Identification of yeast oxidized proteins: chromatographic top-down approach for identification of carbonylated, fragmented and cross-linked proteins in yeast

The effects of oxidative stress on the yeast proteome were studied using hydrogen peroxide as the stress agent. Oxidized proteins were isolated by (1) biotinylation of oxidized proteins with biotin hydrazide, (2) affinity selection using monomeric avidin affinity chromatography, and (3) further fractionated by reversed-phase liquid chromatography (RPLC) on a C(8) column. Oxidized protein fractions from RPLC were then trypsin digested and the peptide cleavage fragments identified by tandem mass spectrometry (MS/MS). Slightly over 400 proteins were identified. Sites of carbonyl formation were found in roughly one fourth of these proteins. Oxidation on other amino acids in carbonylated peptides was seen in 32 cases while carbonylation was absent in 96 of the oxidized proteins observed. Although there are large numbers of potential oxidation sites, oxidation seemed to be restricted to a small area in most of the proteins identified. Sometimes multiple amino acids in the same tryptic peptide were oxidized. A second trend was that more than 8% of the proteins identified appeared in more than one of the RPLC fractions. Based on the position of the peptides identified in the primary structure of protein candidates derived from databases it was concluded that this occurred by fragmentation of a parent protein. It is not clear from the data whether the fragmentation process was of enzymatic or oxidative origin. Finally, peptides from two or more proteins occurred together in more than one reversed phase fraction with 2% of the proteins identified. This data was interpreted to mean that this was the result of protein cross-linking.     

Identification and quantification of protein carbonylation using light and heavy isotope labeled Girard's P reagent

Protein carbonyls are one of the most widely studied markers of oxidative stress. Determining increases in the concentration of protein carbonyls known to be associated with neurodegenerative diseases, heart disease, cancer and ageing. Identification of carbonylation sites in oxidized proteins has been a challenge. Even though recent advances in proteomics has facilitate the identification of carbonylation sites in oxidized proteins, confident identification remains a challenge due to the complicated nature of oxidative damage and the wide range of oxidative modifications. Here, we report the development of a multiplexing strategy that facilitates confident carbonylated peptide identification through a combination of heavy and light isotope coding and a multi-step filtering process. This procedure involves (1) labeling aliquots of oxidized proteins with heavy and light forms of Girard's reagent P (GPR) and combining them in a 1:1 ratio along with (2) LC/MS and MALDI-MS/MS analysis. The filtering process uses LC/MS and MALDI-MS/MS data to rule out false positives by rejecting peptide doublets that do not appear with the correct concentration ratio, retention time, tag number, or resolution. This strategy was used for the identification of heavily oxidized transferrin peptides and resulted in identification 13 distinct peptides. The competency of the method was validated in a complex mixture using oxidized transferrin in a yeast lysate as well as oxidized yeast. Twenty-five percent of the peptides identified in a pure oxidized sample of transferrin were successfully identified from the complex mixture. Analysis of yeast proteome stressed with hydrogen peroxide using this multiplexing strategy resulted in identification of 41 carbonylated peptides from 36 distinct proteins. Differential isotope coding of model peptides at different concentrations followed by mixing at different ratios was used to establish the linear dynamic range for quantification of carbonylated peptides using light and heavy forms of GPR.     

Development of a database of amino acid sequences for human colon carcinoma proteins separated by two-dimensional polyacrylamide gel electrophoresis

