Comprehensive analyses of the proteome and ubiquitome revealed mechanism of high temperature accelerating petal abscission in tree peony

Tree peony(Paeonia suffruticosa Andrews)is a well-known ornamental plant with high economic value,but the short fluorescence is a key obstacle to its ornamental value and industry development.High temperature accelerates flower senescence and abscission,but the associated mechanisms are poorly understood.In this study,the tandem mass tag(TMT)proteome and label-free quantitative ubiquitome from tree peony cut flowers treated with 20℃for 0 h(RT0),20℃or 28℃for 60 h(RT60 or HT60)were examined based on morphological observation,respectively.Totally,6970 proteins and 1545 lysine ubiquitinated(Kub)sites in 844 proteins were identified.Hydrophilic residues(such as glutamate and aspartate)neighboring the Kub sites were in preference,and 36.01%of the Kub sites were located on the protein surface.The differentially expressed proteins(DEPs)and Kub-DEPs in HT60 vs RT60 were mainly enriched in ribosomal protein,protein biosynthesis,secondary metabolites biosynthesis,flavonoid metabolism,carbohydrate catabolism,and auxin biosynthesis and signaling revealed by GO and KEGG analysis,accompanying the increase of endogenous abscisic acid(ABA)accumulation and decrease of endogenous indoleacetic acid(IAA)level.Additionally,the expression patterns of six enzymes(SAMS,ACO,YUC,CHS,ANS and PFK)putatively with Kub modifications were analyzed by proteome and real-time quantitative RT-PCR.The cell-free degradation assays showed PsSAMS and PsACO proteins could be degraded via the 26 S proteasome system in tree peony flowers.Finally,a working model was proposed for the acceleration of flower senescence and abscission by high temperature.In summary,all results contributed to understanding the mechanism of flower senescence induced by high temperature and prolonging fluorescence in tree peony.

In‑depth proteome characterization of endometrium and extraembryonic membranes during implantation in pig

Background Proteome characterization of the porcine endometrium and extraembryonic membranes is important to understand mother-embryo cross-communication.In this study,the proteome of the endometrium and cho-rioallantoic membrane was characterized in pregnant sows(PS)during early gestation(d 18 and 24 of gestation)and in the endometrium of non-pregnant sows(NPS)during the same days using LC-MS/MS analysis.The UniProtKB database and ClueGO were used to obtain functional Gene Ontology annotations and biological and functional networks,respectively.Results Our analysis yielded 3,254 and 3,457 proteins identified in the endometrium of PS and NPS,respectively;of these,1,753 being common while 1,501 and 1,704 were exclusive to PS and NPS,respectively.In addition,we iden-tified 3,968 proteins in the extraembryonic membranes of PS.Further analyses of function revealed some proteins had relevance for the immune system process and biological adhesion in endometrium while the embryonic chorion displayed abundance of proteins related to cell adhesion and cytoskeletal organization,suggesting they dominated the moment of endometrial remodeling,implantation and adhesion of the lining epithelia.Data are available via Pro-teomeXchange with identifier PXD042565.Conclusion This is the first in-depth proteomic characterization of the endometrium and extraembryonic mem-branes during weeks 3 to 4 of gestation;data that contribute to the molecular understanding of the dynamic environ-ment during this critical period,associated with the majority of pregnancy losses.

Mass Spectrometry-based Deep Coverage Proteome:Evaluation of Cellular Protein Extraction Methods

The current study comprehensively evaluates four different protein extraction methods based on urea,sodium dodecyl sulfate(SDS),anionic surfactants(BT),and total RNA extractor(Trizol),aiming to optimize the sample preparation workflow for mass spectrometry-based proteomics.Using HeLa cells as an example,we found that the method employing the mass spectrometry-compatible surfactant BT reagent significantly reduces the total time consumed for protein extraction and minimizes protein losses during the sample preparation process.Further integrating the four protein extraction methods,we identified over 7000 proteins from HeLa cells without relying on pre-fractionation techniques,and 2990 of them were quantified using label-free quantification.It is worth noting that the BT and SDS methods demonstrate higher efficiency in extracting membrane proteins,while the Urea and Trizol methods are more effective in extracting proteins from nuclear and cytoplasmic fractions.In summary,this study provides a novel solution for deep proteome coverage,particularly in the context of cellular protein extraction,by integrating mass spectrometry-compatible surfactants with traditional extraction methods to effectively enhance protein identification numbers.

