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.     

A proteomic approach analysing the <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> response to virulent and avirulent <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> strains

The present work is directed at studying changes at the proteome level in Arabidopsis thaliana leaves in response to Pseudomonas syringae virulent (Pst) and avirulent (Pst avrRpt2) strains. Arabidopsis leaves were sampled from challenged plants at 4, 8 and 24 h post inoculation. Proteins were TCA–acetone–phenol extracted and subjected to 2-DE (5–8 pH range) and MS/MS (MALDI–TOF–TOF) analysis. Out of 800 matched spots on each of the 36 gels analysed, 147 spots were either absent in at least one of the conditions studied (time or treatments; qualitative variable spots) or differentially accumulated between time and treatments (quantitative variable spots). Out of the 24 proteins successfully identified over TAIR10 database, 23 have not been reported previously in similar proteomics studies of the Arabidopsis thaliana–Pseudomonas syringae interaction. The exhaustive statistical analysis performed, including principal component and heat map, showed that 24 h post inoculation can clearly discriminate the challenged plants from the control. The protein change occurred early (4 h post inoculation) following the virulent pathogen infection, whereas the change occurred later (24 h post inoculation) following the avirulent pathogen inoculation. Concerning the variable proteins, three behavioural groups can be observed: group 1 (common protein changes in response to virulent and avirulent pathogen infection), group 2 (protein changes in response to virulent pathogen infection) and group 3 (protein changes in response to avirulent pathogen infection). Differential identified proteins following the pathogen infection belonged to different groups including those of oxidative stress defence, enzymes of metabolic pathways and molecular chaperones.     

Proteomic characterization of the sulfur-reducing hyperthermophilic archaeon Thermococcus onnurineus NA1 by 2-DE/MS–MS

Thermococcus onnurineus NA1, a sulfur-reducing hyperthermophilic archaeon, was isolated from a deep-sea hydrothermal vent area in Papua New Guinea. The strain requires elemental sulfur as a terminal electron acceptor for heterotrophic growth on peptides, amino acids and sugars. Recently, genome sequencing of Thermococcus onnurineus NA1 was completed. In this study, 2-DE/MS–MS analysis of the cytosolic proteome was performed to elucidate the metabolic characterization of Thermococcus onnurineus NA1 at the protein level. Among the 1,136 visualized protein spots, 110 proteins were identified. Enzymes related to metabolic pathways of amino acids utilization, glycolysis, pyruvate conversion, ATP synthesis, and protein synthesis were identified as abundant proteins, highlighting the fact that these are major metabolic pathways in Thermococcus onnurineus NA1. Interestingly, multiple spots of phosphoenolpyruvate synthetase and elongation factor Tu were found on 2D gels generated by truncation at the N-terminus, implicating the cellular regulatory mechanism of this key enzyme by protease degradation. In addition to the proteins involved in metabolic systems, we also identified various proteases and stress-related proteins. The proteomic characterization of abundantly induced proteins using 2-DE/MS–MS enables a better understanding of Thermococcus onnurineus NA1 metabolism.     

Leaf proteomic analysis in cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis>, Crantz) during plant development,from planting of stem cutting to storage root formation

Tuberization in cassava (Manihot esculenta Crantz) occurs simultaneously with plant development, suggesting competition of photoassimilate partitioning between the shoot and the root organs. In potato, which is the most widely studied tuber crop, there is ample evidence suggesting that metabolism and regulatory processes in leaf may have an impact on tuber formation. To search for leaf proteins putatively involved in regulating tuber generation and/or development in cassava, comparative proteomic approaches have been applied to monitor differentially expressed leaf proteins during root transition from fibrous to tuberous. Stringent cross comparison and statistical analysis between two groups with different plant ages using Student’s t test with 95% significance level revealed a number of protein spots whose abundance were significantly altered (P < 0.05) during week 4 to week 8 of growth. Of these, 39 spots were successfully identified by ion trap LC–MS/MS. The proteins span various functional categories from antioxidant and defense, carbohydrate metabolism, cyanogenesis, energy metabolism, miscellaneous and unknown proteins. Results suggested possible metabolic switches in the leaf that may trigger/regulate storage root initiation and growth. This study provides a basis for further functional characterization of differentially expressed leaf proteins, which can help understand how biochemical processes in cassava leaves may be involved in storage root development.     