The tandem use of preparative two-dimensional polyacrylamide gel electrophoresis (2-DE) and electroblotting onto polyvinylidene difluoride membranes has been employed to rapidly isolate a number of proteins from a crude cell extract of a human colon carcinoma cell line (LIM 1863). The immobilized proteins were located by staining with Coomassie Brilliant Blue R-250, and selected protein spots were excised and subjected to Edman degradation. Our results demonstrate that overall sequence yields in the 3-20 pmol range can be achieved on protein spots from four identical 2-DE gels; approximately 150-200 micrograms of total protein was applied to a single 2-DE gel. An approximate two-fold increase in sensitivity of phenylthiohydantoin-amino acid detection (subpicomole range) was achieved by fitting our commercial sequencers with a simple sample transfer device which permitted the analysis of the total phenylthiohydantoin-amino acid derivative. N-Terminal amino acid sequence data was obtained for thirteen electroblotted proteins. All of these sequences positively matched those of proteins of known structure listed in the available protein sequence databases. Approximately 40% of the electroblotted proteins did not yield N-terminal sequence information, presumably because they had blocked N-termini (either naturally or artifactually). Internal amino acid sequence information was obtained from three proteins isolated by preparative 2-DE. This was achieved by in situ digestion of the proteins in the gel matrix with Staphylococcus aureus V8 protease, electrophoresis of the generated peptides in a one-dimensional gel, electrotransfer of the peptides to a polyvinylidene difluoride membrane and microsequence analysis of the electroblotted peptides.     

Reference map of soluble proteins from Streptococcus thermophilus by two-dimensional electrophoresis

Streptococcus thermophilus is a lactic acid bacterium widely used for the production of fermented dairy products. The two-dimensional electrophoresis (2-DE) protein profile was obtained from three independent analyses of 2-DE gels of soluble proteins of the strain PB18. About 270 spots were detected by silver staining and the average molecular weight and isoelectric point of each protein spot were calculated to be 41 600 and 5.2, respectively. Twelve proteins were purified by chromatographic techniques because their concentration was too low for direct sequencing from blots. Eleven were located in the PB18 2-DE profile after silver staining. These preliminary results contribute to the setting up of a two-dimensional image (or reference map) of the proteins from S. thermophilus in order to identify and compare strains of various origin or to follow metabolic process such as stress. Bidimensional autoradiographs of two strains (PB18 and ST105) of S. thermophilus grown in exponential phase at 42 degrees C with [35S]methionine were compared with an image analysis system. Among the eleven located proteins in the 2-DE silver-stained profile, nine were found in PB18 and eight in ST105 autoradiographs. One protein was specific to PB18. The eight proteins could play the role of internal 2-D PAGE markers of p/ and Mr for S. thermophilus.     

适于双向电泳分析的苹果叶片蛋白质提取方法

为了探索适用于双向电泳(2-DE)分析的苹果叶片蛋白质提取方法,比较了三氯乙酸(TCA)/丙酮沉淀法、二硫苏糖醇(DTT)/丙酮法、Tris-HCl提取法和改良的Tris-HCl提取法等4种蛋白质提取方法。以7 cm、pH 3～10的线性固相pH梯度(immobilized pH gradient,IPG)胶条作为第一向电泳,以十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)(12.5%的分离胶)作为第二向电泳,对提取物进行2-DE分离,采用银染显色。结果表明,上述4种方法在2-DE图谱上分别得到140,215,181和616个蛋白质点。其中以改良的Tris-HCl提取法得到的蛋白质点数最多,且背景清晰、图谱上没有明显的横纵条纹。为了进一步验证改良的Tris-HCl提取法的有效性,用18 cm、pH 3～10的线性IPG胶条和12.5%的分离胶对提取的苹果叶片蛋白质进行2-DE分离,考马斯亮蓝R-250染色,共检测到455个蛋白质点,其相对分子质量主要分布在14000～66000范围内,图谱背景清晰,再次证明应用该方法制备的样品适用于双向电泳分析,可用于苹果叶片的蛋白质组学分析。     

Protein pre-fractionation in detergent-polymer aqueous two-phase systems for facilitated proteomic studies of membrane proteins

