Rapid purification method for the 26S proteasome from the filamentous fungus Trichoderma reesei

We have developed a fast and simple two column chromatographic method for the purification of the 26S proteasome from the filamentous fungus Trichoderma reesei that simplifies the overall procedure and reduces the purification time from 5 to 2.5 days. The combination of only the anionic exchange POROS^® HQ column (Applied Biosystems) together with a size exclusion column has not been used previously for proteasome purification. The purified complex was analysed further by two-dimensional electrophoresis (2DE) and examined by transmission electron microscopy (TEM). A total of 102 spots separated by 2DE were identified by mass spectrometry using cross-species identification (CSI) or an in-house custom-made protein database derived from the T. reesei sequencing project. Fifty-one spots out of 102 represented unique proteins. Among them, 30 were from the 20S particle and eight were from the 19S particle. In addition, seven proteasome-interacting proteins as well as several non-proteasome related proteins were identified. Co-purification of the 19S regulatory particle was confirmed by TEM and Western blotting. The rapidity of the purification procedure and largely intact nature of the complex suggest that similar procedure may be applicable to the isolation and purification of the other protein complexes.     

Salt-stress induced changes in the leaf proteome of diploid and tetraploid mandarins with contrasting Na and Cl accumulation behaviour

To understand the genotypic variation of citrus to mild salt stress, a proteomic approach has been carried out in parallel on two citrus genotypes (‘Cleopatra’ and ‘Willow leaf’ mandarins), which differ for Na⁺ and Cl⁻ accumulation, and their cognate autotetraploids (4×). Using two-dimensional electrophoresis approximately 910 protein spots were reproducibly detected in control and salt-stressed leaves of all genotypes. Among them, 44 protein spots showing significant variations at least in one genotype were subjected to mass spectrometry analysis for identification. Salt-responsive proteins were involved in several functions, including photosynthetic processes, ROS scavenging, stress defence, and signalling. Genotype factors affect the salt-responsive pattern, especially that of carbon metabolism. The no ion accumulator ‘Cleopatra’ mandarin genotype showed the highest number of salt-responsive proteins, and up-regulation of Calvin cycle-related proteins. Conversely the ion accumulator ‘Willow leaf’ mandarin showed high levels of several photorespiration-related enzymes. A common set of proteins (twelve spots) displayed higher levels in salt-stressed leaves of 2× and 4× ‘Cleopatra’ and 4× ‘Willow leaf’ mandarin. Interestingly, antioxidant enzymes and heat shock proteins showed higher constitutive levels in 4× ‘Cleopatra’ mandarin and 4× ‘Willow leaf’ mandarin compared with the cognate 2× genotype. This work provides for the first time information on the effect of 8 weeks of salt stress on citrus genotypes contrasting for ion accumulation and their cognate autotetraploids. Results underline that genetic factors have a predominant effect on the salt response, although a common stress response independent from genotype was also found.     

Proteome analysis of <Emphasis Type="Italic">Aspergillus ochraceus</Emphasis>

Genome sequencing for many important fungi has begun during recent years; however, there is still some deficiency in proteome profiling of aspergilli. To obtain a comprehensive overview of proteins and their expression, a proteomic approach based on 2D gel electrophoresis and MALDI-TOF/TOF mass spectrometry was used to investigate A. ochraceus. The cell walls of fungi are exceptionally resistant to destruction, therefore two lysis protocols were tested: (1) lysis via manual grinding using liquid nitrogen, and (2) mechanical lysis via rapid agitation with glass beads using MagNalyser. Mechanical grinding with mortar and pestle using liquid nitrogen was found to be a more efficient extraction method for our purpose, resulting in extracts with higher protein content and a clear band pattern in SDS-PAGE. Two-dimensional electrophoresis gave a complex spot pattern comprising proteins of a broad range of isoelectric points and molecular masses. The most abundant spots were subjected to mass spectrometric analysis. We could identify 31 spots representing 26 proteins, most of them involved in metabolic processes and response to stress. Seventeen spots were identified by de novo sequencing due to a lack of DNA and protein database sequences of A. ochraceus. The proteins identified in our study have been reported for the first time in A. ochraceus and this represents the first proteomic approach with identification of major proteins, when the fungus was grown under submerged culture.     