Proteomic approach to study leaf proteins in a fast-growing tree species, Gmelina arborea Linn. Roxb

Foliar proteome studies have become highly significant for a comprehensive understanding of complex processes associated with plant growth and development. In the present study, we present a proteomic approach to analyze leaf proteins in an important timber-yielding and fast-growing forest tree species, Gmelina arborea Linn. Roxb. (Verbanaceae). Foliar protein analysis involved protein extraction, two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time of flight (MALDI–TOF–TOF). From the 2-DE protein profile of Gmelina leaves, we identified and isolated 150 well-separated protein spots; among these, 64 protein spots were identified by mass spectrometric (MS/MS) analysis. These proteins were classified according to their involvement in basic biological functions, such as photosynthesis, amino acid metabolism, cytoskeleton, cell wall metabolism, stress-related proteins, redox maintenance, electron transport chain, phytohormone metabolism and protein translation and folding. Analytical variance was determined for the protein spots of samples from different plants. The present study is believed to provide a foundation for the use of leaf proteomics in addressing fundamental physiological and biochemical processes associated with growth and productivity of tree species such as Gmelina arborea.     

Analysis of Protein Expression Profiles of <Emphasis Type="Italic">Halobacillus dabanensis</Emphasis> D-8<Superscript>T</Superscript> Under Optimal and High Salinity Conditions

Two-dimensional (2-D) gel electrophoresis was employed to display the expression profiles of proteins of Halobacillus dabanensis D-8^T under 1%, 10%, and 20% salinities. Approximately 700 protein spots could be detected in the 2-D gels by Imagemaster™ 2D Platinum software. The molecular masses of the majority of intracellular proteins were distributed in the range of 17.5 kDa–66 kDa and isoelectric points of 4.0–5.9. In total 133 protein spots were observed with a changed expression level under different salinity conditions. Sixty-two protein spots showed upregulation and 26 new protein spots were found under high salinity conditions, while 25 protein spots were downregulated and 20 spots disappeared. Twenty-seven proteins with a markedly changed expression in hypersaline environments were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF/MS) and MASCOT. A changed expression pattern was observed for proteins related to energy-producing pathways, stress regulators, and proteins involved in the survival of strain D-8^T under high salt challenges. Many proteins play necessary roles in the adaptation to high salt or as a general stress protein, and some proteins are salt-stressed specific proteins that improve the capability of salt-tolerance of strain D-8^T growth under extremely hypersaline condition.     

Proteomic Analysis of Interactions Between the Generalist Herbivore <Emphasis Type="Italic">Spodoptera exigua</Emphasis> (Lepidoptera: Noctuidae) and <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

In this study, comparative proteomics was used to investigate the interaction of Spodoptera exigua and Arabidopsis thaliana. By using 2-D electrophoresis of differentially expressed proteins, combined with high-throughput matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and MALDI-TOF/TOF MS, the changes in the abundance of proteins induced by insect feeding were studied in A. thaliana. More than 1,100 protein spots were reproducibly detected on each gel. The intensities of 30 protein spots in particular changed significantly, showing differences in volume of at least twofold. Among these, 17 protein spots were upregulated, and 13 were downregulated following an 8-h insect feeding period. Nineteen insect-feeding-responsive proteins were identified, all of which were involved in metabolic regulation, binding functions or cofactor requirement of protein, cell rescue, and defense and virulence, as assessed by Munich Information Center for Protein Sequences function category. About 50% of these were involved in metabolism, including transketolase, S-adenosylmethionine synthase 3, 2,3-biphosphoglycerate-independent phosphoglycerate mutase, beta-ureidopropionase, GDP-d-mannose 3′,5′-epimerase, and fatty acid synthase. The identification of insect-feeding-responsive proteins on Arabidopsis provides not only new insights into insect stress but also a good start for further investigation of their functions. Understanding how the plant responses to insects in the proteomic level will provide tools for a better management of insect pest in the field.     

