Proteomic analysis of injured spinal cord tissue proteins using 2-DE and MALDI-TOF MS

Spinal cord injury (SCI) induces a progressive pathophysiology affecting cell survival and neurological integrity via complex and evolving molecular cascades whose interrelationships are not fully understood. Acute injury to the spinal cord undergoes sequential pathological change including hemorrhage, edema, axonal and neuronal necrosis, and demyelination. In the present study, we aimed to establish the proteomic profiles and characterization of the total protein expressed in traumatic injured spinal cord tissue by using 2-DE and matrix assisted laser desorption/ionization-TOF MS (MALDI-TOF MS). We performed proteomic analysis using 2-DE and MS to describe total proteins and differential proteins expression between normal and traumatic injured spinal cord tissues. The study discovered 947 total proteins and analyzed 219 and 270 proteins from normal and injured tissue, respectively. After 24 h of traumatic damage induction, the injured spinal cord tissue up-regulated over 39 proteins including neurofilament light chain, annexin 5, heat shock protein, tubulin beta, peripherin, glial fibrillary acidic protein delta, peroxiredoxin 2, and apolipoprotein A. Twenty-one proteins showed reduction. The majority of the modulated proteins belonged to the 13 functional categories. Proteins that were identified with neural functional category in injured tissue were considered most likely to be involved in wound healing response coupled with neurogenesis and gliogenesis.     

Fractionation and identification of proteins by 2-DE and MS: towards a proteomic analysis of Plasmodium falciparum

Since completion of genome sequencing of the malarial parasite Plasmodium falciparum, proteomic tools for the identification of parasite proteins have become particularly attractive as they allow a more thorough interpretation of these data. Recent advances in 2-D PAGE, MS, and bioinformatics have created great opportunities for mapping and characterization of protein populations. We employed these improvements in a proteomic approach for the analysis of proteins detected in two blood stages of P. falciparum, (i) in the schizont stage and (ii) in the merozoite stage. For the isolation of merozoites, we introduced a new protocol based on the preparation of clustered structures of merozoites upon treatment of cultures with the common cysteine proteinase inhibitor E64. Peptide mass fingerprints of excised and trypsinated protein spots, acquired by MALDI-TOF MS were generated to identify a variety of proteins. Moreover, prefractionation procedures were used to enrich and map low-abundance proteins in protein samples. The data demonstrate that classic proteomic analyses using 2-D PAGE are now feasible for P. falciparum and represent the first step in the direction of creating 2-D reference maps for this medically most relevant protozoon.     

Comparative proteomic analysis of Cd-responsive proteins in wheat roots

Cadmium (Cd) is a major environmental toxicant to plant cells due to its potential inhibitory effects on many physiological processes. To gain a comprehensive understanding of plant response to Cd, wheat seedlings were exposed to a range of Cd concentrations (10, 100 and 200 μM) for 1 week and a combination of physiological and proteomic approaches were used to evidence Cd effects and to access the plant response to Cd toxicity. Root and shoot elongation was decreased, whereas the H₂O₂ and malondialdehyde content in wheat seedlings was increased significantly at higher Cd concentration. Protein profiles analyzed by two-dimensional electrophoresis revealed that 46 protein spots showed 1.5-fold change in protein abundance following Cd exposure; 31 protein spots were up-regulated and 15 protein spots were down-regulated; 25 of them were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. As expected, most of the up-regulated proteins are involved in heavy metal detoxification and antioxidant processes. Enzyme activity analysis revealed that ascorbate peroxidase and glutathione S-transferase activity was stimulated by Cd treatment. Abundance changes of these proteins, together with their putative functions provide us a new insight that can lead to an integrated understanding of the molecular basis of Cd responses in plants.     

