2‐D PAGE and MS analysis of proteins from formalin‐fixed,paraffin‐embedded tissues

In the past decade, encouraging results have been obtained in extraction and analysis of proteins from formalin‐fixed, paraffin‐embedded (FFPE) tissues. However, 2‐D PAGE protein maps with satisfactory proteomic information and comparability to fresh tissues have never been described to date. In the present study, we report 2‐D PAGE separation and MS identification of full‐length proteins extracted from FFPE skeletal muscle tissue. The 2‐D protein profiles obtained from FFPE tissues could be matched to those achieved from frozen tissues replicates. Up to 250 spots were clearly detected in 2‐D maps of proteins from FFPE tissue following standard mass‐compatible silver staining. Protein spots from both FFPE and frozen tissue 2‐D gels were excised, subjected to in situ hydrolysis, and identified by MS analysis. Matched spots produced matched protein identifications. Moreover, 2‐D protein maps from FFPE tissues were successfully subjected to Western immunoblotting, producing comparable results to fresh‐frozen tissues. In conclusion, this study provides evidence that, when adequately extracted, full‐length proteins from FFPE tissues might be suitable to 2‐D PAGE‐MS analysis, allowing differential proteomic studies on the vast existing archives of healthy and pathological‐fixed tissues.     

Proteomic analysis of the bacterial pathogen Bartonella henselae and identification of immunogenic proteins for serodiagnosis

Bartonella henselae is a slow growing, fastidious and facultative intracellular pathogen causing cat scratch disease and vasculoproliferative disorders. To date, knowledge about the pathogenicity of this human pathogenic bacterium is limited and, additionally, serodiagnosis still needs further improvement. Here, we investigated the proteome of B. henselae using 2‐D SDS‐PAGE and MALDI‐TOF‐MS. We provide a comprehensive 2‐D proteome reference map of the whole cell lysate of B. henselae with 431 identified protein spots representing 191 different proteins of which 16 were formerly assigned as hypothetical proteins. To unravel immunoreactive antigens, we applied 2‐D SDS‐PAGE and subsequent immunoblotting using 33 sera of patients suffering from B. henselae infections. The analysis revealed 79 immunoreactive proteins of which 71 were identified. Setting a threshold of 20% seroreactivity, 11 proteins turned out to be immunodominant antigens potentially useful for an improved Bartonella‐specific serodiagnosis. Therefore, we provide for the first time (i) a comprehensive 2‐D proteome map of B. henselae for further proteome‐based studies focussed on the pathogenicity of B. henselae and (ii) an integrated view into the humoral immune responses targeted against this newly emerged human pathogenic bacterium.     

Phenotypic diversity of Edwardsiella tarda isolated from different origins

Aims: To evaluate the diversity of phenotypic characteristics among isolates of Edwardsiella tarda from various origins. Methods and results: A total of 10 E. tarda strains were investigated on biological characteristics including flagella formation, bacterial motility, biofilm formation, extracellular protein and plasmid profiles. All the E. tarda strains (including two previous recognized as nonflagellation strains) were proven to have an average of 1–7 peritrichous flagella with the precise number positively correlated with motility and biofilm formation. All the E. tarda strains exhibited similar protein profiles except ET2034, LMG2793 and ET080814, which lacked the three major bands of approximately 18, 21 and 55 kDa. E. tarda with the same geographic location shared similar plasmid profiles. Conclusions:  Edwardsiella tarda strains exhibited diversities in phenotypic characteristics that may be linked to differences in geographic location or host origin. In addition, the number of flagella is essential for bacterial motility and biofilm formation. Significance and Impact of the Study: This is the first report demonstrating the difference in flagella formation between E. tarda strains, which may broaden the understanding of flagellation trait at intra‐species level. Furthermore, evaluation of virulence‐associated characteristics can provide useful information for unveiling the diverse pathogenic mechanisms of E. tarda.     