Pre-fractionation of a complex mixture of proteins increases the resolution in analytical separations of proteins from cells, tissues or organisms. Here we demonstrate a novel method for pre-fractionation of membrane proteins by a detergent-based aqueous two-phase system. Membrane proteins are strongly under-represented in proteomic studies based on two-dimensional electrophoresis (2-DE). As a model system, we have isolated mitochondria from the yeast Saccharomyces cerevisiae. Mitochondrial proteins were fractionated in an aqueous two-phase system consisting of the polymer poly(ethylene glycol) and either of two commonly used non-ionic detergents, Triton X-114 or dodecyl maltoside (DDM). Soluble proteins partitioned mainly to the polymer phase while membrane proteins were enriched in the detergent phase, as identified from one-dimensional electrophoresis (1-DE) and/or 2-DE followed by mass spectrometric analysis. Pre-fractionation was further enhanced by addition of an anionic detergent, sodium dodecyl sulfate, or a chaotropic salt, NaClO4, and by raising the pH in the system. The two-phase system pre-fractionation was furthermore combined with an alternative two-dimensional high-resolution separation method, namely ion-exchange chromatography and 1-DE. By this approach a larger number of membrane proteins could be identified compared to separation with conventional 2-DE. Thus, pre-fractionation of complex protein mixtures using the aqueous two-phase systems developed here will help to disclose larger proportions of membrane proteins in different proteomes.     

Proteome analysis of mouse brain: two-dimensional electrophoresis profiles of tissue proteins during the course of aging

Mouse brain proteins were isolated from five regions (cerebellum, cerebral cortex, hippocampus, striatum, and cervical spinal cord) at five ages from the 10th week to the 24th month, and separated by two-dimensional gel electrophoresis (2-DE). 2-DE was carried out with an immobilized pH gradient bar in the first dimension, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. Over one thousand protein spots were visualized by silver staining and quantified by image processing. In the analyses, 58 protein spots were distinguishable among the above five brain regions, and 17 proteins were shown to be varied in quantity in the course of aging. Partial amino-terminal sequences and/or internal sequences for a total of 301 protein spots were analyzed. One hundred and eighty proteins appeared to have blocked N-termini and 122 proteins were identified. Twenty-seven new proteins were identified by sequence homology search. A mouse brain proteome database was constructed, which consists of the 2-DE map images and the respective spot data files with 15 related references.     

Quantification of carbonylated proteins in rat skeletal muscle mitochondria using capillary sieving electrophoresis with laser-induced fluorescence detection

Carbonyl-modified proteins are markers of oxidative damage. Here, we report a new method for detecting and quantifying carbonylated proteins by capillary sieving electrophoresis (CSE) with LIF detection (CSE-LIF). Alexa 488 hydrazide is used for the specific labeling of carbonyls while 3-(2-furoyl) quinoline-2-carboxaldehyde (FQ) is used for protein labeling. BSA subjected to metal-catalyzed oxidation is used to optimize the labeling reactions, confirm the separation power of CSE, and characterize the response of the LIF detector. The method is capable of detecting femtomole (fmol) amounts of carbonyls in proteins with molecular masses ranging from 26 to 30 kDa. Using this method, we determined that mitochondrial proteins isolated from skeletal muscle contains 2.1 +/- 0.1 (average +/- SD; n = 3) nmol carbonyl/mg protein. The methodology described here should be compatible with the analysis of single cells and needle biopsies taken from oxidative stress animal models.     

Proteomic analysis reveals metabolic changes during yeast to hypha transition in Yarrowia lipolytica

Fungal dimorphism is important for survival in different environments and has been related to virulence. The ascomycete Yarrowia lipolytica can grow as yeast, pseudomycelial or mycelial forms. We have used a Y. lipolytica parental strain and a Deltahoy1 mutant, which is unable to form hypha, to set up a model for dimorphism and to characterize in more depth the yeast to hypha transition by proteomic techniques. A two-dimensional gel electrophoresis (2-DE) based differential expression analysis of Y. lipolytica yeast and hyphal cells was performed, and 45 differentially expressed proteins were detected; nine with decreased expression in hyphal cells were identified. They corresponded to the S. cerevisiae homologues of Imd4p, Pdx3p, Cdc19, Sse1p, Sol3p, Sod2p, Xpt1p, Mdh1p and to the unknown protein YALIOB00924g. Remarkably, most of these proteins are involved in metabolic pathways, with four showing oxidoreductase activity. Furthermore, taking into account that this is the first report of 2-DE analysis of Y. lipolytica protein extracts, 35 more proteins from the 2D map of soluble yeast proteins, which were involved in metabolism, cell rescue, energy and protein synthesis, were identified.     