Differential proteomics of the plasma of individuals with sepsis caused by Acinetobacter baumannii

This study examines alterations in the plasma proteome in ten adults affected by sepsis caused by Acinetobacter baumannii as compared to paired healthy controls. 2-DE profiles of plasma from patients and paired healthy donors, depleted of the six most abundant proteins, were analysed by the DIGE technique. Protein spot detection and quantification were performed with the Differential In-gel Analysis and Biological Variation Analysis modules of the DeCyder^™ software. Differentially expressed proteins were identified by mass spectrometry (MALDI-TOF/TOF) after colloidal Coomassie blue staining.Almost 900 spots were detected on a unique 2-D gel by the DIGE technique. A total of 269 protein spots of differential abundance were shown to be statistically significant (2.5-fold) with p values of p ≤ 0.01 (135 spots) and p ≤ 0.05 (134 spots) as determined by the t test. Seventy-one spots were submitted to mass spectrometry and about 30% could be successfully identified.This multiplex approach significantly reduced experimental variability, allowing for the confident detection of small differences in protein levels. Results include differentially expressed lipoproteins as well as proteins belonging to inflammatory/coagulation pathways and the kallikrein–kinin system. These data improves the knowledge for future developments in sepsis diagnosis, staging and therapy.     

Detection of glycation sites in proteins by high-resolution mass spectrometry combined with isotopic labeling

The products of nonenzymatic glycation of proteins are formed in a chemical reaction between reducing sugars and the free amino group located either at the N terminus of the polypeptide chain or in the lysine side chain. Glycated proteins and their fragments could be used as markers of the aging process as well as diabetes mellitus and Alzheimer’s disease, making them an object of interest in clinical chemistry. In this article, we propose a new method for the identification of peptide-derived Amadori products in the mixtures obtained by enzymatic hydrolysis of glycated proteins. Two proteins, ubiquitin and human serum albumin (HSA), were modified with an equimolar mixture of glucose and [¹³C₆]glucose and were subjected to enzymatic hydrolysis. The obtained enzymatic digests were analyzed by high-resolution mass spectrometry (HRMS), and the peptide-derived Amadori products were identified on the basis of specific isotopic patterns resulting from ¹³C substitution. The number of glycated peptides in the digest of HSA detected by our procedure was in agreement with the data recently reported in the literature.     

Physiology and proteome responses of two contrasting rice mutants and their wild type parent under salt stress conditions at the vegetative stage

Salinity is one of the major environmental limiting factors that affects growth and productivity of rice (Oryza sativa L.) worldwide. Rice is among the most sensitive crops to salinity, especially at early vegetative stages. In order to get a better understanding of molecular pathways affected in rice mutants showing contrasting responses to salinity, we exploited the power of 2-DE based proteomics to explore the proteome changes associated with salt stress response. Our physiological observations showed that standard evaluation system (SES) scores, Na⁺ and K⁺ concentrations in shoots and Na⁺/K⁺ ratio were significantly different in contrasting mutants under salt stress condition. Proteomics analysis showed that, out of 854 protein spots which were reproducibly detected, 67 protein spots showed significant responses to salt stress. The tandem mass spectrometry analysis of these significantly differentially accumulated proteins resulted in identification of 34 unique proteins. These proteins are involved in various molecular processes including defense to oxidative stresses, metabolisms, photosynthesis, protein synthesis and processing, signal transduction. Several of the identified proteins were emerged as key participants in salt stress tolerance. The possible implication of salt responsive proteins in plant adaptation to salt stress is discussed.     

Proteomic analysis of somatic embryogenesis in <Emphasis Type="Italic">Vitis vinifera</Emphasis>

Two dimensional gel electrophoresis coupled to mass spectrometry has been used to study the somatic embryogenesis in Vitis vinifera, by comparing embryogenic and non embryogenic calluses of the Thompson seedless cv. More than 1,000 spots were reproducibly resolved in colloidal Coomassie brilliant blue stained gels over a pI nonlinear range of 3–10 in the first dimension and using homogeneous 12.5% polyacrylamide gels in the second dimension. The expression pattern of 35 spots differed significantly between the two samples. These spots were processed by mass spectrometry analysis and the protein identity was assigned by using both the non-redundant protein and EST databases. Several responsive proteins, some already known to be involved in the somatic embryogenesis process while others, for the first time put into relation with this process, have been described. Moreover, they have been subdivided in functional categories, and their putative role is discussed in terms of their relevance in the somatic embryogenesis process.     

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.     

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.     

Amino acid sequences of three acyl-binding/lipid-transfer proteins from rape seedlings

The complete amino acid sequence of three acyl-binding/lipid-transfer proteins, AB/LTP I, AB/LTP II and AB/LTP III from germinated rape seeds were determined. AB/LTP I and AB/LTP II consist of 93 residues and the M_r was determined as 9408 by mass spectrometry and calculated as 9406.8 from the sequence. AB/LTP III consists of 92 residues and the M_r was determined as 9424 by mass spectrometry and calculated as 9422.8 from the sequence. The primary structures were determined by automated Edman degradations of the intact proteins and peptides obtained from digestion with trypsin and endoproteinase Asp-N and cyanogen bromide cleavage. Use of ²⁵²Cf plasma-desorption mass spectrometry facilitated the identification and verification of peptides.     