Proteomic comparison between interphase and metaphase of isolated chloroplasts of Cyanidioschyzon merolae (Cyanidiophyceae,Rhodophyta)

With the completion of the Cyanidioschyzon merolae genome project, detailed analysis of organelle proteins with mass spectrometry has now become possible. Chloroplasts of the unicellular red alga Cyanidioschyzon merolae De Luca, Taddei et Varano (Rhodophyta, Cyanidiophyceae) were isolated from synchronized culture and the chloroplast lysates of both interphase and metaphase cells were prepared and subjected to 2D‐polyacrylamide gel electrophoresis. A total of 355 spots (170 identical spots) were recognized and quantified and then analyzed using mass spectrometry. A total of 105 proteins were identified, including 18 proteins for posttranslational functions, 17 photosynthesis‐related proteins, 10 carbohydrate‐related proteins, 15 proteins of unknown functions, and eight proteins predicted to be contaminated from other organelles. On the basis of spot quantity, photosynthesis‐related proteins were most dominant (45.3% in interphase and 56.4% in metaphase). In particular, the proteins forming phycobilisomal complexes were abundant. Comparison of interphase with metaphase revealed that CMG086C (aminomethyltransferase) notably increased in interphase. CMN235C (similar to chlorophyll a/b‐binding protein, CP24) increased in metaphase in agreement with a previously performed microarray analysis. Both CMQ295C (cell division protein FtsH) and CMS004C (plastid division protein FtsZ) increased in interphase. Seven proteins were detected to be interphase‐specific, and 12 proteins were metaphase‐specific. Proteins of unknown functions were poorly characterized by homology search, although thioredoxin‐like domains were predicted in several proteins.     

Immunoproteomic analysis and identification of novel immunogenic proteins from Vibrio harveyi

Aims: The main aim of this study was to screen novel immunogenic proteins of Vibrio harveyi, which could be vaccine candidates. Methods and Results: Whole‐cell proteins of V. harveyi, strain Li01 and Huang01, were first separated by isoelectric focusing, followed by 2D‐PAGE, respectively. Immunogenic proteins were identified by Western blotting, using Epinephelus coioides antisera against V. harveyi strain Li01. Western blot analyses revealed 16 shared immunogenic protein spots in both strains. All of the immunogenic proteins were successfully identified and corresponded to 15 proteins. None of these proteins have been previously reported as immunogenic for V. harveyi. Of the 15 proteins, 11 are specific immunoreactive proteins and four are nonspecific immunoreactive proteins. Furthermore, outer membrane protein N (spot 2) and oligopeptide ATP‐binding cassette (ABC) transporter (spot 3) were used as immunogens to immunize E. coioides for investigation of their protective abilities and activities. The E. coioides immunized with OmpN has abilities to fight against infections caused by V. harveyi Li01 and Huang01. However, vaccination with oligopeptide ABC transporter induces low protective immune response in fish. Conclusions: Eleven novel specific antigens were found, and OmpN could potentially be used as vaccine candidate for the development of novel vaccine against V. harveyi. Significance and Impact of the Study: These data show that immunoproteomics methods can be successfully applied in identifying immunogenic proteins of V. harveyi, which helps to search for the protective antigens in future.     

Proteomic characterization of Phragmites communis in ecotypes of swamp and desert dune

Phragmites communis Trin. (common reed) is a recognized model plant for studying its adaptation to contrasting and harsh environments. To understand the inherent molecular basis for its remarkable resistance to combined stresses, we performed a comprehensive proteomic analysis of the leaf proteins from two ecotypes, i.e. swamp and desert dune, naturally growing in the desert region of northwestern China. First, a proteome reference map of Phragmites was established based on the swamp ecotype. Proteins were resolved by 2‐D/SDS‐PAGE and identified by MALDI‐TOF/TOF MS. In total, 177 spots were identified corresponding to 51 proteins. The major proteins identified are proteins involved in photosynthesis, glutathione and ascorbic acid metabolism as well as protein synthesis and quality control. Second, the 2‐DE profiles of the two ecotypes were compared quantitatively via DIGE analysis. Compared with swamp ecotype, 51 proteins spots are higher‐expressed and 58 protein spots are lower‐expressed by twofold or more in desert dune ecotype. Major differences were found for the proteins involved in light reaction of photosynthesis, protein biosynthesis and quality control and antioxidative reactions. The physiological significance of such differences is discussed in the context of a flow of complex events in relation to plant adaptation to combined environmental stresses.     