Comparative proteomic analysis of canola leaves under salinity stress

Although canola is a moderately salt‐tolerant species, its growth, seed yield, and oil production are markedly reduced under salt stress, particularly during the early vegetative growth stage. To identify the mechanisms of salt responsiveness in canola, the proteins expressed in the second and third newly developed leaves of salt‐tolerant, Hyola 308, and salt‐sensitive, Sarigol, cultivars were analyzed. Plants were exposed to 0, 175, and 350 mM NaCl during the vegetative stage. An increase in the Na content and a reduction in growth were observed in the third leaves compared to the second leaves. The accumulation of Na was more pronounced in the salt‐sensitive compared with the salt‐tolerant genotype. Out of 900 protein spots detected on 2‐DE gels, 44 and 31 proteins were differentially expressed in the tolerant and susceptible genotypes, respectively. Cluster analysis based on the expression level of total and responsive proteins indicated that the second leaves had a discriminator role between the two genotypes at both salinity levels. Using MS analysis, 46 proteins could be identified including proteins involved in responses to oxidative stress, energy production, electron transport, translation, and photosynthesis. Our results suggest that these proteins might play roles in canola adaptation to salt stress.     

Comparative proteomic analysis of salt response proteins in seedling roots of two wheat varieties

A comparative proteomic analysis was made of salt response in seedling roots of wheat cultivars Jing-411 (salt tolerant) and Chinese Spring (salt sensitive) subjected to a range of salt stress concentrations (0.5%, 1.5% and 2.5%) for 2 days. One hundred and ninety eight differentially expressed protein spots (DEPs) were located with at least two-fold differences in abundance on 2-DE maps, of which 144 were identified by MALDI-TOF-TOF MS. These proteins were involved primarily in carbon metabolism (31.9%), detoxification and defense (12.5%), chaperones (5.6%) and signal transduction (4.9%). Comparative analysis showed that 41 DEPs were salt responsive with significant expression changes in both varieties under salt stress, and 99 (52 in Jing-411 and 47 in Chinese Spring) were variety specific. Only 15 and 9 DEPs in Jing-411 and Chinese Spring, respectively, were up-regulated in abundance under all three salt concentrations. All dynamics of the DEPs were analyzed across all treatments. Some salt responsive DEPs, such as guanine nucleotide-binding protein subunit beta-like protein, RuBisCO large subunit-binding protein subunit alpha and pathogenesis related protein 10, were up-regulated significantly in Jing-411 under all salt concentrations, whereas they were down-regulated in salinity-stressed Chinese Spring.     

Comparative proteomic analysis of early salt stress responsive proteins in roots and leaves of rice

Growth and productivity of rice (Oryza sativa L.) are severely affected by salinity. Understanding the mechanisms that protect rice and other important cereal crops from salt stress will help in the development of salt‐stress‐tolerant strains. In this study, rice seedlings of the same genetic species with various salt tolerances were studied. We first used 2DE to resolve the expressed proteome in rice roots and leaves and then used nanospray liquid chromatography/tandem mass spectrometry to identify the differentially expressed proteins in rice seedlings after salt treatment. The 2DE assays revealed that there were 104 differentially expressed protein spots in rice roots and 59 in leaves. Then, we identified 83 proteins in rice roots and 61 proteins in rice leaves by MS analysis. Functional classification analysis revealed that the differentially expressed proteins from roots could be classified into 18 functional categories while those from leaves could be classified into 11 functional categories. The proteins from rice seedlings that most significantly contributed to a protective effect against increased salinity were cysteine synthase, adenosine triphosphate synthase, quercetin 3‐O‐methyltransferase 1, and lipoxygenase 2. Further analysis demonstrated that the primary mechanisms underlying the ability of rice seedlings to tolerate salt stress were glycolysis, purine metabolism, and photosynthesis. Thus, we suggest that differentially expressed proteins may serve as marker group for the salt tolerance of rice.     

A proteomic approach to analyze salt-responsive proteins in rice leaf sheath

To examine the response of rice to salt stress, changes in protein expression were analyzed using a proteomic approach. To investigate dose- and time-dependent responses, rice seedlings were exposed to 50, 100 and 150 mM NaCl for 6 to 48 h. Proteins were extracted from leaf sheath and separated by two-dimensional polyacrylamide gel electrophoresis. Eight proteins showed 1- to 3-fold up-regulation in leaf sheath, in response to 50 mM NaCl for 24 h. Among these, three proteins were unidentified (LSY081, LSY262 and LSY363) while five proteins were identified as fructose bisphosphate aldolases, photosystem II (PSII) oxygen evolving complex protein, oxygen evolving enhancer protein 2 (OEE2) and superoxide dismutase (SOD). The maximum expression levels of seven proteins were at 24 h. Their expression declined after 48 h of 50 mM NaCl treatment. In contrast, SOD maintained its elevated expression throughout these conditions. The increased expression of proteins seen in the 50 mM NaCl treatment group was less pronounced in the groups receiving 100 or 150 mM NaCl for 24 h. The expression of SOD was a common response to cold, drought, salt and abscisic acid (ABA) stresses while the expression of LSY081, LSY363 and OEE2 was enhanced by salt and ABA stresses. LSY262 was expressed in leaf sheath and root, while fructose bisphosphate aldolases, PSII oxygen evolving complex protein and OEE2 were expressed in leaf sheath and leaf blade. LSY363 was expressed in leaf sheath but was below the level of detection in leaf blade and root. These results indicate that specific proteins expressed in specific regions of rice show a coordinated response to salt stress.     