蛋白质组学技术筛选和鉴定白化黑线仓鼠皮肤白化相关差异蛋白质

目的比较黑线仓鼠及其白化突变系背部皮肤蛋白表达的差异,寻找差异蛋白质,从蛋白质水平探讨白化病的发生机制。方法应用双向凝胶电泳技术分离出差异蛋白质,用质谱法分析其结构与组成,通过蛋白质数据库确定差异蛋白的功能。结果从64个表达差异蛋白斑点中发现33个显著差异的蛋白点,其中又有14个差异点匹配到了有意义的蛋白质。14个差异点共鉴定出11个差异蛋白质,这些差异蛋白质按功能可分为4类:(1)糖代谢相关蛋白;(2)运输蛋白;(3)细胞骨架蛋白;(4)其他蛋白。结论黑线仓鼠与其白化突变系背部皮肤蛋白表达存在明显差异,其中一些蛋白与白化病发生相关,并可能成为白化病致病机理研究的分子标志物和药物治疗靶向位点。     

Proteome analysis of wheat leaf under salt stress by two-dimensional difference gel electrophoresis (2D-DIGE)

Salt stress is a major abiotic stress that limits agricultural productivity in many regions of the world. To understand the molecular basis of the salt stress response in wheat (Triticum aestivum L.), a proteomic approach was used to identify the salt stress-responsive proteins in an elite Chinese wheat cultivar, Zhengmai 9023, which exhibits a high yield, superior gluten quality and better biotic resistance. Three-week-old seedlings were treated with NaCl of four different concentrations (1.0%, 1.5%, 2.0%, and 2.5%). The total proteins from the leaves of untreated and NaCl-treated plants were extracted and separated by two-dimensional difference gel electrophoresis (2D-DIGE). A total of 2358 protein spots were detected on the gels, among which 125 spots showed a significant change in protein abundance, and 83 differentially expressed spots were localised on preparative gels. Using Q-TOF mass spectrometry, 52 salt-responsive spots were identified, which were classified into six functional categories that included transport-associated proteins, detoxifying enzymes, ATP synthase, carbon metabolism, protein folding, and proteins with unknown biological functions. Of the 52 differentially expressed proteins, 26 were up-regulated, 21 were down-regulated, and five spots showed multi-expression patterns. In particular, some important proteins for salt tolerance were found to be up-regulated in Zhengmai 9023 under salt stress, such as H⁺-ATPases, glutathione S-transferase, ferritin and triosephosphate isomerase.     

Protein profile analysis of salt-responsive proteins in leaves and roots in two cultivars of creeping bentgrass differing in salinity tolerance

Knowledge of stress-responsive proteins is critical for further understanding the molecular mechanisms of stress tolerance. The objectives of this study were to establish a proteomic map for a perennial grass species, creeping bentgrass (A. stolonifera L.), and to identify differentially expressed, salt-responsive proteins in two cultivars differing in salinity tolerance. Plants of two cultivars (‘Penncross’ and ‘Penn-A4’) were irrigated daily with water (control) or NaCl solution to induce salinity stress in a growth chamber. Salinity stress was obtained by adding NaCl solution of 2, 4, 6, and 8 dS m⁻¹ in the soil daily for 2-day intervals at each concentration, and then by watering soil with 10 dS m⁻¹ solution daily for 28 days. For proteomic map, using two-dimensional electrophoresis (2-DE), approximately 420 and 300 protein spots were detected in leaves and roots, respectively. A total of 148 leaf protein spots and 40 root protein spots were excised from the 2-DE gels and subjected to mass spectrometry analysis. In total, 106 leaf protein spots and 24 root protein spots were successfully identified. Leaves had more salt-responsive proteins than roots in both cultivars. The superior salt tolerance in ‘Penn-A4’, indicated by shoot extension rate, relative water content, and cell membrane stability during the 28-day salinity stress could be mainly associated with its higher level of vacuolar H⁺-ATPase in roots and UDP-sulfoquinovose synthase, methionine synthase, and glucan exohydrolase in leaves, as well as increased accumulation of catalase and glutathione S-transferase in leaves. Our results suggest that salinity tolerance in creeping bentgrass could be in part controlled by an alteration of ion transport through vacuolar H⁺-ATPase in roots, maintenance of the functionality and integrity of thylakoid membranes, sustained polyamine biosynthesis, and by the activation of cell wall loosening proteins and antioxidant defense mechanisms.     

