Search

HSP70s constitute a family of chaperones, some isoforms of which appear to play a role in sperm function. Notably, global proteomic studies analysing proteins deregulated in asthenozoospermia, a main cause of male infertility characterized by low sperm motility, showed the deregulation of some HSP70 isoforms. However, to date, no clear trend has been established since the variations in the abundance of HSP70 isoforms differed between studies. The HSPA2 isoform has been reported to play a key role for fertilization, but its deregulation and possible relocation during capacitation, a maturation process making the spermatozoon capable to fertilize an oocyte, is debated in the literature. The aim of the present study was to investigate the fate of all sperm HSP70 isoforms during capacitation and in relation to sperm motility. Using Multiple-Reaction Monitoring (MRM) mass spectrometry, we showed that the relative abundance of all detected isoforms was stable between non-capacitated and capacitated spermatozoa. Immunofluorescence using two different antibodies also demonstrated the stability of HSP70 isoform localization during capacitation. We also investigated spermatozoa purified from 20 sperm samples displaying various levels of total and progressive sperm motility. We showed that the abundance of HSP70 isoforms is not correlated to sperm total or progressive motility and does not vary significantly between astheno-teratozoospermic, normozoospermic, and teratozoospermic samples.

Poly(hydroxybutyrate-co-hydroxyhexanoate) [P(HB-co-HHx)] and poly(hydroxybutyrate-co-hydroxyvalerate-co-hydroxyhexanoate) [P(HB-co-HV-co-HHx)] demonstrate interesting mechanical and thermal properties as well as excellent biocompatibility, making them suitable for multiple applications and notably biomedical purposes. The production of such polymers was described in Rhodospirillum rubrum, a purple nonsulfur bacteria in a nutrient-lacking environment where the HHx synthesis is triggered by the presence of hexanoate in the medium. However, the production of P(HB-co-HHx) under nutrient-balanced growth conditions in R. rubrum has not been described so far, and the assimilation of hexanoate is poorly documented. In this study, we used proteomic analysis and a mutant fitness assay to demonstrate that hexanoate assimilation involve β-oxidation and the ethylmalonyl-coenzyme A (CoA) (EMC) and methylbutanoyl-CoA (MBC) pathways, both being anaplerotic pathways already described in R. rubrum. Polyhydroxyalkanoate (PHA) production is likely to involve the de novo fatty acid synthesis pathway. Concerning the polymer composition, HB is the main component of the polymer, probably as acetyl-CoA and butyryl-CoA are intermediates of hexanoate assimilation pathways. When no essential nutrient is lacking in the medium, the synthesis of PHA seems to help maintain the redox balance of the cell. In this framework, we showed that the fixation of CO(2) is required to sustain the growth. An increase in the proportion of HHx in the polymer was observed when redox stress was engendered in the cell under bicarbonate-limiting growth conditions. The addition of isoleucine or valerate in the medium also increased the HHx content of the polymer and allowed the production of a terpolymer of P(HB-co-HV-co-HHx). IMPORTANCE The use of purple bacteria, which can assimilate volatile fatty acids, for biotechnological applications has increased, since they reduce the production costs of added-value compounds such as PHA. P(HB-co-HHx) and P(HB-co-HV-co-HHx) have demonstrated interesting properties, notably for biomedical applications. In a nutrient-lacking environment, R. rubrum is known to synthesize such polymers when hexanoate is used as the carbon source. However, their production in R. rubrum in non-nutrient-lacking growth conditions has not been described so far, and the assimilation of hexanoate is poorly documented. As the carbon source and its assimilation directly impact the polymer composition, we studied under non-nutrient-lacking growth conditions the assimilation pathway of hexanoate and PHA production in R. rubrum. Proteomic analysis and mutant fitness assays allowed us to explain PHA production and composition. An increase in HHx content of the polymer and production of P(HB-co-HV-co-HHx) was possible using the knowledge gained on metabolism under hexanoate growth conditions.

