Rapid typing of Moraxella catarrhalis subpopulations based on outer membrane proteins using mass spectrometry

Moraxella catarrhalis is a major mucosal pathogen of the human respiratory tract both in children and in adults. Two subpopulations of this organism have been described that differ in 16S rRNA gene sequence and virulence traits. Three 16S rRNA types have been defined. 2-DE followed by protein identification by MS revealed significant differences in the outer membrane protein (OMP) patterns of each M. catarrhalis 16S rRNA type. Approximately 130 features were detected on the 2-DE map of each M. catarrhalis 16S rRNA type. However, only 50 features were expressed by all strains. Furthermore, direct profiling of isolated OMP using MALDI-TOF MS resulted in a characteristic spectral fingerprint for each 16S rRNA type. Fingerprints remained identical when intact cells instead of isolated OMP were analyzed. This finding suggests that the source of desorbed ions is the outer membrane. Based on the fingerprint we were able to assign 18 well-characterized clinical M. catarrhalis isolates to the correct subpopulation. Therefore, MALDI-TOF of intact M. catarrhalis provides a rapid and robust tool for M. catarrhalis strain typing that could be applied in epidemiological studies.     

Differential proteomic analysis of Aeromonas salmonicida outer membrane proteins in response to low iron and in vivo growth conditions

Aeromonas salmonicida subsp. salmonicida is the etiological agent of furunculosis, a serious infectious disease of salmonids. Bacterial phenotypes are known to change in vivo compared to the in vitro state. Proteomic analysis of in vivo phenotypes is usually not possible due to insufficient biomass. Using an in vivo growth chamber model, the pathogenic fish bacterium A. salmonicida was cultured in pure culture in vivo in its host, the Atlantic salmon, to obtain sufficient biomass to allow proteomic analysis. Growth of A. salmonicida under in vitro iron-restricted conditions resulted in the expression of outer membrane proteins of 73, 76 and 85 kDa, which were not present when grown under in vitro iron-replete conditions. Mass spectrometry analysis identified the 73 kDa protein as a colicin receptor, the 76 kDa protein as an outer membrane heme receptor, and the 85 kDa protein as a ferric siderophore receptor. When cultured in vivo, A. salmonicida up-regulated the identical 73, 76 and 85 kDa proteins. The results of this study also suggest, at least with respect to the outer membrane proteins, that the in vitro iron-restricted growth model largely reproduces the results obtained from growth of A. salmonicida within the peritoneal cavity of salmon.     

Immunopotentiation of outer membrane protein through anti-idiotype Pasteurella multocida vaccine in rabbits

Pasteurella multocida was isolated from cattle affected with haemorrhagic septicaemia and characterized on the basis of morphological, cultural and biochemical tests. Bacterial outer membrane proteins (OMPs) were extracted with 1% Sarkosyl method. P. multocida anti-idiotype vaccine prepared from OMPs (21.3 mg per 100 ml), was evaluated and compared with bacterin supplemented with 10% OMPs and plain alum-adsorbed bacterin in rabbit models. It was observed that OMPs-anti-idiotype vaccine induced high levels of antibody titres (geomean titres -GMT) detected using indirect haemagglutination (IHA) test. The OMPs anti-idiotype antibody titres of 168.9 GMT were obtained to 42.2 GMT in OMPs supplemented bacterin on 21 days post vaccination, while the plain bacterin had the least titre of 27.9 GMT. The OMPs-anti-idiotype vaccine provoked better immunogenic response in terms of highest GMT titres and long lasting effect in rabbits and 100% protection against the challenge with homologous strain of P. multocida,while 88% protection was obtained in rabbits, given OMPs supplemented bacterin.     