Characterization of Oxidative Stress in Chicken Heart Tissue by High-Performance Liquid Chromatography with Ultraviolet‐Visible (HPLC–UV–Vis) Detection

Oxidative stress can lead to tissue damage in the body, resulting in conditions that may lead to heart failure. Carbonyl proteins and malondialdehyde are common markers for determining oxidative stress. In this study, a high-performance liquid chromatography with ultraviolet‐visible (HPLC–UV–Vis) method was developed for both carbonyl proteins and malondialdehyde and compared with common spectrophotometric methods. The HPLC–UV–Vis method for determining carbonyl proteins showed a linear correlation in a range of 0.05–1.6 µg^.mL^?1 (correlation coefficient: 0.9939). The limits of detection and quantitation were determined to be 1.7 and 5.1?pmol on-column, respectively. The HPLC–UV–Vis method for malondialdehyde exhibited a linear correlation in the range of 0.04–13.6 µg^.mL^?1 (correlation coefficient: 0.9982) with limits of detection and quantitation of 1.5 and 4.5?pmol on-column, respectively. As a proof of concept, the applicability of the methods for both marker substances was investigated in chicken heart tissue as a complex matrix sample. To simulate oxidative stress, the tissue was treated with hydrogen peroxide as the oxidizing agent. Samples prepared with hydrogen peroxide showed increased carbonyl protein and malondialdehyde levels when compared to non-oxidized samples and samples treated additionally with the antioxidant Trolox. Regarding oxidative stress, similar results were achieved when compared with common spectrophotometric methods. However, HPLC–UV–Vis presented higher reproducibility and accuracy. The developed methods for both markers enable rapid and simple investigation of possible antioxidant effects on tissue samples.     

Proteome analysis of differential protein expression in infarcted rat heart after verapamil treatment

To explore the protein-level mechanism of action verapamil in acute myocardial infarcted rats, the myocardial proteome was analyzed by two-dimensional electrophoresis (2-DE). Compared with the sham-operated group and the infarcted group, the result shows that 8 protein expressions in the verapamil treated group were up-regulated, and 7 protein expressions in this group were down-regulated significantly. Using MALDI-TOF-MS, 15 proteins with significant changes were identified through a database search. These proteins can be divided into 4 groups by their biological function: (1) Energy metabolism and mitochondrial function related proteins; (2) oxidative stress-induced proteins; (3) cytoskeletal Proteins; (4) other proteins. The findings show that the myocardial protective effects of verapamil in the myocardial damage process are related to the recovery of energy supply as well as anti-oxidative stress property. __________ Translated from Chemical Journal of Chinese Universities, 2008, 29 (3) (in Chinese)     

Protein changes in post mortem sea bass (Dicentrarchus labrax) muscle monitored by one- and two-dimensional gel electrophoresis

This study was devoted to the identification of specific peptides and proteins which can be used as indicators of freshness in fish. The post mortem evolution of protein patterns in farmed sea bass muscle was monitored by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis (2-DE) after 0, 2, 4, and 6 days cold storage. SDS-electrophoresis, of total proteins and proteins soluble in low-ionic-strength solutions, revealed the gradual disappearance of a protein band of 16 kDa immediately after fish death. 2-DE allowed the classification of fish samples according to post mortem time. Three spots of interest, which disappeared progressively, were identified on the 2-DE patterns. Further research is required to establish the identity of these polypeptides and to evaluate their expression and post mortem evolution in another fish species.     

Acid-labile surfactant improves in-sodium dodecyl sulfate polyacrylamide gel protein digestion for matrix-assisted laser desorption/ionization mass spectrometric peptide mapping

Mass spectrometry (MS) together with genome database searches serves as a powerful tool for the identification of proteins. In proteome analysis, mixtures of cellular proteins are usually separated by sodium dodecyl sulfate (SDS) polyacrylamide gel-based two-dimensional gel electrophoresis (2-DE) or one-dimensional gel electrophoresis (1-DE), and in-gel digested by a specific protease. In-gel protein digestion is one of the critical steps for sensitive protein identification by these procedures. Efficient protein digestion is required for obtaining peptide peaks necessary for protein identification by MS. This paper reports a remarkable improvement of protein digestion in SDS polyacrylamide gels using an acid-labile surfactant, sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate (ALS). Pretreatment of gel pieces containing protein spots separated by 2-DE with a small amount of ALS prior to trypsin digestion led to increases in the digested peptides eluted from the gels. Consistently, treatment of gel pieces containing silver-stained standard proteins and those separated from tissue extracts resulted in the detection of increased numbers of peptide peaks in spectra obtained by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOFMS). Hence the present protocol with ALS provides a useful strategy for sensitive protein identification by MS.     