Plasmids carrying metal resistance genes (MRGs) have been suggested to be key ecological players in the adaptation of metal-impacted microbial communities, making them promising drivers of bio-remediation processes. However, the impact of metals on plasmid-mediated spread of MRGs through selection, plasmid loss, and transfer is far from being fully understood. In the present study, we used two-member bacterial communities to test the impact of lead on the dispersal of the IncP plasmid pKJK5 from a Pseudomonas putida KT2440 plasmid donor and two distinct recipients, Variovorax paradoxus B4 or Delftia acidovorans SPH-1 after 4 and 10 days of mating. Two versions of the plasmid were used, carrying or not carrying the lead resistance pbrTRABCD operon, to assess the importance of fitness benefit and conjugative potential for the dispersal of the plasmid. The spread dynamics of metal resistance conveyed by the conjugative plasmid were dependent on the recipient and the lead concentration: For V. paradoxus, the pbr operon did not facilitate neither lead resistance nor variation in plasmid spread. The growth gain brought by the pbr operon to D. acidovorans SPH-1 and P. putida KT2440 at 1 mM Pb enhanced the spread of the plasmid. At 1.5 mM Pb after 4 days, the proteomics results revealed an oxidative stress response and an increased abundance of pKJK5-encoded conjugation and partitioning proteins, which most likely increased the transfer of the control plasmid to D. acidovorans SPH-1 and ensured plasmid maintenance. As a consequence, we observed an increased spread of pKJK5-gfp. Conversely, the pbr operon reduced the oxidative stress response and impeded the rise of conjugation- and partitioning-associated proteins, which slowed down the spread of the pbr carrying plasmid. Ultimately, when a fitness gain was recorded in the recipient strain, the spread of MRG-carrying plasmids was facilitated through positive selection at an intermediate metal concentration, while a high lead concentration induced oxidative stress with positive impacts on proteins encoding plasmid conjugation and partitioning.

Background The great metabolic versatility of the purple non-sulfur bacteria is of particular interest in green technology. Rhodospirillum rubrum S1H is an α-proteobacterium that is capable of photoheterotrophic assimilation of volatile fatty acids (VFAs). Butyrate is one of the most abundant VFAs produced during fermentative biodegradation of crude organic wastes in various applications. While there is a growing understanding of the photoassimilation of acetate, another abundantly produced VFA, the mechanisms involved in the photoheterotrophic metabolism of butyrate remain poorly studied. Results In this work, we used proteomic and functional genomic analyses to determine potential metabolic pathways involved in the photoassimilation of butyrate. We propose that a fraction of butyrate is converted to acetyl-CoA, a reaction shared with polyhydroxybutyrate metabolism, while the other fraction supplies the ethylmalonyl-CoA (EMC) pathway used as an anaplerotic pathway to replenish the TCA cycle. Surprisingly, we also highlighted a potential assimilation pathway, through isoleucine synthesis and degradation, allowing the conversion of acetyl-CoA to propionyl-CoA. We tentatively named this pathway the methylbutanoyl-CoA pathway (MBC). An increase in isoleucine abundance was observed during the early growth phase under butyrate condition. Nevertheless, while the EMC and MBC pathways appeared to be concomitantly used, a genome-wide mutant fitness assay highlighted the EMC pathway as the only pathway strictly required for the assimilation of butyrate. Conclusion Photoheterotrophic growth of Rs. rubrum with butyrate as sole carbon source requires a functional EMC pathway. In addition, a new assimilation pathway involving isoleucine synthesis and degradation, named the methylbutanoyl-CoA (MBC) pathway, could also be involved in the assimilation of this volatile fatty acid by Rs. rubrum.

The ability of cyanobacterium Arthrospira sp. to assimilate waste nitrogen sources (ammonium and urea) makes it an important candidate for wastewater management. The aim of this work was to evaluate a cultivation approach based on continuous-transitional-feeding regime (nitrate-ammonium-nitrate) in a photobioreactor to assess the effects of ammonium salts on Arthrospira sp. PCC 8005 metabolism. Using a comprehensive biochemical, proteomic and stoichiometric profiling of biomass, this study demonstrated that the proposed cultivation approach could increase the proteins and pigments yields by 20-30%, compared to the respective yields obtained from wild-type Arthrospira sp. strain A light-energy-transfer model was used to predict the biomass and oxygen productivities of Arthrospira sp. cultivated under transitional-feeding regime. 95 ± 2% match was observed between the experimental and simulated productivities. This study thus opened new avenues for use of ammonium rich wastewater for commercial production of high value pigments, biofuel and bioplastics using Arthrospira sp.

The aim of the present study was to evaluate the effects of varying concentrations of different nitrogen sources (individually or in combination) on the biochemical, transcriptomic and proteomic profiles of Arthrospira sp. PCC 8005 under batch and continuous modes. In batch mode, while ammonium showed a repressive effect on nitrate-assimilation pathway of the cyanobacteria; better growth and nutrient uptake rate were observed in presence of urea than nitrate. The inhibitory effect of ammonium was further confirmed by the continuous photobioreactor study wherein the nutrient feed was transiently replaced from nitrate to ammonium (28mM turbiostat regime). The changes in lipid, exopolysaccharide, transcriptomic and proteomic profiles of cyanobacteria on transition from nitrate to ammonium indicated at an onset of nutrient stress.