Proteomics in the liver of gilthead sea bream (Sparus aurata) to elucidate the cellular response induced by the intake of maslinic acid

Maslinic acid (MA) is a pentacyclic triterpene used as a feed additive to stimulate growth, protein‐turnover rates, and hyperplasia in fish. To further our understanding of cellular mechanisms underlying the action of MA, we have used 2‐DE coupled with MS to identify proteins differentially expressed in the livers of juvenile gilthead sea bream (Sparus aurata) grown under fish‐farm conditions and fed with a 100 mg/kg MA‐enriched diet (MA₁₀₀). After the comparison of the protein profiles from MA₁₀₀ fed fish and from control, 49 protein spots were found to be altered in abundance (≥2‐fold). Analysis by MALDI‐TOF/TOF allowed the unambiguous identification of 29 spots, corresponding to 19 different proteins. These proteins were: phosphoglucomutase, phosphoglucose isomerase, S‐adenosyl methionine‐dependent methyltransferase class I, aldehyde dehydrogenase, catalase, 6‐phosphogluconate dehydrogenase, fumarylacetoacetate hydrolase, 4‐hydroxyphenylpyruvic dioxygenase, methylmalonate‐semialdehyde dehydrogenase, lysozyme, urate oxidase, elongation factor 2, 60 kDa heat‐shock protein, 58 kDa glucose‐regulated protein, cytokeratin E7, type‐II keratin, intermediate filament proteins, 17‐β‐hydroxysteroid dehydrogenase type 4, and kinase suppressor of Ras1. Western blot analysis of kinase suppressor of Ras1, glucose 6‐phosphate dehydrogenase, elongation factor 2, 60 kDa heat‐shock protein, and catalase supported the proteome evidence. Based on the changes found in the protein‐expression levels of these proteins, we proposed a cellular‐signalling pathway to explain the hepatic‐cell response to the intake of a diet containing MA.     

Exploring the proteome of an echinoderm nervous system: 2-DE of the sea star radial nerve cord and the synaptosomal membranes subproteome

We describe the first proteomic characterization of the radial nerve cord (RNC) of an echinoderm, the sea star Marthasterias glacialis. The combination of 2-DE with MS (MALDI-TOF/TOF) resulted in the identification of 286 proteins in the RNC. Additionally, 158 proteins were identified in the synaptosomal membranes enriched fraction after 1-DE separation. The 2-DE RNC reference map is available via the WORLD-2DPAGE Portal (http://www.expasy.ch/world-2dpage/) along with the associated protein identification data which are also available in the PRIDE database. The identified proteins constitute the first high-throughput evidence that seems to indicate that echinoderms nervous transmission relies primarily on chemical synapses which is similar to the synaptic activity in adult mammal's spinal cord. Furthermore, several homologous proteins known to participate in the regeneration events of other organisms were also identified, and thus can be used as targets for future studies aiming to understand the poorly uncharacterized regeneration capability of echinoderms. This "echinoderm missing link" is also a contribution to unravel the mystery of deuterostomian CNS evolution.     

Differential proteomic analysis of proteins in wheat spikes induced by Fusarium graminearum

Scab, caused by Fusarium graminearum, is a serious spike disease in wheat. To identify proteins in resistant wheat cultivar Wangshuibai induced by F. graminearum infection, proteins extracted from spikes 6, 12 and 24 h after inoculation were separated by 2-DE. Thirty protein spots showing 3-fold change in abundance when compared with treatment without inoculation were characterized by MALDI-TOF MS and matched to proteins by querying the mass spectra in protein databases or the Triticeae EST translation database. Based on their volume profiles, these proteins were classified into four categories. The first one fell off rapidly at the initial inoculation and then rose at 12 or 24 hai, the second one decreased considerably after inoculation and remained at low level, the third one rose at the initial inoculation and then declined at 12 or 24 hai, the forth one showed steady increase after inoculation and maintained at a high level. Many of the proteins identified in the first two categories are related to carbon metabolism and photosynthesis. While most of proteins identified in the last two categories are related to stress defense of plants, indicating that proteins associated with the defense reactions were activated or translated shortly after inoculation.     