Proteomic characterization of the abdominal ganglion of Aplysia californica-A protein resource for neuroscience

Aplysia californica (AC) is a widely used model for testing learning and memory. Although ESTs have been generated, proteomics studies on AC proteins are limited. Studies at the protein level, however, are mandatory, not only due to the fact that studies at the nucleic acid level are not allowing conclusions about PTMs. A gel-based proteomics method was therefore applied to carry out protein profiling in abdominal ganglia from AC. Abdominal ganglia were extirpated, proteins extracted and run on 2DE with subsequent in-gel digestion with trypsin, chymotrypsin, and partially by subtilisin. Peptides were identified using a nano-LC-ESI-LTQ-FT-mass spectrometer. MS/MS data were analyzed by searching the NCBI nonredundant public AC EST database and the NCBI nonredundant public AC protein database. A total of 477 different proteins represented by 363 protein spots were detected and were assigned to different protein pathways as for instance signaling (receptors, protein kinases, and phosphatases), metabolism, protein synthesis, handling and degradation, cytoskeleton and structural, oxido-redox, heat shock and chaperone, hypothetical, predicted and unnamed proteins. The generation of a protein map of soluble proteins shows the existence of so far hypothetical and predicted proteins and is allowing and challenging further work at the protein level, in particular in the field of neuroscience.     

Proteomic analysis of salinity-stressed <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> revealed differential suppression and induction of a large number of important housekeeping proteins

Salinity stress is one of the most common abiotic stresses that hamper plant productivity worldwide. Successful plant adaptations to salt stress require substantial changes in cellular protein expression. In this work, we present a 2-DE-based proteomic analysis of a model unicellular green alga, Chlamydomonas reinhardtii, subjected to 300 mM NaCl for 2 h. Results showed that, in addition to the protein spots that showed partial up- or down-regulation patterns, a number of proteins were exclusively present in the proteome of the control cells, but were absent from the salinity-stressed samples. Conversely, a large number of proteins exclusively appeared in the proteome of the salinity-stressed samples. Of those exclusive proteins, we could successfully identify, via LC–MS/MS, 18 spots uniquely present in the control cells and 99 spots specific to NaCl-treated cells. Interestingly, among the salt-exclusive protein spots, we identified several important housekeeping proteins like molecular chaperones and proteins of the translation machinery, suggesting that they may originate from post-translational modifications rather than from de novo biosynthesis. The possible role and the salt-specific modification of these proteins by salinity stress are discussed.     

An improved method of protein isolation and proteome analysis with <Emphasis Type="Italic">Saccharina japonica</Emphasis> (Laminariales) incubated under different pH conditions

The brown alga Saccharina japonica is abundant on rocky coasts of Far East Asia, including Korea, Japan, and China. S. japonica produces high levels of compounds used in the food, cosmetic, and pharmaceutical industries. Thus, many studies have focused on the biosynthesis, extraction, purification, and application of carbohydrates, as well as biochemical features that yield cellular proteins. However, total protein isolation has proved difficult, due to viscous polysaccharides on the surface of S. japonica. To extract total proteins cleanly from S. japonica, we examined various lysis buffers and detergents for effective cell lysis and removal of polysaccharide. Lysis solution D (7 M urea, 4% [3-(3-cholami-dopropyl dimethylammonio) propanesulfonate], 2 M thio-urea, 100 mM dithiothreitol, 4% pharmalyte, 4% polyvinylpyrrolidone) achieved a comparatively high yield of protein extraction, with 12 mg of proteins purified per 1 g of dry weight of S. japonica. Proteins isolated using lysis solution D and subjected to two-dimension polyacrylamide gel electrophoresis generated more than 200 protein spots. Of these, 60 spots were analyzed by matrix-assisted laser desorption ionization-time of flight/mass spectrometry (MALDI-TOF/MS) and MALDI-TOF/MS/MS. A database search revealed that these proteins include glyceraldehyde-3-phosphate dehydrogenase, tryptophan synthase α chain, 6-phosphogluconate dehydrogenase (6PGD), actin, phosphoglycerate kinase, elongation factor Tu, kinesin, fucoxanthin-chlorophyll a–c binding protein F precursor and ATP synthase subunit β. Many protein spots were unidentified. When S. japonica was incubated at different pH, tryptophan synthase α chain and variant surface glycoprotein 7 precursor were highly expressed at pH 7.5 and 9.5, respectively, whereas 6PGD and kinesin showed low expression at pH 9.5.     

Proteomic analysis of human skeletal muscle (<Emphasis Type="Italic">m. vastus lateralis</Emphasis>) proteins: Identification of 89 gene expression products

Proteins from bioptates and autoptates of human skeletal muscle m. vastus lateralis were separated by O’Farrell two-dimensional gel electrophoresis (2DE). MALDI-TOF MS and MS/MS enabled identification of 89 protein spots as expression products of 55 genes. A modification of the O’Farrell’s method including non-equilibrium electrophoresis in a pH gradient allowed detection — among major sarcomeric, mitochondrial, and cytosolic proteins — of several proteins, such as PDZ- and LIM domain-containing ones (pI > 8.70), fragments of known proteins, and a stable complex of heavy and light ferritin chains. The data underlie further studies of human skeletal muscle proteins in terms of molecular mechanisms of some physiological and pathological processes.     