This study was conducted with the aim of embedding circular economies (waste recycling) with photosynthetic biorefineries, for production of commercially viable by-products. Since nitrogen source constitute the major input costs for commercial Arthrospira sp. production, the use of nitrogen rich wastewater for Arthrospira sp. cultivation could significantly reduce their production costs. This study evaluated the effects of high concentrations (8.5–120 mM) of alternative nitrogen sources (urea, ammonium and nitrite) on the biochemical, pigment and proteomic profile of Arthrospira sp., under batch and continuous conditions. Arthrospira sp. cells fed with urea were quantified with modified biochemical and proteomic profile compared to the nitrate fed cells. No inhibitory effect of urea was observed on the biomass even at 120 mM. Nitrite fed cells exhibited comparable biochemical and proteomic profiles as nitrate fed cells. These results clearly indicated at the possibility of using urea rich wastewater streams for profitable cultivation of Arthrospira sp.

Purple nonsulfur bacteria represent a promising resource for biotechnology because of their great metabolic versatility. Rhodospirillum rubrum has been widely studied regarding its metabolism of volatile fatty acid, mainly acetate. As the glyoxylate shunt is unavailable in Rs. rubrum , the citramalate cycle pathway and the ethylmalonyl-coenzyme A (CoA) pathway are proposed as alternative anaplerotic pathways for acetate assimilation. However, despite years of debate, neither has been confirmed to be essential. Here, using functional genomics, we demonstrate that the ethylmalonyl-CoA pathway is required for acetate photoassimilation. Moreover, an unexpected reversible long-term adaptation is observed, leading to a drastic decrease in the lag phase characterizing the growth of Rs. rubrum in the presence of acetate. Using proteomic and genomic analyses, we present evidence that the adaptation phenomenon is associated with reversible amplification and overexpression of a 60-kb genome fragment containing key enzymes of the ethylmalonyl-CoA pathway. Our observations suggest that a genome duplication and amplification phenomenon is not only involved in adaptation to acute stress but can also be important for basic carbon metabolism and the redox balance. IMPORTANCE Purple nonsulfur bacteria represent a major group of anoxygenic photosynthetic bacteria that emerged as a promising resource for biotechnology because of their great metabolic versatility and ability to grow under various conditions. Rhodospirillum rubrum S1H has notably been selected by the European Space Agency to colonize its life support system, called MELiSSA, due to its capacity to perform photoheterotrophic assimilation of volatile fatty acids (VFAs), mainly acetate. VFAs are valuable carbon sources for many applications, combining bioremediation of contaminated environments with the generation of added-value products. Acetate is one of the major volatile fatty acids generated as a by-product of fermentation processes. In Rs. rubrum , purple nonsulfur bacteria, the assimilation of acetate is still under debate since two different pathways have been proposed. Here, we clearly demonstrate that the ethylmalonyl-CoA pathway is the major anaplerotic pathway for acetate assimilation in this strain. Interestingly, we further observed that gene duplication and amplification, which represent a well-known phenomenon in antibiotic resistance, also play a regulatory function in carbon metabolism and redox homeostasis.

Cyanobacteria are photosynthetic prokaryotes that play a crucial role in the Earth’s nitrogen and carbon cycles. Nitrogen availability is one of the most important factors in cyanobacterial growth. Interestingly, filamentous non-diazotrophic cyanobacteria, such asArthrospirasp. PCC 8005, have developed survival strategies that enable them to adapt to nitrogen deprivation. Metabolic studies recently demonstrated a substantial synthesis and accumulation of glycogen derived from amino acids during nitrogen starvation. Nevertheless, the regulatory mechanism of this adaptation is poorly understood. To the best of our knowledge, this study is the first proteomic and cellular analysis ofArthrospirasp. PCC 8005 under nitrogen depletion. Label-free differential proteomic analysis indicated the global carbon and nitrogen reprogramming of the cells during nitrogen depletion as characterized by an upregulation of glycogen synthesis and the use of endogenous nitrogen sources. The degradation of proteins and cyanophycin provided endogenous nitrogen when exogenous nitrogen was limited. Moreover, formamides, cyanates and urea were also potential endogenous nitrogen sources. The transporters of some amino acids and alternative nitrogen sources such as ammonium permease 1 were induced under nitrogen depletion. Intriguingly, althoughArthrospirais a non-diazotrophic cyanobacterium, we observed the upregulation of HetR and HglK proteins, which are involved in heterocyst differentiation. Moreover, after a long period without nitrate, only a few highly fluorescent cells in each trichome were observed, and they might be involved in the long-term survival mechanism of this non-diazotrophic cyanobacterium under nitrogen deprivation.