2-DE proteomic analysis of HSP70 in mollusc Chamelea gallina

Bidimensional electrophoresis (2-DE) protocols were adapted on Chamelea gallina digestive glands studies by the analysis of Heat Shock Proteins (HSP) compared with monodimensional electrophoresis (1-DE) results. Because polycyclic aromatic hydrocarbons (PAH) act on HSPs, C. gallina specimens were exposed to 0.5 mg/L of benzo[a]pyrene (B[a]P) for 24 h, 7 and 12 days. Immunoblotting after 1-DE showed a single band of 70 kDa significantly induced after 7 days of B[a]P exposure. After 2-DE, eight major high-resolved spots between 17 and 98 kDa were revealed. Three spots fell within the range of 62–98 kDa and of 5–6 pI, parameters which could include HSP70. Two spots of 77 and 72 kDa, obtained after 2-DE immunoblotting, could correspond to constitutive HSC70 and to inducible HSP70 forms respectively. Changes observed in inducible and in constitutive forms might be related to an adaptation to stress and to a normal protein synthesis capability, respectively. Employment of 2-DE and relationship between HSP70 and HSC70 may be useful to clarify their role in molluscs subjected to stress events.     

Comparative 2-DE proteomic analysis of date palm (Phoenix dactylifera L.) somatic and zygotic embryos

Two-dimensional gel electrophoresis coupled to mass spectrometry has been used to compare the proteome of date palm (Phoenix dactylifera L. cv. Deglet Nour) zygotic and somatic embryos. Proteins were trichloroacetic acid–acetone–phenol extracted, quantified, and resolved by 2-DE in the 5 to 8 pH range. Total protein content and number of resolved spots were higher in zygotic (110 ± 14.5 mg/g DW; 349 spots) than in somatic (70.96 ± 4.8 mg/g DW; 210 spots) embryos. The 2-DE map of both systems showed qualitative (263) and quantitative (72) differences. Statistical analysis of spot intensity was performed by PCA, obtaining two accurate groupings of the samples and determining the most discriminating spots. Samples were also clustered using Euclidean distance with average linkage algorithm of the Genesis software package. Sixty-three variable spots were subjected to mass spectrometry analysis, resulting in 23 identifications. Identified proteins were classified in the following functional categories; glycolysis (8 proteins), citrate cycle (1), ATP synthesis (1), carbohydrate biosynthesis (2), amino acids metabolism (1), stress related (4), storage (3), and with no function assigned for three of them. Most of the somatic embryo specific proteins identified belonged to glycolysis pathways, whereas those of the zygotic embryo to storage and stress-related proteins. Differences are discussed in terms of metabolism and biology of both types of embryos.     

Analysis of protein changes during grape berry ripening by 2-DE and MALDI-TOF

Grape berry, a nonclimacteric fruit, during ripening turns from green, hard and acidic to coloured, soft and sweet. Many studies have focused on dynamic changes of mRNA levels, metabolites, sugars or individual proteins, but this is the first report of a proteomic approach applied to the screening of the most prominent variations that take place during berry ripening. Vitis vinifera cv. 'Nebbiolo Lampia' berries were collected at 10-day intervals, starting 1 month after flowering to complete ripe stage; total protein extracts from deseeded berries were separated by 2-DE. A total of 730 spots were detected in the 2-DE gels. 118 protein spots, differentially expressed during berry development, were subjected to MALDI-TOF analysis. Ninety-three of them were identified, corresponding to 101 proteins. The majority of proteins were linked to metabolism, energy and protein synthesis and fate. In comparison to published surveys of major berry proteins, fewer proteins related to stress response and more proteins related to cell structure were differentially expressed. Our data confirm a general decrease of glycolysis during ripening, and an increase of PR proteins in the range of 20-35 kDa. They furthermore suggest that oxidative stress decreases during ripening while extensive cytoskeleton rearrangement takes place in this period.     