Intermittent administration of morphine alters protein expression in rat nucleus accumbens

Repeated exposure to drugs of abuse causes time-dependent neuroadaptive changes in the mesocorticolimbic system of the brain that are considered to underlie the expression of major behavioral characteristics of drug addiction. We used a 2-D gel-based proteomics approach to examine morphine-induced temporal changes in protein expression and/or PTM in the nucleus accumbens (NAc) of morphine-sensitized rats. Rats were pretreated with saline [1 mL/kg subcutaneously (s.c.)] or morphine (10 mg/kg, s.c.) once daily for 14 days and the animals were decapitated 1 day later. The NAc was extracted and proteins resolved by 2-DE. Several protein functional groups were found to be regulated in the morphine-treated group, representing cytoskeletal proteins, proteins involved in neurotransmission, enzymes involved in energy metabolism and protein degradation, and a protein that regulates translation.     

MS characterization of qualitative protein polymorphisms in the spinal cords of inbred mouse strains

The spinal cord proteomes of two inbred mouse strains with different susceptibility to experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis, were investigated by 2‐DE and MALDI‐MS. A proteome map comprising 304 different protein species was established. Using 2‐D fluorescence difference gel electrophoresis, a comparison of the mouse strains revealed 26 qualitatively polymorphic proteins with altered electrophoretic mobility. MS analyses and DNA sequencing were applied to characterize their structural differences and 14 single amino acid substitutions were identified. Moreover, analysis of selectively enriched phosphopeptides from the neurofilament heavy polypeptide of both mouse strains revealed a high degree of diversity in the phosphorylated C‐terminal domains of this protein. The described approach is capable to structurally characterize qualitative protein polymorphisms, whereas their functional significance remains to be elucidated. For some proteins formerly associated with experimental autoimmune encephalomyelitis and/or multiple sclerosis structural polymorphisms are described here, which may be subjected to further investigations. In addition, this work should be of general interest for proteomic analysis of inbred strains, because it shows potentials and constraints in the use of 2‐DE analysis and MALDI‐MS to detect and characterize structural protein polymorphisms.     

An improved method of sample preparation on AnchorChip targets for MALDI-MS and MS/MS and its application in the liver proteome project

An improved method for sample preparation for MALDI-MS and MS/MS using AnchorChip targets is presented. The method, termed the SMW method (sample, matrix wash), results in better sensitivity for peptide mass fingerprinting as well as for sequencing by MS/MS than previously published methods. The method allows up-concentration and desalting directly on the mass spectrometric target and should be amenable for automation. A draw back caused by extensive oxidation of methionine and tryptophan in the SMW method can be alleviated by the addition of n-octyl glucopyranoside and DTT to the sample solution. The method was validated for protein identification from a 2-DE based liver proteome study. The SMW method resulted in identification of many more proteins and in most cases with a better score than the previously published methods.     

Proteomic analysis of a meningococcal outer membrane vesicle vaccine prepared from the group B strain NZ98/254

In the absence of a suitable carbohydrate-based vaccine, outer membrane vesicle (OMV) vaccines have been used to disrupt outbreaks of serogroup B meningococcal disease for more than 20 years. Proteomic technology provides physical methods with the potential to assess the composition and consistency of these complex vaccines. 2-DE, combined with MS, were used to generate a proteome map of an OMV vaccine, developed to disrupt a long-running outbreak of group B disease in New Zealand. Seventy four spots from the protein map were identified including the outer membrane protein (OMP) antigens: PorA, PorB, RmpM and OpcA. Protein identification indicates that, in addition to OMPs, OMV vaccines contain periplasmic, membrane-associated and cytoplasmic proteins. 2-D-DIGE technology highlighted differences between preclinical development batches of vaccines from two different manufacturers.     