Herpesviruses are highly prevalent viruses associated with numerous pathologies both in animal and human populations. Until now, most of the strategies used to prevent or to cure these infections have been unsuccessful because these viruses have developed numerous immune evasion mechanisms. Therefore, a better understanding of their complex lifecycle is needed. In particular, while the genome of numerous herpesviruses has been sequenced, the exact composition of virions remains unknown for most of them. Mass spectrometry has recently emerged as a central method and has permitted fundamental discoveries in virology. Here, we review mass spectrometry-based approaches that have recently allowed a better understanding of the composition of the herpesvirus virion. In particular, we describe strategies commonly used for proper sample preparation and fractionation to allow protein localization inside the particle but also to avoid contamination by nonstructural proteins. A collection of other important data regarding post-translational modifications or the relative abundance of structural proteins is also described. This review also discusses the poorly studied importance of host proteins in herpesvirus structural proteins and the necessity to develop a quantitative workflow to better understand the dynamics of the structural proteome. In the future, we hope that this collaborative effort will assist in the development of new strategies to fight these infections.

Paratuberculosis is a chronic enteritis caused in domestic and wild ruminant species by Mycobacterium avium subsp. paratuberculosis (MAP) that is responsible for major economic losses to the agricultural industry. To date, no satisfactory therapeutic, vaccine, or diagnostic tools are available, globally impairing all control programs. In this study, we have undertaken a large-scale postgenomic analysis of MAP proteins, to identify specific antigens that could potentially improve the diagnosis of paratuberculosis. Two complementary approaches were implemented, the first one consisting in the systematic proteomic identification of proteins present in MAP culture filtrates (CFs), followed by the selection of MAP-specific proteins by BLAST query on available mycobacterial genomes. The resulting database represents the first established secretome of MAP and a useful source of potentially specific antigens. The second approach consisted in the immunoproteomic analysis of both MAP extracts and CFs, using sera from MAP-infected and uninfected cattle. Combining results obtained with both approaches resulted in the identification of 25 candidate diagnostic antigens. Five of these were tested in an ELISA assay for their diagnostic potential, on a limited panel of field sera, and the combination of three of them competed in performance with available commercial assays, reaching a test sensitivity of 94.74% and specificity of 97.92%.

The vomeronasal organ is a chemosensory organ present in most vertebrates and involved in chemical communication. In the last decade, the deciphering of the signal transduction process of this organ has progressed. However, less is known about the vomeronasal organ ligands and their structure-function relationships. Snakes possess a highly developed vomeronasal system that is used in various behaviors such as mating, predator detection, or prey selection, making this group a suitable model for study of the vomeronasal chemoreception. In this work, we used a proteomics approach to identify and characterize proteins from frog cutaneous mucus proteome involved in prey recognition by snakes of the genus Thamnophis. Herein we report the purification and characterization of two proteins isolated from the frog skin secretome that elicit the vomeronasal organ-mediated predatory behavior of Thamnophis marcianus. These proteins are members of the parvalbumin family, which are calcium-binding proteins generally associated to muscular and nervous tissues. This is the first report that demonstrates parvalbumins are not strictly restricted to intracellular compartments and can also be isolated from exocrine secretions. Purified parvalbumins from frog muscle and mucus revealed identical chemoattractive properties for T. marcianus. Snake bioassay revealed the Ca(2+)/Mg(2+) dependence of the bioactivity of parvalbumins. So parvalbumins appear to be new candidate ligands of the vomeronasal organ.