Environmental concentration of ammonia nitrogen induced marked changes in proteome of clam Ruditapes philippinarum in dose-and time-dependent manner

Previous studies have revealed that ammonia nitrogen has several adverse effects on clam Ruditapes philippinarum.However,knowledge is lacking regarding the related proteins involved in the toxicological responses,which is vital to elucidate the underlying mechanism of ammonia nitrogen.In this study,clams R.philippinarum were exposed to ammonia nitrogen for 21 d at two environmentally relevant concentrations.The tandem mass tags approach(TMT)was applied to assay the differentially expressed proteins(DEPs)in clam gill tissues on the 3 rd and 21 st day.Finally,a total of 7263 proteins were identified.Bioinformatics analyses revealed that clam protein profiles changed in dose-and time dependent manner after ammonia nitrogen exposure.We inferred that the clams may face heavy challenges after ammonia exposure,such as unbalanced gender ratio,lysosomal disease,energy lack,neurological disorders,altered glutamine metabolism,increased lipid synthesis,and impaired immunity.Variation profiles of enzyme activities of glutaminase and glutamine synthase provided direct evidence to verify the related inference from proteome data.Most of the inferred toxic effects merit further study.This study identified important proteins related to ammonia nitrogen toxicity in the clam and indicated the severe stress of marine ammonia pollution on the healthy development of mollusc aquaculture.

Identification of transition factors in myotube formation from proteome and transcriptome analyses

Muscle fibers are the main component of skeletal muscle and undergo maturation through the formation of myotubes.During early development,a population of skeletal muscle satellite cells(SSCs)proliferate into myoblasts.The myoblasts then undergo further differentiation and fusion events,leading to the development of myotubes.However,the mechanisms involved in the transition from SSCs to myotube formation remain unclear.In this study,we characterized changes in the proteomic and transcriptomic expression profiles of SSCs,myoblasts(differentiation for 2 d)and myotubes(differentiation for 10 d).Proteomic analysis identified SLMAP and STOM as potentially associated with myotube formation.In addition,some different changes in MyoD,MyoG,Myosin7 and Desmin occurred after silencing SLMAP and STOM,suggesting that they may affect changes in the myogenic marker.GO analysis indicated that the differentiation and migration factors SVIL,ENSCHIG00000026624(AQP1)and SERPINE1 enhanced the transition from SSCs to myoblasts,accompanied by changes in the apoptotic balance.In the myoblast vs.myotube group,candidates related to cell adhesion and signal transduction were highly expressed in the myotubes.Additionally,CCN2,TGFB1,MYL2 and MYL4 were identified as hub-candidates in this group.These data enhance our existing understanding of myotube formation during early development and repair.

Phosphorus Starvation Tolerance in Rice Through Combined Physiological,Biochemical,and Proteome Analyses