Comparative proteome analysis of the embryo proper and yolk sac membrane of day 11.5 cultured rat embryos

BACKGROUND: Proteomic analysis of cultured postimplantation rat embryos is expected to be useful for investigation into embryonic development. Here we analyzed protein expression in cultured postimplantation rat embryos by two-dimensional electrophoresis (2-DE) and mass-spectrometric protein identification. METHODS: Rat embryos were cultured from day 9.5 for 48 h or from day 10.5 for 24 h. Proteins of the embryo proper and yolk sac membrane were isolated by 2-DE and differentially analyzed with a 2-D analysis software. Selected protein spots in the 2-DE gels were identified by matrix-assisted laser desorption/ionization-time of flight tandem mass spectrometric analysis and protein database search. RESULTS: About 800 and 1,000 protein spots were matched through the replicate 2-DE gels each from one embryo in the embryo proper and yolk sac membrane, respectively, and virtually the same protein spots were observed irrespective to the length of culture period. From protein spots specific to the embryo proper (126 spots) and yolk sac membrane (304 spots), proteins involved in tissue-characteristic functions, such as morphogenesis and nutritional transfer, were identified: calponin, cellular retinoic acid binding protein, cofilin, myosin, and stathmin in the embryo proper, and Ash-m, dimerization cofactor of hepatocyte nuclear factor, ERM-binding phosphoprotein, cathepsin, and legumain in the yolk sac membrane. CONCLUSION: Proteomic analysis of cultured postimplantation rat embryos will be a new approach in developmental biology and toxicology at the protein level.     

Genetic changes in muscle protein following hybridization between Haliotis diversicolor reeve Japan and Taiwan populations revealed using a proteomic approach

Protein expression patterns were compared in a Japan and Taiwan population of Haliotis diversicolor and in a hybrid between them using 2DE and MALDI‐TOF‐TOF analyses. Using the software PDQuest, 924 ± 7 protein spots were detected in the Japan population (RR), 861 ± 11 in the Taiwan population (TT), and 882 ± 9 in the F₁ hybrid (TR). RR and TR were clustered together, but the distance between RR and TT was the maximum using hierarchical cluster analysis. A total of 46 gel spots were identified and a total of 15 spots matched with abalone proteins (a 33.6% identification rate). Hybrid exhibiting additivity or overdominance accounted for 73.9% of these 46 identified proteins. The 46 differentially expressed proteins were shown to be involved in major biological processes, including muscle contraction and regulation, energy metabolism, and stress response. The proteins involved in energy metabolism included adenosine triphosphate (ATP) synthase β subunit, fructose 1, 6‐bisphosphate aldolase, triosephosphate isomerase, enolase, arginine kinase, and tauropine dehydrogenase. These proteins exhibited additivity in their offspring. The proteins involved in stress responses included HSP Hsp70 (exhibiting overdominance in the offspring) and Cu/Zn‐superoxide dismutase (exhibiting additivity). These results suggested that proteomic approach is suitable for analysis of heterosis and functional prediction of abalone hybridization.     

Proteomic analysis of colonic mucosa in a rat model of irritable bowel syndrome

Irritable bowel syndrome (IBS) is one of the most common functional disorders of the gastrointestinal tract. It is characterized by abdominal pain and changes in bowel habits. Various studies have investigated the pathophysiologic processes underlying IBS, but the mechanism remains poorly understood. In the present study, we established an IBS model and identified differentially expressed proteins in colon tissue of IBS rats compared with healthy controls by 2‐D gel electrophoresis, MALDI‐TOF‐MS, and Western blot analysis. Our results showed that 13 of the 1396 protein spots on 2‐D gel were differently expressed between the IBS and control groups. Ontological analysis of these proteins revealed primary roles in catalytic activity (protein disulfide‐isomerase A3, glyoxalase I, cathepsin S, α‐enolase), structural support (cytokeratin 8), antioxidant activity (peroxiredoxin‐6), protein binding (transgelin, serpin peptidase inhibitor B5), and signal transduction (40S ribosomal protein SA). Protein disulfide‐isomerase A3 and cytokeratin 8 overexpression in IBS were confirmed by Western blot. The findings indicate that multiple proteins are involved in IBS processes that influence intestinal tract immunity, inflammation, and nerve regulation. Our study provides useful candidate genes and proteins for further investigation.     