Profiling of Caenorhabditis elegans proteins using two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-time of flight-mass spectrometry

The nematode Caenorhabditis elegans (C. elegans) is the first animal whose whole 97 Mb genome sequence, encoding ca. 19000 open reading frames (ORF's), has been essentially determined. We tried to establish a 2-DE map of the nematode proteome by means of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). A soluble protein fraction of mixed stages of the worm, wild-type strain N2, was applied to 2-D PAGE. After Coomassie Brilliant Blue (CBB) staining, 1200 spots were detected and 140 major spots were excised from the gel and subjected to in-gel digestion with Achromobacter protease I (lysyl endopeptidase). Resulting peptides were analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) followed by peptide mass fingerprinting for protein identification. With this approach we have obtained a two-dimensional electrophoresis (2-DE) protein map in which 69 spots were localized as landmarks for comparison of expression profiles to elucidate the basis of various biological events.     

Phenotyping apolipoprotein E*3-leiden transgenic mice by two-dimensional polyacrylamide gel electrophoresis and mass spectrometric identification

Apolipoprotein E (ApoE) plays an important role in cholesterol and triglyceride metabolism, being one of the major structural components of chylomicrons and very low density lipoprotein (VLDL) remnants. ApoE functions as a ligand in the receptor-mediated uptake of these remnants from the blood by the liver. A variant form of ApoE, apolipoprotein E*3-Leiden, shows reduced affinity for the low density lipoprotein (LDL) receptor, and results in the dominant expression of type III hyperlipoproteinemia. Two-dimensional electrophoresis (2-DE) has been used to characterise protein expression in serum samples from control and transgenic mice expressing the human ApoE*3-Leiden mutation, fed a cholesterol-rich diet, and transgenic mice fed a normal diet. For the identification of proteins, single silver-stained spots were excised from the 2-DE gels and subjected to in-gel enzymatic digestion. Extracted peptides were analysed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). This proteomic approach has enabled the ApoE*3-Leiden variant to be positioned in a 2-DE separation of serum proteins, and has identified changes in the expression of haptoglobin, indicating that this protein may provide a marker for the potential onset of atherosclerosis.     

A proteome database of human primary T helper cells

We have established the first public database of human primary T helper cell proteome using two-dimensional electrophoresis (2-DE) and matrix assisted laser desorption/ionization-time of flight-mass spectrometry. For the database, CD4+ human T cells were activated with anti-CD3+anti-CD28 antibodies and metabolically labeled with [35S]methionine for 24 h. Cells were lysed and proteins were separated by 2-DE. About 1500 protein spots were detected in the resulting 2-DE gels with silver staining, and 2000 spots with autoradiography. We have identified 91 proteins from the 2-DE gels using peptide mass fingerprinting, and annotated them to our database. The identified proteins are also linked to SWISS-PROTand NCBI protein databases. Our database is available via the Internet at http://www3.btk.utu.fi:8080/Genomics/Proteomics/Database.     