Phosphorus(P) deficiency limits the growth,development,and productivity of rice.To better understand the underlying mechanisms in P-deficiency tolerance and the role of Pup1 QTL in enhancing P use efficiency(PUE) for the development of P-efficient rice cultivars,a pair of contrasting rice genotypes(Pusa-44 and NIL-23) was applied to investigate the morpho-physio-biochemical and proteomic variation under P-starvation stress.The rice genotypes were grown hydroponically in a PusaRich medium with adequate P(16 mg/kg,+P) or without P(0 mg/kg,-P) for 30 d.P-starvation manifested a significant reductions in root and shoot biomass,shoot length,leaf area,total chlorophyll,and P,nitrogen and starch contents,as well as protein kinase activity.The stress increased root-to-shoot biomass ratio,root length,sucrose content,and acid phosphatase activity,particularly in the P-tolerant genotype(NIL-23).Comparative proteome analysis revealed several P metabolism-associated proteins(including OsCDPKs,OsMAPKs,OsCPKs,OsLecRK2,and OsSAPks) to be expressed in the shoot of NIL-23,indicating that multiple protein kinases were involved in P-starvation/deficiency tolerance.Moreover,the up-regulated expression of OsrbcL,OsABCG32,OsSUS5,OsPoll-like B,and ClpC2 proteins in the shoot,and OsACA9,OsACA8,OsSPS2F,OsPP2C15,and OsBiP3 in the root of NIL-23,indicated their role in P-starvation stress control through the Pup1 QTL. Thus,our findings indicated that-P stress-responsive proteins,in conjunction with morpho-physio-biochemical modulations,improved PUE and made NIL-23 a P-deficiency tolerant genotype due to the introgression of the Pup1 QTL in the Pusa-44 background.

The sperm-interacting proteome in the bovine isthmus and ampulla during the periovulatory period

Background Spermatozoa interact with oviduct secretions before fertilization in vivo but the molecular players of this dialog and underlying dynamics remain largely unknown.Our objectives were to identify an exhaustive list of sperm-interacting proteins(SIPs)in the bovine oviduct fluid and to evaluate the impact of the oviduct anatomical region(isthmus vs.ampulla)and time relative to ovulation(pre-ovulatory vs.post-ovulatory)on SIPs number and abundance.Methods Pools of oviduct fluid(OF)from the pre-ovulatory ampulla,pre-ovulatory isthmus,post-ovulatory ampulla,and post-ovulatory isthmus in the side of ovulation were collected from the slaughterhouse.Frozen-thawed bull sperm were incubated with OF or phosphate-buffered saline(control)for 60 min at 38.5℃.After protein extraction and digestion,sperm and OF samples were analyzed by nanoLC-MS/MS and label-free protein quantification.Results A quantitative comparison between proteins identified in sperm and OF samples(2333 and 2471 proteins,respectively)allowed for the identification of 245 SIPs.The highest number(187)were found in the pre-ovulatory isthmus,i.e.,time and place of the sperm reservoir.In total,41 SIPs(17%)were differentially abundant between stages in a given region or between regions at a given stage and 76 SIPs(31%)were identified in only one region×stage condition.Functional analysis of SIPs predicted roles in cell response to stress,regulation of cell motility,fertilization,and early embryo development.Conclusion This study provides a comprehensive list of SIPs in the bovine oviduct and evidences dynamic spatiotemporal changes in sperm-oviduct interactions around ovulation time.Moreover,these data provide protein candidates to improve sperm conservation and in vitro fertilization media.

Subinhibitory Levels of Fluoroquinolones Result in Enrichment of the Membrane Proteome of Staphylococcus aureus

Staphylococcus aureus is a common marine foodborne pathogen.In this study,antibiotics ciprofloxacin and enrofloxacin were used to induce drug-resistance in S.aureus.The differentially expressed proteins(DEPs)were analyzed and compared with those in the bacteria cultured without antibiotics.The primary proteomic alterations were in the levels of cell membrane components and proteins related to lysine and folic acid biosynthesis,which were all significantly up-regulated.The minimal inhibitory concentrations(MIC)for both test drugs were elevated to 10μg m L^(-1)following serial passaging.These results indicated that,for both ciprofloxacin and enrofloxacin,drug-resistance were developed even in the subinhibitory levels and the primary response was a major alteration in the cell membrane proteome.These changes were similar to those observed in S.aureus cultured with super-MIC levels of these antibiotics.The current study provides a theoretical basis for in-depth study of the related changes of marine foodborne pathogens in subinhibitory concentrations that are commonly found in situ.

