic species of Leptospira cause leptospirosis, a bacterial zoonotic disease with a global distribution affecting over one million people annually. Reservoir hosts of leptospirosis, including rodents, dogs, and cattle, exhibit little to no signs of disease but shed large numbers of organisms in their urine. Transmission occurs when mucosal surfaces or abraded skin come into contact with infected urine or urine-contaminated water or soil. Whilst little is known about how Leptospira adapt to and persist within a reservoir host, in vitro studies suggest that leptospires alter their transcriptomic and proteomic profiles in response to environmental signals encountered during mammalian infection. We applied the dialysis membrane chamber (DMC) peritoneal implant model to compare the whole cell proteome of in vivo derived leptospires with that of leptospires cultivated in vitro at 30°C and 37°C by 2-dimensional difference in-gel electrophoresis (2-D DIGE). Of 1,735 protein spots aligned across 9 2-D DIGE gels, 202 protein spots were differentially expressed ( p < 0.05, fold change >1.25 or < -1.25) across all three conditions. Differentially expressed proteins were excised for identification by mass spectrometry. Data are available via ProteomeXchange with identifier PXD006995. The greatest differences were detected when DMC-cultivated leptospires were compared with IV30- or IV37-cultivated leptospires, including the increased expression of multiple isoforms of Loa22, a known virulence factor. Unexpectedly, 20 protein isoforms of LipL32 and 7 isoforms of LipL41 were uniformly identified by DIGE as differentially expressed, suggesting that unique post-translational modifications (PTMs) are operative in response to mammalian host conditions. To test this hypothesis, a rat model of persistent renal colonization was used to isolate leptospires directly from the urine of experimentally infected rats. Comparison of urinary derived leptospires to IV30 leptospires by 2-D immunoblotting confirmed that modification of proteins with trimethyllysine and acetyllysine occurs to a different degree in response to mammalian host signals encountered during persistent renal colonization. These results provide novel insights into differential protein and PTMs present in response to mammalian host signals which can be used to further define the unique equilibrium that exists between pathogenic leptospires and their reservoir host of infection.

The key to successful cryopreservation lies in the induction of tolerance towards dehydration/desiccation and freezing. The accumulation of osmo-active compounds, which can be induced by drought and cold stress, is therefore important. In the present study, three-week old shoots from in vitro plantlets of the cultivated potato Solanum tuberosum and its frost-resistant relative Solanum commersonii were submitted to osmotic stress (by using sucrose) and chilling (6°C). After 14days of exposure, shoot tips were sampled in order to gain an insight into changes of the proteome and soluble sugars. Also, the effect of these treatments on growth performance behaviour and on the success of cryopreservation was evaluated. Identified proteins that changed in abundance due to stress were associated with stress response. Additionally, carbohydrate analyses in both species, after exposure to chilling, also indicated species-related differences; this observation could point towards a better-adapted physiological state of the donor plants of S. commersonii prior to the cryoprocedure and therefore a better recovery of the meristems., Biological Significance: To our knowledge, this is the first study in which cryopreservation experiments are combined with the observation of the responses to abiotic stress exposure involving the potato species S. commersonii and S. tuberosum. These two species are known to have a different cold-acclimation behaviour, which seems to be closely related to their tolerance towards cryopreservation. Furthermore, common and differential responses to abiotic stresses were observed in the two species indicating that some pathways could be crucial not only in the plant's response to stress but also in tolerance towards cryopreservation., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Concern has been expressed about the overuse of biocides in farm animal production and food industries. Biocide application can create selective pressures that lead to increased tolerance to one or more of these compounds and are concomitant with the emergence of cross-resistance to antibiotics. A triclosan sensitive Salmonella enterica serovar Typhimurium and the isogenic triclosan tolerant mutant were studied at the proteomic level in order to elucidate cellular mechanisms that facilitate biocide tolerance. 2-D differential fluorescent gel electrophoresis (DIGE) compared protein profiles of parent and mutant Salmonella, in the presence and absence of triclosan. Differentially expressed proteins were identified by mass spectrometry and divided into two groups: Group A describes proteins differentially expressed between susceptible and triclosan tolerant Salmonella and includes the known triclosan target FabI which contained a mutation at the triclosan target binding site. Group B identified proteins differentially expressed in response to triclosan exposure and defines a general cell defence network. Only four proteins were common to both groups highlighting the diverse range of pathways employed by Salmonella to counteract biocides. These data suggest that sub-lethal concentrations of triclosan induce discernible changes in the proteome of exposed Salmonella and provide insights into mechanisms of response and tolerance., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The overall study of post-translational modifications (PTMs) of proteins is gaining strong interest. Beside phosphorylation and glycosylation, truncations of the nascent polypeptide chain at the N- or C-terminus are by far the most common types of PTMs. Nevertheless, little attention has been paid to the development of approaches that allow a systematic analysis of these proteolytic processing events. Here we present a protocol that allows the identification of the C-terminal sequence of proteins separated by two-dimensional polyacrylamide gel electrophoresis (2DE). For each purified protein, a peptide mixture is generated by cleavage of the protein with cyanogen bromide. During incubation with carboxypeptidases only the original C-terminal fragment forms a ladder. Ladder readout is performed using MALDI mass spectrometry. 2DE-separated proteins from Shewanella oneidensis were chosen as a model system to investigate the effectiveness of the approach.

In this protocol, we describe an approach in which two-dimensional electrophoresis (2DE)-separated proteins are guanidinated in-gel prior to enzymatic cleavage. In contrast to previously described techniques, this procedure allows the extracted tryptic peptides to be N-terminally sulfonated without any further sample purification. The protocol was applied on a proteomic study of 2DE-separated proteins from Halorhodospira halophila, an extremophilic eubacterium with an unsequenced genome at the moment of analysis.

Because protein identifications rely on matches with sequence databases, high-throughput proteomics is currently largely restricted to those species for which comprehensive sequence databases are available. The identification of proteins derived from organisms with unsequenced genomes mainly depends on homology searching. Here, we report the use of a simplified, gel-based, chemical derivatization strategy for de novo sequence analysis using a MALDI-TOF/TOF mass spectrometer. This approach allows the determination of de novo peptide sequences of up to 20 amino acid residues in length. The protocol was applied on a proteomic study of 2-D PAGE-separated proteins from Halorhodospira halophila, an extremophilic eubacterium with yet unsequenced genome. Using three different homology-based search algorithms, we were able to identify more than 30 proteins from this organism using subpicomole quantities of protein.

Here we report a novel approach in which gel-separated proteins are guanidinated in-gel prior to enzymatic cleavage. In contrast to previously described techniques, this procedure allows the extracted tryptic peptides to be N-terminal sulfonated without any further sample purification. The derivatized peptides were subsequently fragmented using a matrix-assisted laser desorption/ionization time of flight/time of flight instrument. The approach facilitates the de novo sequence analysis and allows obtaining longer stretches of amino acid sequence information. We demonstrate that the obtained information can be used to identify proteins using a sequence similarity search algorithm. The technique was compared to the standard peptide mass fingerprint approach, applied either in-gel or in solution, using a number of sodium dodecyl sulfate-polyacrylamide gel electrophoresis separated model proteins. Finally, the new protocol was applied on a proteomic study of two-dimensional PAGE separated proteins from Shewanella oneidensis. More than 50 proteins from this organism were identified using sub-picomol quantities of protein, and peptide sequences of up to 20 amino acid residues in length have been determined.