Proteome analysis of sorbitol fermentation specific protein in Vibrio cholerae by 2-DE and MS

Vibrio cholerae can be differentiated into epidemic and non-epidemic strains by sorbitol fermentation speed, but little research has been done on its mechanisms. In this study, we investigated differential protein expression of the two strains in response to sorbitol metabolism. V. cholerae strains were cultured in media with and without sorbitol, respectively. Proteins were separated by 2-DE, and those that showed different expression in the two media were identified by MALDI-TOF MS. Fifteen proteins in epidemic strains and 11 proteins in non-epidemic strains showed a different expression in sorbitol medium. Among them, 4 proteins were common to epidemic and non-epidemic strains. Gene sequence analysis showed that some mutations occurred in these proteins between the two strains. Potential functions of these proteins included sugar uptake, amino acid uptake, electron transport, sulfate and thiosulfate transport.     

Comparative 2-DE proteomic analysis of date palm (Phoenix dactylifera L.) somatic and zygotic embryos

Two-dimensional gel electrophoresis coupled to mass spectrometry has been used to compare the proteome of date palm (Phoenix dactylifera L. cv. Deglet Nour) zygotic and somatic embryos. Proteins were trichloroacetic acid–acetone–phenol extracted, quantified, and resolved by 2-DE in the 5 to 8 pH range. Total protein content and number of resolved spots were higher in zygotic (110 ± 14.5 mg/g DW; 349 spots) than in somatic (70.96 ± 4.8 mg/g DW; 210 spots) embryos. The 2-DE map of both systems showed qualitative (263) and quantitative (72) differences. Statistical analysis of spot intensity was performed by PCA, obtaining two accurate groupings of the samples and determining the most discriminating spots. Samples were also clustered using Euclidean distance with average linkage algorithm of the Genesis software package. Sixty-three variable spots were subjected to mass spectrometry analysis, resulting in 23 identifications. Identified proteins were classified in the following functional categories; glycolysis (8 proteins), citrate cycle (1), ATP synthesis (1), carbohydrate biosynthesis (2), amino acids metabolism (1), stress related (4), storage (3), and with no function assigned for three of them. Most of the somatic embryo specific proteins identified belonged to glycolysis pathways, whereas those of the zygotic embryo to storage and stress-related proteins. Differences are discussed in terms of metabolism and biology of both types of embryos.     

A reference map and identification of porcine testis proteins using 2-DE and MS

The development of the testis is essential for maturation of male mammals. A complete understanding of proteins expressed in the testis will provide biological information on many reproductive dysfunctions in males. The purposes of this study were to apply a proteomic approach to investigating protein composition and to establish a 2-D PAGE reference map for porcine testis proteins. MALDI-TOF MS was performed for protein identification. When 1 mg of total proteins was assayed by 2-D PAGE and stained with colloidal CBB, more than 400 proteins with a pI of pH 3-10 and M(r) of 10-200 kDa could be detected. Protein expression varied among individuals, with CV between 4.7 and 131.5%. A total of 447 protein spots were excised for identification, among which 337 spots were identified by searching the mass spectra against the NCBInr database. Identification of the remaining 110 spots was unsuccessful. A 2-D PAGE-based porcine testis protein database has been constructed on the basis of the results and will be published on the WWW. This database should be valuable for investigating the developmental biology and pathology of porcine testis.     

Comparative Proteomics of Chromium-Transformed Beas-2B Cells by 2D-DIGE and MALDI-TOF/TOF MS

Continuation of prolonged treatment against arsenicosis with conventional chelating therapy is a global challenge. The present study was intended to evaluate the defensive effect of arjunolic acid against arsenic-induced oxidative stress and female reproductive dysfunction. Wistar strain adult female rats were given sodium arsenite (10 mg/kg body weight) in combination with arjunolic acid (10 mg/kg body weight) orally for two estrous cycles. Electrozymographic analysis explored that arjunolic acid co-treatment counteracted As³⁺-induced ROS production in uterine tissue by stimulating the activities of endogenous enzymatic antioxidants. Arjunolic acid was able to enhance the protection against mutagenic uterine DNA breakage, necrosis, and ovarian–uterine tissue damages in arsenicated rats by improving the ovarian steroidogenesis. The mechanisms might be coupled with the augmentation of antioxidant defense system, partly through the elimination of arsenic with the involvement of S-adenosyl methionine pool where circulating levels of vitamin B₁₂, folic acid, and homocysteine play critical roles as evidenced from our present investigation.     