Comprehensive and deep profiling of the plasma proteome with protein corona on zeolite NaY

Proteomic characterization of plasma is critical for the development of novel pharmacodynamic biomarkers.However,the vast dynamic range renders the profiling of proteomes extremely challenging.Here,we synthesized zeolite NaY and developed a simple and rapid method to achieve comprehensive and deep profiling of the plasma proteome using the plasma protein corona formed on zeolite NaY.Specifically,zeolite NaY and plasma were co-incubated to form plasma protein corona on zeolite NaY(NaY-PPC),followed by conventional protein identification using liquid chromatography-tandem mass spectrometry.NaY was able to significantly enhance the detection of low-abundance plasma proteins,minimizing the“masking”effect caused by high-abundance proteins.The relative abundance of middleand low-abundance proteins increased substantially from 2.54%to 54.41%,and the top 20 highabundance proteins decreased from 83.63%to 25.77%.Notably,our method can quantify approximately 4000 plasma proteins with sensitivity up to pg/mL,compared to only about 600 proteins identified from untreated plasma samples.A pilot study based on plasma samples from 30 lung adenocarcinoma patients and 15 healthy subjects demonstrated that our method could successfully distinguish between healthy and disease states.In summary,this work provides an advantageous tool for the exploration of plasma proteomics and its translational applications.

Global and Comparative Proteome Analysis of Nitrogen-Stress Responsive Proteins in the Root, Stem and Leaf of Brassica napus

Nitrogen(N)is one of the basic nutrients and signals for plant development and deficiency of it would always limit the productions of crops in the field.Quantitative research on expression of N-stress responsive proteins on a proteome level remains elusive.In order to gain a deep insight into the proteins responding to nitrogen stress in rapeseed(Brassica napus L.),comparative proteomic analysis was performed to investigate changes of protein expression profiles from the root,stem and leaf under different N concentrations,respectively.More than 200 differential abundance proteins(DAPs)were detected and categorized into groups according to annotations,including“binding and catalytic activity”,“involved in primary metabolism and cellular processes”,“stress-response”and so on.Variation in chlorophyll(Chl)content and antioxidant activities further revealed that oxidative stress raised with the increase of N concentration.Bioinformatics analysis based on the expression level of total proteins suggested these DAPs might play important roles in adaptation to N-stress conditions.Generally,these results provides a new aspect into N-stress responding proteins in Brassica plants.

Study on the Optimization of Two-dimensional Electrophoresis Technology System for Rapeseed Proteome

[Objective] The research aimed to establish the two-dimensional electrophoresis（2-DE）technology which was suitable for the rapeseed proteome research.[Method] Xiangyou 17 was as the material.The sample preparation method,gel concentration and loading amount,etc.in 2-DE technology were optimized.[Result] The best extraction method of total protein of rapeseed was TCA-acetone method,and the protein spots on 2-DE map were the most.When IPG strip（pH 3-10）and 12% gel were used,and the loading amount was 250 μg,the two-dimensional electrophoresis map with the clear background,good repeatability and high protein spot resolution was obtained.[Conclusion] The research laid the foundation for carrying out the rapeseed proteomics research.

Commentary on: Mobley AJ, Lam YW, Lau KM, Pais VM, Lesperance JO, Steadman B, et al. Monitoring the serological Proteome Colon, the latest modality in prostate cancer detection. J Urol 2004; 172: 331-7.

This manuscript examines the utility, utilizing the Ciphergen Protein Biosystem II, to develop a fingerprint for the diagnosis of prostate cancer. The investigators compared samples from control individuals as well as those with prostate cancer. In doing so, they utilize several chip platforms on which to examine the resulting