Gammaherpesvirinae, such as the human Epstein-Barr virus (EBV) and the Kaposi’s sarcoma associated herpesvirus (KSHV) are highly prevalent pathogens that have been associated with several neoplastic diseases. As EBV and KSHV are host-range specific and replicate poorly in vitro, animal counterparts such as Murid herpesvirus-4 (MuHV-4) have been widely used as models. In this study, we used MuHV-4 in order to improve the knowledge about proteins that compose gammaherpesviruses virions. To this end, MuHV-4 extracellular virions were isolated and structural proteins were identified using liquid chromatography tandem mass spectrometry-based proteomic approaches. These analyses allowed the identification of 31 structural proteins encoded by the MuHV-4 genome which were classified as capsid (8), envelope (9), tegument (13) and unclassified (1) structural proteins. In addition, we estimated the relative abundance of the identified proteins in MuHV-4 virions by using exponentially modified protein abundance index analyses. In parallel, several host proteins were found in purified MuHV-4 virions including Annexin A2. Although Annexin A2 has previously been detected in different virions from various families, its role in the virion remains controversial. Interestingly, despite its relatively high abundance in virions, Annexin A2 was not essential for the growth of MuHV-4 in vitro. Altogether, these results extend previous work aimed at determining the composition of gammaherpesvirus virions and provide novel insights for understanding MuHV-4 biology.

Background Pseudomonas putida exerts a filamentous phenotype in response to environmental stress conditions that are encountered during its natural life cycle. This study assessed whether P. putida filamentation could confer survival advantages. Filamentation of P. putida was induced through culturing at low shaking speed and was compared to culturing in high shaking speed conditions, after which whole proteomic analysis and stress exposure assays were performed. Results P. putida grown in filament-inducing conditions showed increased resistance to heat and saline stressors compared to non-filamented cultures. Proteomic analysis showed a significant metabolic change and a pronounced induction of the heat shock protein IbpA and recombinase RecA in filament-inducing conditions. Our data further indicated that the associated heat shock resistance, but not filamentation, was dependent of RecA. Conclusions This study provides insights into the altered metabolism of P. putida in filament-inducing conditions, and indicates that the formation of filaments could potentially be utilized by P. putida as a survival strategy in its hostile, recurrently changing habitat.

Facioscapulohumeral muscular dystrophy (FSHD) is a progressive muscle disorder linked to a contraction of the D4Z4 repeat array in the 4q35 subtelomeric region. This deletion induces epigenetic modifications that affect the expression of several genes located in the vicinity. In each D4Z4 element, we identified the double homeobox 4 (DUX4) gene. DUX4 expresses a transcription factor that plays a major role in the development of FSHD through the initiation of a large gene dysregulation cascade that causes myogenic differentiation defects, atrophy and reduced response to oxidative stress. Because miRNAs variably affect mRNA expression, proteomic approaches are required to define the dysregulated pathways in FSHD. In this study, we optimized a differential isotope protein labeling (ICPL) method combined with shotgun proteomic analysis using a gel-free system (2DLC-MS/MS) to study FSHD myotubes. Primary CD56(+) FSHD myoblasts were found to fuse into myotubes presenting various proportions of an atrophic or a disorganized phenotype. To better understand the FSHD myogenic defect, our improved proteomic procedure was used to compare predominantly atrophic or disorganized myotubes to the same matching healthy control. FSHD atrophic myotubes presented decreased structural and contractile muscle components. This phenotype suggests the occurrence of atrophy-associated proteolysis that likely results from the DUX4-mediated gene dysregulation cascade. The skeletal muscle myosin isoforms were decreased while non-muscle myosin complexes were more abundant. In FSHD disorganized myotubes, myosin isoforms were not reduced, and increased proteins were mostly involved in microtubule network organization and myofibrillar remodeling. A common feature of both FSHD myotube phenotypes was the disturbance of several caveolar proteins, such as PTRF and MURC. Taken together, our data suggest changes in trafficking and in the membrane microdomains of FSHD myotubes. Finally, the adjustment of a nuclear fractionation compatible with mass spectrometry allowed us to highlight alterations of proteins involved in mRNA processing and stability.

For more than three decades, proteomics have been a crucial tool for deciphering the intricate molecular systems governing biology. O'Farrel was the first to utilise 2 dimensional gel electrophoresis (2DE) to perform actual complex proteomic analyses (O'Farrell, 1975). 2DE has very quickly emerged at the forefront of this rapidly growing field of research and has allowed for thousands of studies in widely varied domains. The development of 2D Fluorescence Gel Electrophoresis (2D DIGE) has provided more accurate and reliable proteins quantification due to the simultaneous migration on a same gel of samples to be compared, avoiding gel-to-gel variation. More recently, technological improvements in liquid chromatography and mass spectrometry have made it possible to develop so called "gel-free proteomics" in which, after total proteome enzymatic digestion, the produced peptides are separated with a high resolution chromatographic system and identified using tandem mass spectrometry. A gel-free approach presents a number of advantages over 2DE, such as a higher sensitivity, an easier automation of procedures to provide a better reproducibility and a reduced influence of intrinsic protein characteristics (pI, molecular weight, etc.). Nevertheless, a high complementarity between 2DE and gel-free approaches has been extensively reported (Finamore et al., 2010; Charro et al., 2011; Matallana-Surget et al., submitted), which suggests that both methods will continue to be considered together for a long time. Furthermore, 2DE also presents some advantages over a gel-free workflow approach in particular contexts. Indeed, 2DE presents the important benefit of allowing for the detection of protein isoforms, which is still complicated using gel-free approaches.