The Novel Responses of Ethambutol Against <Emphasis Type="Italic">Mycobacterium smegmatis</Emphasis> mc<Superscript>2</Superscript>155 Revealed by Proteomics Analysis

Ethambutol (EMB), one of the effective anti-mycobacterial drugs, inhibits the biosynthesis of mycobacterium cell wall. To elucidate the molecular mechanism of EMB against tuberculosis (TB), Mycobacterium smegmatis mc²155 was employed as a model of mycobacterial system in this study. We compared the protein profiles on M. smegmatis mc²155 treated by EMB and untreated using fluorescence difference two-dimensional gel electrophoresis (2-D DIGE). A total of 40 differential protein spots were selected and 22 proteins were identified by HPLC-nano ESI–MS/MS analysis, including 16 over-expressed proteins and 6 under-expressed proteins. These proteins mainly affected energy metabolism, as well as synthesis and modification of macromolecules. The expressions of correspondent genes were confirmed by RT-PCR. This investigation provided some clues for searching potential drug targets.     

A proteomics approach for the analysis of hemolymph proteins involved in the immediate defense response of the soft tick, <Emphasis Type="Italic">Ornithodoros savignyi</Emphasis>, when challenged with <Emphasis Type="Italic">Candida albicans</Emphasis>

A proteomics approach was employed to identify proteins secreted into the hemolymph of Ornithodorus savignyi ticks 2 h after immune-challenge with the yeast, Candida albicans. Profiling of the proteins present in hemolymph of unchallenged ticks versus ticks challenged with heat-killed yeast revealed five proteins to be differentially expressed. The modulated protein spots were subjected to tandem mass spectrometry (MS/MS) analysis, but could not be positively identified. These proteins can be assigned to the immune response as they were not induced after aseptic injury. In an attempt to identify hemolymph proteins that recognize and bind to yeast cells, hemolymph obtained from both unchallenged and challenged ticks was incubated with C. albicans. Elution of the bound proteins followed by SDS–PAGE analysis indicated that three proteins (97, 88 and 26 kDa) present in both unchallenged and challenged hemolymph samples bind to yeast cells. The constant presence of these three proteins in tick hemolymph leads us to believe that they may be involved in non-self recognition and participate in yeast clearance from tick plasma. The analyzed yeast-binding proteins could also not be positively identified, suggesting that all the tick immune proteins investigated in this study are novel.     

Proteomic analysis of the GlnR-mediated response to nitrogen limitation in Streptomyces coelicolor M145

GlnR is the global regulator of nitrogen assimilation in Streptomyces coelicolor M145 and other actinobacteria. Two-dimensional polyacrylamide gel electrophoresis analyses were performed to identify new GlnR target genes by proteomic comparison of wild-type S. coelicolor M145 and a ΔglnR mutant. Fifty proteins were found to be differentially regulated between S. coelicolor M145 and the ΔglnR mutant. These spots were identified by nanoHPLC–ESI-MS/MS and classified according to their cellular role. Most of the identified proteins are involved in amino acid biosynthesis and in carbon metabolism, demonstrating that the role of GlnR is not restricted to nitrogen metabolism. Thus, GlnR is supposed to play an important role in the global metabolic control of S. coelicolor M145.     

Differential Proteomic Analysis of Temperature-Induced Autolysis in Mycelium of <Emphasis Type="Italic">Pleurotus tuber</Emphasis>-<Emphasis Type="Italic">regium</Emphasis>

Autolysis is an important physiological process found in fungal cultivation. However, there is hitherto no report on the autolysis of Pleurotus tuber-regium. We have investigated the enzymes secreted by temperature-induced (40°C as treatment versus 10°C as control) autolysis of the mycelium of P. tuber-regium grown in submerged cultivation. A comparison between the intracellular proteins (inside the mycelium) and the extracellular proteins (in the culture medium) of the treatment and control by proteomic analysis involving 2D PAGE and MALDI–TOF–MS was made. Twenty-two up-regulated protein spots were detected and eight proteins were identified. They included proteasome which participates in the ubiquitin–proteasome pathway; β-1,3-glucanosyltransferase and tubulin which are involved in the renewal and repair of cell wall; protease and endoglucanase which promote the natural degradation of cell wall and cytoplasm; 14-3-3 protein which takes part in cell signal transduction; and two putative proteins presumably relate to the autolysis process. These identified proteins suggest partially the metabolic processes of the autolysis in the P. tuber-regium mycelium.     