Proteome of human colon cancer stem cells:A comparative analysis

AIM： To isolate and identify the biological characteristics of human colon cancer stem cells （SW1116 cells） and further study their proteome. METHODS： SW1116 cells were isolated and cultured with a serum-free medium （SFM）. Sphere formation was assayed to observe the formation of colon cancer stem cell spheres. SW1116 cells were inoculated into a serum-containing medium for observing their differentiation characteristics. Proliferation curve and cross-resistance of SWl116 cells to different drugs were detected by MTT. Percentage of SP cells in SW1116 cells was detected with Hoechst33342 staining. Telomerase activity in SW1116cells was checked by polymerase chain reaction （PCR）-enzyme linked immunosorbent assay. Expressions of stem cell relevant genes and proteins were detected by reverse transcription-PCR and Western blot, respectively. Total protein was isolated from SW1116 cells by two-dimensional gel electrophoresis （2-DE） and differentially expressed proteins were identified by tandem mass spectrometry （MALDI-TOF/TOF）. RESULTS： The isolated SW1116 cells presented as spheroid and suspension growths in SFM with a strong self-renewal, proliferation, differentiation and drug-resistance ability. The percentage of SP cells in SW1116 cells was 38.9%. The SW1116 cells co-expressed the CD133 and CD29 proteins. The telomerase activity in SW1116 cells was increased. The expressions of different stem cell relevant genes and proteins were detected. The proteomic analysis showed that the 26 protein spots were differently expressed in SW1116 cells and 10 protein spots were identified as ubiquitin fusion- degradation l-like protein, nuclear chloride channel protein, tubulin 13, Raichu404X, stratifin, F-actin cap- ping protein α-1 subunit, eukaryotic translation elongation factor 1 delta isoform 2, hypothetical protein, glyceraldehyde-3-phosphate dehydrogenase and guanine nucleotide binding protein 13 polypeptide 2-like 1, respectively. CONCLUSION： SW1116 cells are biologically characterized by self-renewal, proliferation and differentiation, and the differently expressed proteins in SW1116 cells may be essential for isolating cancer stem cells.

Comparative analysis between endometrial proteomes of pregnant and non-pregnant ewes during the peri-implantation period

Background： Early pregnancy failure has a profound impact on both human reproductive health and animal production. 2/3 pregnancy failures occur during the peri-implantation period; however, the underlying mechanism（s） remains unclear. Well-organized modification of the endometrium to a receptive state is critical to establish pregnancy Aberrant endometrial modification during implantation is thought to be largely responsible for early pregnancy loss. Result： In this study, using well-managed recipient ewes that received embryo transfer as model, we compared the endometrial proteome between pregnant and non-pregnant ewes during implantation period. After embryo transfer, recipients were assigned as pregnant or non-pregnant ewes according to the presence or absence of an elongated conceptus at Day 17 of pregnancy. By comparing the endometrial proteomic profiles between pregnant and non-pregnant ewes, we identified 94 and 257 differentially expressed proteins （DEPs） in the endometrial caruncular and intercaruncular areas, respectively. Functional analysis showed that the DEPs were mainly associated with immune response, nutrient transport and utilization, as well as proteasome-mediated proteolysis. Conclusion： These analysis imply that dysfunction of these biological processes or pathways of DEP in the endometrium is highly associated with early pregnancy loss. In addition, many proteins that are essential for the establishment of pregnancy showed dysregulation in the endometrium of non-pregnant ewes. These proteins, as potential candidates, may contribute to early pregnancy loss.

Screening of Serum Biomarkers for Distinguishing between Latent and Active Tuberculosis Using Proteome Microarray

Objective To identify potential serum biomarkers for distinguishing between latent tuberculosis infection（LTBI） and active tuberculosis（TB）. Methods A proteome microarray containing 4,262 antigens was used for screening serum biomarkers of 40 serum samples from patients with LTBI and active TB at the systems level. The interaction network and functional classification of differentially expressed antigens were analyzed using STRING 10.0 and the TB database, respectively. Enzyme-linked immunosorbent assays（ELISA） were used to validate candidate antigens further using 279 samples. The diagnostic performances of candidate antigens were evaluated by receiver operating characteristic curve（ROC） analysis. Both antigen combination and logistic regression analysis were used to improve diagnostic ability. Results Microarray results showed that levels of 152 Mycobacterium tuberculosis（Mtb）-antigenspecific IgG were significantly higher in active TB patients than in LTBI patients（P 〈 0.05）, and these differentially expressed antigens showed stronger associations with each other and were involved in various biological processes. Eleven candidate antigens were further validated using ELISA and showed consistent results in microarray analysis. ROC analysis showed that antigens Rv2031 c, Rv1408, and Rv2421 c had higher areas under the curve（AUCs） of 0.8520, 0.8152, and 0.7970, respectively. In addition, both antigen combination and logistic regression analysis improved the diagnostic ability. Conclusion Several antigens have the potential to serve as serum biomarkers for discrimination between LTBI and active TB.