Proteomics reveals selective regulation of proteins in response to memory-related serotonin stimulation in Aplysia californica ganglia

The marine mollusk Aplysia californica (Aplysia) is a powerful model for learning and memory due to its minimalistic nervous system. Key proteins, identified to be regulated by the neurotransmitter serotonin in Aplysia, have been successfully translated to mammalian models of learning and memory. Based upon a recently published large‐scale analysis of Aplysia proteomic data, the current study investigated the regulation of protein levels 24 and 48 h after treatment with serotonin in Aplysia ganglia using a 2‐D gel electrophoresis approach. Protein spots were quantified and protein‐level changes of selected proteins were verified by Western blotting. Among those were Rab GDP dissociation inhibitor alpha (RabGDIα), synaptotagmin‐1 and deleted in azoospermia‐associated protein (DAZAP‐1) in cerebral ganglia, calreticulin, RabGDIα, DAZAP‐1, heterogeneous nuclear ribonucleoprotein F (hnRNPF), RACK‐1 and actin‐depolymerizing factor (ADF) in pleural ganglia and DAZAP‐1, hnRNPF and ADF in pedal ganglia. Protein identity of the majority of spots was confirmed by a gel‐based mass spectrometrical method (FT‐MS). Taken together, protein‐level changes induced by the learning‐related neurotransmitter serotonin in Aplysia ganglia are described and a role for the abovementioned proteins in synaptic plasticity is proposed.     

EVIDENCE FOR A SPECIALIZED LOCALIZATION OF THE CHLOROPLAST ATP‐SYNTHASE SUBUNITS α, β, AND γ IN THE EYESPOT APPARATUS OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE)1

The eyespot apparatus (EA) of Chlamydomonas reinhardtii P. A. Dang. consists of two layers of carotenoid‐rich lipid globules subtended by thylakoids. The outermost globule layer is additionally associated with the chloroplast envelope membranes and the plasma membrane. In a recent proteomic approach, we identified 202 proteins from isolated EAs of C. reinhardtii via at least two peptides, including, for example, structural components, signalling‐related proteins, and photosynthetic‐related membrane proteins. Here, we have analyzed the proteins of the EA with regard to their topological distribution using thermolysin to find out whether the arrangement of globules and membranes provides protection mechanisms for some of them. From about 230 protein spots separated on two‐dimensional gels, the majority were degraded by thermolysin. Five major protein spots were protected against the action of this protease. These proteins and some that were degradable were identified by mass spectrometry. Surprisingly, the thermolysin‐resistant proteins represented the α and β subunits of the soluble CF₁ complex of the chloroplast ATP synthase. Degradable proteins included typical membrane proteins like LHCs, demonstrating that thermolysin is not in general sterically prevented by the EA structure from reaching membrane‐associated proteins. A control experiment showed that the CF₁ complex of thylakoids is efficiently degraded by thermolysin. Blue native PAGE of thermolysin‐treated EAs followed by SDS‐PAGE revealed that the α and β subunits are present in conjunction with the γ subunit in a thermolysin‐resistant complex. These results provide strong evidence that a significant proportion of these ATP‐synthase subunits have a specialized localization and function within the EA of C. reinhardtii.     

Highly sensitive saturation labeling reveals changes in abundance of cell cycle‐associated proteins and redox enzyme variants during oocyte maturation in vitro

Oocyte maturation is a complex process and a critical issue in assisted reproduction techniques (ART) in humans and other mammals. We used a sensitive 2‐D DIGE saturation labeling approach including an internal pooled standard for quantitative proteome profiling of immature versus in vitro matured bovine oocytes in six independent samples. The study comprised 48 2D gel images representing 24 DIGE experiments. From 250 ng sample analyzed per gel, quantitative analysis revealed an average of 2244 spots in pH 4–7 images and 1291 spots in pH 6–9 images. Thirty‐eight spots with different intensities were detected in total. Spots of a preparative gel from 2200 oocytes were identified by nano‐LC‐MS/MS analysis. The ten spots which could be unambiguously identified include the Ca²⁺‐binding protein translationally controlled tumor protein, enzymes of the Krebs and pentose phosphate cycles, clusterin, 14‐3‐3 ?, elongation factor‐1 gamma, and redox enzymes such as polymorphic forms of GST Mu 5 and peroxiredoxin‐3. The cellular distribution of two proteins was determined by confocal laser scanning microscopy. The interesting protein candidates identified by this study may help to improve the in vitro maturation process in order to increase the rate of successful in vitro fertilization and other ART in cattle and other mammals.     