Proteome Analysis of Aniline-Induced Proteins in Acinetobacter lwoffii K24

Acinetobacter lwoffii K24 is a soil bacterium that can use aniline as a sole carbon and nitrogen source (by β-ketoadipate pathway genes (cat genes)) and has two copies of catABC gene separately located on the chromosome. In order to identify aniline-induced proteins, two-dimensional electrophoresis (2-DE) was applied to soluble protein fractions of A. lwoffii K24 cultured in aniline and succinate media. In the range of pH3–10, more than 370 spots were detected on the silver stained gels. Interestingly, more than 20 spots were selectively induced on aniline-cultured bacteria. Twenty-three protein spots of A. lwoffii K24 were analyzed by N-terminal microsequencing and internal microsequencing with in-gel digestion. Of 20 aniline induced protein spots, we identified six β-ketoadipate pathway genes, one subunit of amino group transfer (putative subunit of aniline oxygenase), malate dehydrogenase, putative ABC transporter, putative hydrolase, HHDD isomerase, and five unknown proteins. Especially in case of two catechol 1,2-dioxygenases (CDI₁ and CDI₂), more than three isotypes were detected on the 2D gel. This study showed that the proteome analysis of A. lwoffii K24 may be helpful for identification of genes induced by aniline and understanding of their function in the cell. Received: 2 April 2001 / Accepted: 14 May 2001     

Alteration of DBP Levels in CSF of Patients with MS by Proteomics Analysis

Objective The diagnosis of multiple sclerosis (MS) is still challenging recently due to the lack of a specific diagnostic test. Proteomics analysis was applied to biomarkers discovery and their pathways study. Methods First, the proteins of CSF from MS patients and control group were analyzed individually with 2D-DIGE technology (two-dimensional difference gel electrophoresis). Then, protein spots were found out with DeCyder6.0 software which showed different expression levels in the gel images between the two groups. The information regarding these proteins was collected based on MALDI-TOF/MS and related database searches. Lastly, interaction between these proteins was further analyzed by using Metacore software. Results There were 13 proteins that showed more than 1.5-fold difference in expression levels between the two groups. Furthermore, the identification made by MALDI-TOF/MS revealed that one of the most significant differential proteins was DBP (vitamin D-binding protein), which decreased in the experimental group. This result was confirmed by ELISA (P < 0.01). Moreover, network between the 13 proteins were partially got, which showed some biological interactions. Conclusion These results support a correlation between the level of DBP and MS. DBP may be a potential useful biomarker for diagnosis or a medicine target for treatment of MS.     

Identification of (η6-arene)ruthenium(II) protein binding sites in E. coli cells by combined multidimensional liquid chromatography and ESI tandem mass spectrometry: specific binding of [(η6- p -cymene)RuCl2(DMSO)] to stress-regulated proteins and to helicases

An automated multidimensional protein identification technology, which combines biphasic liquid chromatography with electrospray ionisation tandem mass spectrometry (MS/MS), was employed to analyse tryptic peptides from Escherichia coli cells treated with the antiproliferation agent [(η⁶-p-cymene)RuCl₂(DMSO)], where DMSO is dimethyl sulfoxide. MS/MS spectra were recorded for molecular ions generated by neutral loss of p-cymene from intensive peptide ions coordinated by the (η⁶-p-cymene)Ru^II fragment. Matching of the MS/MS spectra of the ruthenated peptides to spectra of proteins in the E. coli database enabled the identification of five protein targets for [(η⁶-p-cymene)RuCl₂(DMSO)]. One of these is the constitutive cold-shock protein cspC, which regulates the expression of genes encoding stress-response proteins, and three of the other targets, ppiD, osmY and sucC, are proteins of the latter type. The DNA damage-inducible helicase dinG was likewise established as a protein target. Aspartate carboxylate functions were identified as the probable Ru binding sites in cspC, ppiD and dinG, and threonine and lysine side chains in osmY and sucC, respectively. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.