Proteome analysis of alfalfa roots in response to water deficit stress

To evaluate the response of alfalfa to water deficit （WD） stress, WD-induced candidates were investigated through a proteomic approach. Alfalfa seedlings were exposed to WD stress for 12 and 15 days respectively, followed by 3 days re-watering. Water deficit increased H202 content, lipid peroxidation, DPPH （1,1-diphenyl-2-picrylhydrazyl）-radical scavenging activity, and the free proline level in alfalfa roots. Root proteins were extracted and separated by two-dimentional polyacrylamide gel electrophoresis （2-DE）. A total of 49 WD-responsive proteins were identified in alfalfa roots; 25 proteins were reproducibly found to be up-regulated and 24 were down-regulated. Two proteins, namely cytosolic ascorbate peroxidase （APx2） and putative F-box protein were newly detected on 2-DE maps of WD-treated plants. We identified several proteins including agamous-like 65, albumin b-32, inward rectifying potassium channel, and auxin-independent growth promoter. The identified proteins are involved in a variety of cellular functions including calcium signaling, abacisic acid （ABA） biosynthesis, reactive oxygen species （ROS） regulation, transcription/translation, antioxidant/detoxification/stress defense, energy metabolism, signal transduction, and storage. These results indicate the potential candidates were responsible for adaptive response in alfalfa roots.

Molecular mechanisms of growth depression in broiler chickens(Gallus Gallus domesticus)mediated by immune stress:a hepatic proteome study

Background:Immunological stress decreases feed intake,suppresses growth and induces economic losses.However,the underlying molecular mechanism remains unclear.Label-free liquid chromatography and mass spectrometry(LC-MS)proteomics techniques were employed to investigate effects of immune stress on the hepatic proteome changes of Arbor Acres broilers(Gallus Gallus domesticus)challenged with Escherichia coli lipopolysaccharide(LPS).Results:Proteomic analysis indicated that 111 proteins were differentially expressed in the liver of broiler chickens from the immune stress group.Of these,28 proteins were down-regulated,and 83 proteins were up-regulated in the immune stress group.Enrichment analysis showed that immune stress upregulated the expression of hepatic proteins involved in defense function,amino acid catabolism,ion transport,wound healing,and hormone secretion.Furthermore,immune stress increased valine,leucine and isoleucine degradation pathways.Conclusion:The data suggests that growth depression of broiler chickens induced by immune stress is triggered by hepatic proteome alterations,and provides a new insight into the mechanism by which immune challenge impairs poultry production.