Long term anoxia in rainbow trout investigated by 2-DE and MS/MS

Twenty-four hours of N(2) induced anoxia induced global perturbations on protein expression in rainbow trout hypodermal fibroblasts cell line. Anoxia was obtained by depleting the medium of O(2) by flushing with N(2), and protein changes were studied by 2-DE coupled with MS providing quantitative measurements of a large number of proteins in one single study. The anoxic insult changed the level of 33 protein spots: 22 of these were up-regulated compared to the control situation and 11 were down-regulated. Using MS/MS sequencing 19 of the 33 protein spots that changed were identified, corresponding to a success rate of more than 50%. The identified proteins included two proteins involved in energy metabolism namely phosphoglycerate mutase and isocitrate dehydrogenase. In addition we observed the up-regulation of a cluster of proteins that contribute to cytoskeleton function. These are calpain, EB1, and Rho GDP dissociation inhibitor (GDI). The up-regulation of Rho GDI was shown to develop in a time dependent manner with no significant increase for up to 8 h of anoxia. In conclusion, this study provides a thorough investigation of the effect of anoxia in a cell line from rainbow trout.     

MALDI-TOF MS用于肺炎克雷伯菌同源性分析的初步研究

目的评价基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption/ionization-time of flight mass spectrometry,MALDI-TOF MS)对肺炎克雷伯菌(Klebsiella pneumoniae,KPN)同源性分析的能力。方法对2013年2-4月青岛大学附属医院重症监护病房(ICU)分离的7株KPN和其他科室分离的13株KPN进行溯源性回顾分析,采用方法为脉冲场凝胶电泳(pulsed-field gel electrophoresis,PFGE)、多位点序列分型(multilocus sequence typing,MLST)和MALDI-TOF MS。结果 PFGE和MLST结果一致,MALDI-TOF MS同源性分析中将20株菌株分为2类,Ⅰ类为来自ICU的7株菌,Ⅱ类为其他科室的13株菌,与前2种方法得到的结果基本一致。结论 MALDI-TOF MS技术能够准确鉴定肺炎克雷伯菌且可对其进行同源性分析,较其他同源性分析方法快捷、方便,可满足临床对院感工作的需求。     

MALDI-TOF MS proteomic phenotyping of filamentous and other fungi from clinical origin

Major changes in medical, intensive care and organ transplantation practices are drastically increasing the risk of fungal opportunistic infections. We designed and set-up a MALDI-TOF MS-based assay to identify the most isolated and emerging therapy-refractory/uncommon fungi from cystic fibrosis (CF) and immunocompromised patients. Two-hundred and thirty isolates from 10 different genera (Aspergillus, Emericella, Fusarium, Geosmithia, Neosartorya, Penicillium, Pseudallescheria, Scedosporium, Talaromyces, Fomitopsis), investigated during routine diagnostic efforts, were correlated to 22 laboratory-adapted reference MALDI-TOF MS "proteomic phenotypes". A growth time-course at 30°C on Sabouraud agar medium was performed for the 22 "phenotypes" at 48, 72, 96 and 120h points. The best peptide extraction conditions for full recovery of conidia- or asci-producing multihyphal morph structures and the highest intra- and inter-class profiling correlation were identified for the 120h point spectra dataset, from which an engineered library derived (pre-analytical phase). Fingerprinting classifiers, selected by Wilcoxon/Kruskal-Wallis algorithm, were computed by Genetic Algorithm, Support Vector Machine, Supervised Neuronal Network and Quick Classifier model construction. MS identification (ID) of clinical isolates was referred to genotyping (GT) and, retrospectively, compared to routine morphotyping (MT) IDs (analytical phase). Proteomic phenotyping is revolutionizing diagnostic mycology as fully reflecting species/morph varieties but often overcoming taxonomic hindrance.