Identification of antigenic Edwardsiella tarda surface proteins and their role in pathogenesis

Edwardsiella tarda causes an infectious fish disease called edwardsiellosis. Several outer membrane proteins (OMPs) are associated with virulence factors and are attractive as vaccine candidates. In this study, 4 immuno-reactive OMPs of E. tarda were detected using anti-sera from flounder infected with E. tarda. Using matrix-assisted laser desorption/ionization mass spectrometry analyses, 2 of the 4 OMPs were identified as OmpA and murein lipoprotein (Lpp), which are highly conserved surface proteins in gram-negative bacteria. For further characterization of these surface proteins, we generated ompA- and lpp-inactivated mutants by insertion of a kanamycin cassette in the corresponding genes, and named these mutants E. tarda CK99 and CK164, respectively. As expected, immuno-reactive OmpA and Lpp proteins were absent in E. tarda CK99 and CK164, respectively, confirming that OmpA and Lpp are antigenic surface proteins. Interestingly, the LD₅₀ value of E. tarda CK164 in fish (2.0 × 10⁸ colony-forming unit [CFU]/fish) was greater than that of the parental strain (3.0 × 10⁷ CFU/fish). The LD₅₀ of E. tarda CK99 did not differ from that of its parental strain. After administering attenuated E. tarda CK164 to fish, we monitored the E. tarda-specific immune response profile. We observed that the E. tarda-specific serum IgM titer increased in a time-dependent manner, and was much higher than the value observed after the administration of a heat-killed E. tarda control. Moreover, fish vaccinated with E. tarda CK164 were 100% protected when challenged by CK41, a pathogenic strain. Our results suggest that E. tarda CK164 can potentially be used for developing an effective live attenuated vaccine for edwardsiellosis that can be applied in the aquaculture industry.     

Preliminary analysis of the cauliflower mitochondrial proteome

Purified cauliflower (Brassica oleracea var. botrytis) mitochondrial proteins fractionated into soluble, membrane, integral membrane and peripheral membrane samples were analyzed by 2D- PAGE (isoelectric focusing/ SDS polyacrylamide gel electrophoresis). 2D gels patterns were compared using the Imager Master 2D Elite software. 561 silver stained protein spots were resolved after electrophoresis under standard conditions of a whole protein extract. In the soluble fraction a prevalent number of more intense protein spots was observed. The cauliflower protein 2D patterns resembled Arabidopsis thaliana 2D patterns. The two protein spots selected which occupied a similar isoelectric point positions on both gels represented the same proteins as revealed by ESI-MS analysis of cauliflower proteins. The third selected spot belongs to unidentified proteins. The comparative analysis of mitochondrial suborganellar fractions proved the usefulness of this approach.     

Proteomic analysis of Arabidopsis thaliana (L.) Heynh responses to a generalist sucking pest (Myzus persicae Sulzer)

Herbivorous insects can cause severe cellular changes to plant foliage following infestations, depending on feeding behaviour. Here, a proteomic study was conducted to investigate the influence of green peach aphid (Myzus persicae Sulzer) as a polyphagous pest on the defence response of Arabidopsis thaliana (L.) Heynh after aphid colony establishment on the host plant (3 days). Analysis of about 574 protein spots on 2‐DE gels revealed 31 differentially expressed protein spots. Twenty out of these 31 differential proteins were selected for analysis by mass spectrometry. In 12 of the 20 analysed spots, we identified seven and nine proteins using MALDI‐TOF‐MS and LC‐ESI‐MS/MS, respectively. Of the analysed spots, 25% contain two proteins. Different metabolic pathways were modulated in Arabidopsis leaves according to aphid feeding: most corresponded to carbohydrate, amino acid and energy metabolism, photosynthesis, defence response and translation. This paper has established a survey of early alterations induced in the proteome of Arabidopsis by M. persicae aphids. It provides valuable insights into the complex responses of plants to biological stress, particularly for herbivorous insects with sucking feeding behaviour.     