Comparative Proteome Analysis of Human Lung Squamous Carcinoma Tissue

Objective： To establish the two-dimensional electrophoresis profiles with high resolution and reproducibility from human lung squamous carcinoma tissue and paired normal tumor-adjacent bronchial epithelial tissue, and to identify differential expression tumor-associated proteins by using proteome analysis. Methods： Comparative proteome analysis with 20 human lung squamous carcinoma tissues and the paired normal bronchial epithelial tissues adjacent to tumors was carried out. The total proteins of human lung squamous carcinoma tissue and paired normal tumor-adjacent bronchial epithelial tissue were separated by means of immobilized pH gradient-based two-dimensional gel electrophoresis （2-DE） and silver staining. The differential expression proteins were analyzed and then identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry （MALDI-TOF-MS）. Results： （1） Well-resolved, reproducible 2-DE patterns of human lung squamous carcinoma and adjacent normal bronchial epithelial tissues were obtained. For tumor tissue, average spots of 3 gels were 1567±46, and 1436±54 spots were matched with an average matching rate of 91.6%. For control, average spots of 3 gels were 1349±58, and 1228±35 spots were matched with an average matching rate of 91.03%. The average position deviation of matched spots was 0.924±0.128 mm in IEF direction, and 1.022±0.205 mm in SDS-PAGE direction; （2） A total of 1178±56 spots were matched between the eleetrophoretie maps of 20 human lung squamous carcinoma tissues and paired normal tumor-adjacent bronchial epithelial tissues. Seventy-six differentially expressed proteins were screened; （3） Sixty-eight differential proteins were identified by PMF, some proteins were the products of oneogenes, and others involved in the regulation of cell cycle and signal transduetion; （4） In order to validate the reliability of the identified results, the expression of 3 proteins mdm2, c-jun and EGFR, which was correlated with lung squamous carcinoma, was detected by immunohistoehemieal staining and Western blot analysis. The results revealed that mdm2, c-jun and EGFR were up-regulated in lung squamous carcinomas, whereas they were down-regulated in adjacent normal bronchial epithelial tissues, normal lung tissues and inflammatory pseudotumor, which was consistent with our proteome analysis results. Conclusion： The well-resolved, reproducible 2-DE patterns of human lung squamous carcinoma and adjacent normal bronchial epithelial tissues were established and 68 differential proteins were characterized by applying comparative proteome analysis successfully. These results will provide scientific foundation for screening the molecular biomarker used to diagnose and treat lung squamous carcinoma, as well as to improve the patient＇s prognosis and provide new clue for the research of lung squamous carcinogenic mechanism.

Proteome Analysis of Inhibitory Effect of Gadolinium on Sinorhizobium fredii

The inhibitory effect of gadolinium on Sinorhizobium fredii USDA 205 was studied on a global scale using twodimensional gel electrophoresis and MALDI-TOF MS. The results indicated that 22 proteins were significantly affected by 1 mmol · L^-1 Gd^3 ＋ treatment when compared with an untreated control. Among these proteins, nine were up-regulated and thirteen were down-regulated. The differently expressed proteins were classified into 8 functional categories based on their functions, including transporters, proteins for cellular defence, and proteins involved in metabolism.

Proteome Analysis of the Adaptation of a Phenol-Degrading Bacterium Acinetobacter sp. EDP3 to the Variation of Phenol Loadings

Strain EDP3 was isolated from an industrial-activated sludge. It belonged to the gamma group of Pro-teobacteria with an identity of 97.0% to Acinetobacter calcoaceticus according to the 16S rRNA gene sequences. It can tolerate up to 1000mg·L^-1 phenol at room temperature with a much longer lag phase. This indicates that higher phenol concentration has induced some physiological and genotypic changes in the bacterium. The aim of this study is, therefore, to investigate these responses to phenol concentration variations in strain EDP3. Proteome analysis is conducted by means of a two-dimensional polyacrylamide gel electrophoresis （2D PAGE） and matrix-assisted laser desorption ionization time of flight mass spectrometry （MALDI-TOF-MS） was conducted to obtain a deeper insight into the adaptive responses inside the bacterium. Comparative analysis of the proteome profiles of strain EDP3. grown in 400mg·L^-1 and 1000mg·L^-1 phenol allowed us to identify that among all the proteins up-regulated under the higher phenol concentration, oxidative stress proteins were dominant. The synthesis of a heat shock protein, 60000 chaperonin GroEL, was also amplified. In addition, the expression of one membrane protein, adenosine 5＇-triphosphate （ATP）-binding cassette （ABC） type sugar transporter, was found up-regulated. The inhibition of adenosine 5＇-triphosphate （ATP） and RNA/protein synthesis was also observed.