Among differential proteomic methods based on stable isotopic labeling, isotope-coded protein labeling (ICPL) is a recent non-isobaric technique devised to label primary amines found in proteins. ICPL overcomes some of the disadvantages found in other chemical-labeling techniques, such as iTRAQ or ICAT. However, previous analyses revealed that more than 30% of the proteins identified in regular ICPL generally remain unquantified. In this study, we describe a modified version of ICPL, named Post-digest ICPL, that makes it possible to label and thus to quantify all the peptides in a sample (bottom-up approach). Optimization and validation of this Post-digest ICPL approach were performed using a standard protein mixture and complex protein samples. Using this strategy, the number of proteins that were identified and quantified was greatly increased in comparison with regular ICPL and cICAT approaches. The pros and cons of this improvement are discussed. This complementary approach to traditional ICPL was applied to the analysis of modification of protein abundances in the model bacterium Cupriavidus metallidurans CH34 after cultivation under simulated microgravity. In this context, two different systems - a 2-D clinorotation and 3-D random positioning device - were used and the results were compared and discussed.

Mass spectrometry (MS)-based approaches are used increasingly to address diverse questions in proteomics research, enabling one to comprehensively analyze complex samples of proteins on a global le...

Beside bioreactor modeling studies, the molecular characterization of life-support organisms appeared to be essential to complete their global behavior picture, in particular, culture conditions. Using a combination of LC-MS/MS approaches with gel-free and gel-based peptides fractionation steps, we identified 932 proteins from the alpha-proteobacterium Rhodospirillum rubrum S1H. In addition, abundance data were retrieved using the recently developed emPAI approach which takes into account the number of sequenced peptides per protein. This work has also allowed identification of new and specific proteins for the Rhodospirillaceae family.

The filamentous cyanobacterium Arthrospira platensis has a long history of use as a food supply and it has been used by the European Space Agency in the MELiSSA project, an artificial microecosystem which supports life during long-term manned space missions. This study assesses progress in the field of cyanobacterial shotgun proteomics and light/dark diurnal cycles by focusing on Arthrospira platensis. Several fractionation workflows including gel-free and gel-based protein/peptide fractionation procedures were used and combined with LC-MS/MS analysis, enabling the overall identification of 1306 proteins, which represents 21% coverage of the theoretical proteome. A total of 30 proteins were found to be significantly differentially regulated under light/dark growth transition. Interestingly, most of the proteins showing differential abundance were related to photosynthesis, the Calvin cycle and translation processes. A novel aspect and major achievement of this work is the successful improvement of the cyanobacterial proteome coverage using a 3D LC-MS/MS approach, based on an immobilized metal affinity chromatography, a suitable tool that enabled us to eliminate the most abundant protein, the allophycocyanin. We also demonstrated that cell growth follows a light/dark cycle in A. platensis. This preliminary proteomic study has highlighted new characteristics of the Arthrospira platensis proteome in terms of diurnal regulation.

UVB oxidizes proteins through the generation of reactive oxygen species. One consequence of UVB irradiation is carbonylation, the irreversible formation of a carbonyl group on proline, lysine, arginine or threonine residues. In this study, redox proteomics was performed to identify carbonylated proteins in the UVB resistant marine bacterium Photobacterium angustum. Mass-spectrometry was performed with either biotin-labeled or dinitrophenylhydrazide (DNPH) derivatized proteins. The DNPH redox proteomics method enabled the identification of 62 carbonylated proteins (5% of 1221 identified proteins) in cells exposed to UVB or darkness. Eleven carbonylated proteins were quantified and the UVB/dark abundance ratio was determined at both the protein and peptide levels. As a result we determined which functional classes of proteins were carbonylated, which residues were preferentially modified, and what the implications of the carbonylation were for protein function. As the first large scale, shotgun redox proteomics analysis examining carbonylation to be performed on bacteria, our study provides a new level of understanding about the effects of UVB on cellular proteins, and provides a methodology for advancing studies in other biological systems.