Increased oxidative-modifications of cytosolic proteins in 3,4-methylenedioxymethamphetamine (MDMA, ecstasy)-exposed rat liver

It is well established that 3,4‐methylenedioxymethamphetamine (MDMA, ecstasy) causes acute liver damage in animals and humans. The aim of this study was to identify and characterize oxidative modification and inactivation of cytosolic proteins in MDMA‐exposed rats. Markedly increased levels of oxidized and nitrated cytosolic proteins were detected 12 h after the second administration of two consecutive MDMA doses (10 mg/kg each). Comparative 2‐DE analysis showed markedly increased levels of biotin‐N‐methylimide‐labeled oxidized cytosolic proteins in MDMA‐exposed rats compared to vehicle‐treated rats. Proteins in the 22 gel spots of strong intensities were identified using MS/MS. The oxidatively modified proteins identified include anti‐oxidant defensive enzymes, a calcium‐binding protein, and proteins involved in metabolism of lipids, nitrogen, and carbohydrates (glycolysis). Cytosolic superoxide dismutase was oxidized and its activity significantly inhibited following MDMA exposure. Consistent with the oxidative inactivation of peroxiredoxin, MDMA activated c‐Jun N‐terminal protein kinase and p38 kinase. Since these protein kinases phosphorylate anti‐apoptotic Bcl‐2 protein, their activation may promote apoptosis in MDMA‐exposed tissues. Our results show for the first time that MDMA induces oxidative‐modification of many cytosolic proteins accompanied with increased oxidative stress and apoptosis, contributing to hepatic damage.     

Analysis of membrane–protein complexes of the marine sulfate reducer Desulfobacula toluolica Tol2 by 1D blue native‐PAGE complexome profiling and 2D blue native‐/SDS‐PAGE

Sulfate‐reducing bacteria (SRB) obtain energy from cytoplasmic reduction of sulfate to sulfide involving APS‐reductase (AprAB) and dissimilatory sulfite reductase (DsrAB). These enzymes are predicted to obtain electrons from membrane redox complexes, i.e. the quinone‐interacting membrane‐bound oxidoreductase (QmoABC) and DsrMKJOP complexes. In addition to these conserved complexes, the genomes of SRB encode a large number of other (predicted) membrane redox complexes, the function and actual formation of which is unknown. This study reports the establishment of 1D Blue Native‐PAGE complexome profiling and 2D BN‐/SDS‐PAGE for analysis of the membrane protein complexome of the marine sulfate reducer Desulfobacula toluolica Tol2. Analysis of normalized score profiles of >800 proteins in combination with hierarchical clustering and identification of 2D BN‐/SDS‐PAGE separated spots demonstrated separation of membrane complexes in their native form, e.g. ATP synthase. In addition to the QmoABC and DsrMKJOP complexes, other complexes were detected that constitute the basic membrane complexome of D. toluolica Tol2, e.g. transport proteins (e.g. sodium/sulfate symporters) or redox complexes involved in Na⁺‐based bioenergetics (RnfABCDEG). Notably, size estimation indicates dimer and quadruple formation of the DsrMKJOP complex in vivo. Furthermore, cluster analysis suggests interaction of this complex with a rhodanese‐like protein (Tol2_C05230) possibly representing a periplasmic electron transfer partner for DsrMKJOP.     

A rapid and simple procedure for the depletion of abundant storage proteins from legume seeds to advance proteome analysis: A case study using Glycine max

2‐D analysis of plant proteomes containing thousands of proteins has limited dynamic resolution because only abundant proteins can be detected. Proteomic assessment of the non‐abundant proteins within seeds is difficult when 60–80% is storage proteins. Resolution can be improved through sample fractionation using separation techniques based upon different physiological or biochemical principles. We have developed a fast and simple fractionation technique using 10 mM Ca²⁺ to precipitate soybean (Glycine max) seed storage globulins, glycinin and β‐conglycinin. This method removes 87±4% of the highly abundant seed proteins from the extract, allowing for 541 previously inconspicuous proteins present in soybean seed to be more detectable (volume increase of ≥50%) using fluorescent detection. Of those 541 enhanced spots, 197 increased more than 2.5‐fold when visualized with Coomassie. The majority of those spots were isolated and identified using peptide mass fingerprinting. Fractionation also provided detection of 63 new phosphorylated protein spots and enhanced the visibility of 15 phosphorylated protein spots, using 2‐D electrophoretic separation and an in‐gel phosphoprotein stain. Application of this methodology toward other legumes, such as peanut, bean, pea, alfalfa and others, also containing high amounts of storage proteins, was examined, and is reported here.