Application of proteomics for determining protein markers for wool quality traits

The technique of two-dimensional electrophoresis (2-DE) has been under investigation for its usefulness in identifying protein markers for wool quality traits in sheep. However, before this could be achieved, unique problems relating to the detection and quantitation of wool proteins needed to be overcome so that 2-DE protein maps could be examined using computational programs like Melanie II. Four protein staining regimes were examined. Colloidal Coomassie Blue G-250 was found to be superior to Coomassie Blue R-250 and gave satisfactory staining of all protein classes. Silver staining detects minor strings of keratinous proteins, but unfortunately it negatively stains intermediate filament proteins, the major high sulphur proteins (HSPs) and the high glycine tyrosine proteins and the latter two classes can only be seen by overstaining the background of the gel. In contrast, labeling reduced keratins with [14C]iodoacetamide, followed by autoradiography detection, results in a protein map with low background and all protein spots stained positively. 2-DE has been used to obtain wool protein maps of Lincoln/Merino chimeric sheep to examine wool originating from two genotypes grown with different crimp frequencies within the same fleece. Between fleece, variations have also been examined. Work to date suggests that several major HSPs may be associated with the fibre curvature trait known as crimp frequency. From matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectral mapping, one of these proteins has been identified as being from the B2A family from the HSP class.     

Evaluation of several two-dimensional gel electrophoresis techniques in cardiac proteomics

Two-dimensional gel electrophoresis (2-DE) is currently the best method for separating complex mixtures of proteins, and its use is gradually becoming more common in cardiac proteome analysis. A number of variations in basic 2-DE have emerged, but their usefulness in analyzing cardiac tissue has not been evaluated. The purpose of the present study was to systematically evaluate the capabilities and limitations of several 2-DE techniques for separating proteins from rat heart tissue. Immobilized pH gradient strips of various pH ranges, parameters of protein loading and staining, subcellular fractionation, and detection of phosphorylated proteins were studied. The results provide guidance for proteome analysis of cardiac and other tissues in terms of selection of the isoelectric point separating window for cardiac proteins, accurate quantitation of cardiac protein abundance, stabilization of technical variation, reduction of sample complexity, enrichment of low-abundant proteins, and detection of phosphorylated proteins.     

Thermal denaturation produced degenerative proteins and interfered with MS for proteins dissolved in lysis buffer in proteomic analysis

In 1-DE, proteins were traditionally mixed with the standard Laemmli buffer and boiled for several minutes. Recently, proteins dissolved in lysis buffer were used to produce better-resolved 2-DE gels, but thermal denaturation procedure still remained in some proteomic analysis. To determine the detailed effects of thermal denaturation on SDS-PAGE and MS, both 1-DE and 2-DE were performed using proteins heated at 100°C for different periods of time, and 17 protein bands/spots were positively identified by MALDI TOF/TOF MS/MS. Protein profiles on both 1-DE and 2-DE gels changed obviously and more polydisperse bands/spots were observed with increased heating time for over-heated samples. Based on these observations, an alternative protein marker-producing method was designed by directly dissolving protein standards without BSA into lysis buffer. This new kind of protein marker could be stored at room temperature for a long time, thus was more convenient for using and shipping. The identification of 17 proteins via MS and comparison of their identities revealed MASCOT-searched scores, number of both matched peptides, total searched peptides and sequence coverage became progressively lower with increasing denaturation intensity, probably due to the interference of thermal denaturation on trypsin cleavage efficiency and produced redundant modified peptides. Therefore, it was concluded that thermal denaturation not only changed the protein profiles and produced more polydisperse protein bands/spots, but also heavily interfered with the subsequent MS analysis, hence not recommended in future proteomic analysis for proteins dissolved in lysis buffer.     

维拉帕米作用下急性心梗大鼠比较蛋白质组学研究

以急性心梗大鼠为研究对象, 应用双向凝胶电泳法(2-DE)分析比较了维拉帕米作用下急性心梗大鼠心肌蛋白表达的差异, 从蛋白质水平探讨了维拉帕米心肌保护作用的发生机制. 结果表明, 与假手术组及模型组相比, 维拉帕米给药组心肌组织中有8个蛋白点表达显著上调, 7个蛋白点表达显著下调. 采用质谱(MALDI-TOF-MS)分析结合数据库检索, 共鉴定了其中的15种蛋白质, 可按功能分为如下4类: (1) 能量代谢及线粒体功能相关蛋白; (2) 氧化应激相关蛋白; (3) 细胞骨架蛋白; (4) 其它蛋白. 研究结果表明, 维拉帕米的心肌保护作用与恢复心肌损伤过程中的能量供应及对抗氧化应激等作用有关.