Evaluation of protein N-glycosylation in 2-DE: Erythropoietin as a study case

The structure, function, and physico-chemical properties of many proteins are determined by PTM, being glycosylation the most complex. This study describes how a combination of typical proteomics methods (2-DE) combines with glycomics strategies (HPLC, MALDI-TOF-MS, exoglycosidases sequencing) to yield comprehensive data about single spot-microheterogeneity, providing meaningful information for the detection of disease markers, pharmaceutical industry, antidoping control, etc. Recombinant erythropoietin and its hyperglycosylated analogue darbepoetin-alpha were chosen as showcases because of their relevance in these fields and the analytical challenge they represent. The combined approach yielded good results in terms of sample complexity (mixture glycoforms), reproducibility, sensitivity ( approximately 25 pmoles of glycoprotein/spot), and identification of the underlying protein. Heterogeneity was present in all spots but with a clear tendency; spots proximal to the anode contained the highest amount of tetra-antennary tetra-sialylated glycans, whereas the opposite occurred for spots proximal to the cathode with the majority of the structures being undersialylated. Spot microheterogeneity proved a consequence of the multiple glycosylation sites as they contributed directly to the number of possibilities to account for a discrete charge in a single spot. The interest of this combined glycoproteomics method resides in the efficiency for detecting and quantifying subtle dissimilarities originated from altered ratios of identical glycans including N-acetyl-lactosamine repeats, acetylation, or antigenic epitopes, that do not significantly contribute to the electrophoretic mobility, but affect the glycan microheterogeneity and the potential underlying related functionality.     

Proteome map of the neural complex of the tunicate Ciona intestinalis,the closest living relative to vertebrates

Ciona intestinalis (the common sea squirt) is the closest living chordate relative to vertebrates with cosmopolitan presence worldwide. It has a relatively simple nervous system and development, making it a widely studied alternative model system in neuroscience and developmental biology. The use of Ciona as a model organism has increased significantly after the draft genome was published. In this study, we describe the first proteome map of the neural complex of C. intestinalis. A total of 544 proteins were identified based on 1DE and 2DE FTMS/ITMSMS analyses. Proteins were annotated against the Ciona database and analyzed to predict their molecular functions, roles in biological processes, and position in constructed network pathways. The identified Ciona neural complex proteome was found to map onto vertebrate nervous system pathways, including cytoskeleton remodeling neurofilaments, cell adhesion through the histamine receptor signaling pathway, γ‐aminobutyric acid‐A receptor life cycle neurophysiological process, glycolysis, and amino acid metabolism. The proteome map of the Ciona neural complex is the first step toward a better understanding of several important processes, including the evolution and regeneration capacity of the Ciona nervous system.     

Proteomic analysis of the cold stress response in the moss,Physcomitrella patens

Cold stress has adverse effects on plant growth and development. Plants respond and acclimate to cold stress through various biochemical and physiological processes, thereby acquiring stress tolerance. To better understand the basis for tolerance, we carried out a proteomic study in the model moss, Physcomitrella patens, characterizing gametophore proteins with 2‐DE and mass spectroscopy. Following exposure to 0°C for up to 3 days, out of the more than 1000 protein spots reproducibly resolved, only 45 changed in abundance by at least 1.5‐fold. Of these, 35 were identified by tryptic digestion and mass spectroscopy. Photosynthetic proteins decreased, whereas many catabolic proteins increased. In addition, cold stress up‐regulated a variety of signaling, cytoskeleton, and defense proteins and few proteins in these classes were down‐regulated. Up‐regulated proteins include the 14‐3‐3‐like protein, actin, HSP70s, lipoxygenases, and cytochrome P450 proteins. These results point to pathways that are important for the mechanism of cold stress response in P. patens and by extension to the entire plant kingdom.