Novel Techniques for Identification and Characterization of Proteins Loaded on Gels in Femtomole Amounts

A combination of techniques is presented allowing gel-purified protein identification in the femtomole range using matrix-assisted-laser-desorption-ionization mass spectrometry. The proteins are detected in the primary gel by a sensitive negative staining procedure, transferred, and concentrated in a secondary gel matrix. There, they are digested in the presence of H₂ ¹⁸O and their sequences are predicted (1) by peptide mass fingerprinting, (2) by comparing the post-source-decay (PSD) spectra with theoretical spectra of candidate isobaric peptides using a computer algorithm called MassFrag, and (3) by a manual readout of the ¹⁸O/¹⁶O-labeled fragmentation ions in the PSD spectra.     

Study of proteins associated with the Eimeria tenella refractile body by a proteomic approach

Refractile bodies (RB), whose function is still unknown, are specific structures of Eimeriidae parasites. In order to study their proteome, RB were purified from Eimeria tenella sporozoites by a new procedure using a reversible fixation followed by centrifugation. RB proteins were resolved by two-dimensional electrophoresis. Around 76 and 89 spots were detected on RB two-dimensional gels using gradients in the 3-10 and 4-7 range, respectively. RB proteins were located mainly between pH 5 and 7. RB gels were then compared with previously established maps of the entire sporozoite proteome. Proteins appearing in new spots were identified by mass spectrometry. Thirty protein isoforms were located in RB. Added to the already known RB proteins such as Eimepsin and SO7', the new RB proteins were defined as haloacid dehalogenase, hydrolase, subtilase, lactacte dehydrogenase or ubiquitin family proteins. The RB proteome analysis confirmed the hypothesis that this structure is a reservoir for proteins necessary to invasion but also suggests that RB have energetic and metabolic functions.     

Abscisic Acid Refines the Synthesis of Chloroplast Proteins in Maize (Zea mays) in Response to Drought and Light

To better understand abscisic acid (ABA) regulation of the synthesis of chloroplast proteins in maize (Zea mays L.) in response to drought and light, we compared leaf proteome differences between maize ABA-deficient mutant vp5 and corresponding wild-type Vp5 green and etiolated seedlings exposed to drought stress. Proteins extracted from the leaves of Vp5 and vp5 seedlings were used for two-dimensional electrophoresis (2-DE) and subsequent matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). After Coomassie brilliant blue staining, approximately 450 protein spots were reproducibly detected on 2-DE gels. A total of 36 differentially expressed protein spots in response to drought and light were identified using MALDI-TOF MS and their subcellular localization was determined based on the annotation of reviewed accession in UniProt Knowledgebase and the software prediction. As a result, corresponding 13 proteins of the 24 differentially expressed protein spots were definitely localized in chloroplasts and their expression was in an ABA-dependent way, including 6 up-regulated by both drought and light, 5 up-regulated by drought but down-regulated by light, 5 up-regulated by light but down-regulated by drought; 5 proteins down-regulated by drought were mainly those involved in photosynthesis and ATP synthesis. Thus, the results in the present study supported the vital role of ABA in regulating the synthesis of drought- and/or light-induced proteins in maize chloroplasts and would facilitate the functional characterization of ABA-induced chloroplast proteins in C₄ plants.     

Proteomic Analysis of Methylarginine-Containing Proteins in HeLa Cells by Two-Dimensional Gel Electrophoresis and Immunoblotting with a Methylarginine-Specific Antibody

Protein arginine methylation is found in many nucleic acid binding proteins affecting numerous cellular functions. In this study we identified methylarginine-containing proteins in HeLa cell extracts by two-dimensional electrophoresis and immunoblotting with a methylarginine-specific antibody. Protein spots with matched protein stain and blotting signals were analyzed by mass spectrometry. The identities of 12 protein spots as 11 different proteins were suggested. Known methylarginine-containing proteins such as hnRNP A2/B1, hnRNP A1, hnRNP G and FUS were identified, indicating the feasibility of our approach. However, four highly abundant metabolic enzymes that might co-electrophorese with methylarginine-containing proteins were also identified. Other nucleic acid binding proteins hnRNP M, hnRNP I and NonO protein were identified. Recombinant hnRNP M and a peptide with the RGG sequence in hnRNP M could be further methylated in vitro. The immunoblotting results of immunoprecipitated hnRNP I and NonO protein are consistent with arginine methylation in both proteins. In this study we identified methylarginine-containing proteins in HeLa cells through proteomic approaches and the method is fast and robust for further applications.     

Modified two-dimensional electrophoresis for identification and autoradiography of ribosomal proteins reacted withp-chloromercuribenzoate

Ribosomal proteins labelled with [¹⁴C]PCMB, a reversibly reacting sulfhydryl reagent, lose most of their radioactivity when separated by the conventional two-dimensional polyacrylamide gel electrophoresis. Therefore, the two-dimensional method was modified to insure stability of the PCMB-protein complex during the procedure. Proteins ofEscherichia coli ribosomes were separated by the modified method, and those which reacted with [¹⁴C]PCMB were identified by counting of stained spots and by autoradiography of dried gels.