Identification of enzymes and activity from two-dimensional gel electrophoresis

Identification of proteins with enzymatic activity by mass spectrometry (MS) and concomitant determination of function by screening enzyme activity from two-dimensional gel electrophoresis (2DE) is one of the challenges of gel-based proteomics. In this protocol, proteins are extracted from spinal cord tissue followed by 2DE with in-gel digestion and identification by matrix-assisted laser desorption/ionization. Protein spots identified as possible enzyme of interest are punched, eluted by SDS-containing Tris buffer and renatured by buffers under reductive conditions. Enzyme activity is determined using micromethods. Within about 4 weeks, a structural and functional map can be generated and MS identification can be validated, complementing immunochemical methods. 2DE separation can be seen as a prepurification step and therefore allows activity assays from minute amounts of protein as provided in a 2DE gel spot; the method may be an alternative to the time-consuming use of recombinant enzyme techniques.     

Proteome analysis of hairy root from Panax ginseng C.A. Meyer using peptide fingerprinting, internal sequencing and expressed sequence tag data

As an initial step to the comprehensive proteomic analysis of Panax ginseng C. A. Meyer, protein mixtures extracted from the cultured hairy root of Panax ginseng were separated by two-dimensional polyacrylamide gel electrophoresis (2-DE). The protein spots were analyzed and identified by peptide finger printing and internal amino acid sequencing by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and electrospray ionization quadrupole-time of flight mass spectrometry (ESI Q-TOF MS), respectively. More than 300 protein spots were detected on silver stained two-dimensional (2-D) gels using pH 3-10, 4-7, and 4.5-5.5 gradients. Major protein spots (159) were analyzed by peptide fingerprinting or de novo sequencing and the functions of 91 of these proteins were identified. Protein identification was achieved using the expressed sequence tag (EST) database from Panax ginseng and the protein database of plants like Arabidopsis thaliana and Oryza sativa. However, peptide mass fingerprinting by MALDI-TOF MS alone was insufficient for protein identification because of the lack of a genome database for Panax ginseng. Only 17 of the 159 protein spots were verified by peptide mass fingerprinting using MALDI-TOF MS whereas 87 out of 102 protein spots, which included 13 of the 17 proteins identified by MALDI-TOF MS, were identified by internal amino acid sequencing using tandem mass spectrometry analysis by ESI Q-TOF MS. When the internal amino acid sequences were used as identification markers, the identification rate exceeded 85.3%, suggesting that a combination of internal sequencing and EST data analysis was an efficient identification method for proteome analysis of plants having incomplete genome data like ginseng. The 2-D patterns of the main root and leaves of Panax ginseng differed from that of the cultured hairy root, suggesting that some proteins are exclusively expressed by different tissues for specific cellular functions. Proteome analysis will undoubtedly be helpful for understanding the physiology of Panax ginseng.     

Proteome profiling of human epithelial ovarian cancer cell line TOV-112D

A proteome profiling of the epithelial ovarian cancer cell line TOV-112D was initiated as a protein expression reference in the study of ovarian cancer. Two complementary proteomic approaches were used in order to maximise protein identification: two-dimensional gel electrophoresis (2DE) protein separation coupled to matrix assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and one-dimensional gel electrophoresis (1DE) coupled to liquid-chromatography tandem mass spectrometry (LC MS/MS). One hundred and seventy-two proteins have been identified among 288 spots selected on two-dimensional gels and a total of 579 proteins were identified with the 1DE LC MS/MS approach. This proteome profiling covers a wide range of protein expression and identifies several proteins known for their oncogenic properties. Bioinformatics tools were used to mine databases in order to determine whether the identified proteins have previously been implicated in pathways associated with carcinogenesis or cell proliferation. Indeed, several of the proteins have been reported to be specific ovarian cancer markers while others are common to many tumorigenic tissues or proliferating cells. The diversity of proteins found and their association with known oncogenic pathways validate this proteomic approach. The proteome 2D map of the TOV-112D cell line will provide a valuable resource in studies on differential protein expression of human ovarian carcinomas while the 1DE LC MS/MS approach gives a picture of the actual protein profile of the TOV-112D cell line. This work represents one of the most complete ovarian protein expression analysis reports to date and the first comparative study of gene expression profiling and proteomic patterns in ovarian cancer.     

Quantitative profiling of drug-associated proteomic alterations by combined 2-nitrobenzenesulfenyl chloride (NBS) isotope labeling and 2DE/MS identification

The identification of drug-responsive biomarkers in complex protein mixtures is an important goal of quantitative proteomics. Here, we describe a novel approach for identifying such drug-induced protein alterations, which combines 2-nitrobenzenesulfenyl chloride (NBS) tryptophan labeling with two-dimensional gel electrophoresis (2DE)/mass spectrometry (MS). Lysates from drug-treated and control samples are labeled with light or heavy NBS moiety and separated on a common 2DE gel, and protein alterations are identified by MS through the differential intensity of paired NBS peptide peaks. Using NBS/2DE/MS, we profiled the proteomic alterations induced by tamoxifen (TAM) in the estrogen receptor (ER) positive MCF-7 breast cancer cell line. Of 88 protein spots that significantly changed upon TAM treatment, 44 spots representing 23 distinct protein species were successfully identified with NBS-paired peptides. Of these 23 TAM-altered proteins, 16 (70%) have not been previously associated with TAM or ER activity. We found the NBS labeling procedure to be both technically and biologically reproducible, and the NBS/2DE/MS alterations exhibited good concordance with conventional 2DE differential protein quantitation, with discrepancies largely due to the comigration of distinct proteins in the regular 2DE gels. To validate the NBS/2DE/MS results, we used immunoblotting to confirm GRP78, CK19, and PA2G4 as bona fide TAM-regulated proteins. Furthermore, we demonstrate that PA2G4 expression can serve as a novel prognostic factor for disease-free survival in two independent breast cancer patient cohorts. To our knowledge, this is the first report describing the proteomic changes in breast cancer cells induced by TAM, the most commonly used selective estrogen receptor modulator (SERM). Our results indicate that NBS/2DE/MS may represent a more reliable approach for cellular protein quantitation than conventional 2DE approaches.     

Identification of proteins from two-dimensional polyacrylamide gels using a novel acid-labile surfactant

Protein identification by peptide mass mapping usually involves digestion of gel-separated proteins with trypsin, followed by mass measurement of the resulting peptides by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Positive identification requires measurement of enough peptide masses to obtain a definitive match with sequence information recorded in protein or DNA sequence databases. However, competitive binding and ionization of residual surfactant introduced during polyacrylamide gel electrophoresis (PAGE) can inhibit solid-phase extraction and MS analysis of tryptic peptides. We have evaluated a novel, acid-labile surfactant (ALS) as an alternative to sodium dodecylsulfate (SDS) for two-dimensional (2-D) PAGE separation and MALDI-MS mapping of proteins. ALS was substituted for SDS at the same concentration in buffers and gels used for 2-D PAGE. Manual and automated procedures for spot cutting and in-gel digestion were used to process Coomassie stained proteins for MS analysis. Results indicate that substituting ALS for SDS during PAGE can significantly increase the number of peptides detected by MALDI-MS, especially for proteins of relatively low abundance. This effect is attributed to decomposition of ALS under acidic conditions during gel staining, destaining, peptide extraction and MS sample preparation. Automated excision and digestion procedures reduce contamination by keratin and other impurities, further enhancing MS identification of gel separated proteins.     

Nonredundant mass spectrometry: a strategy to integrate mass spectrometry acquisition and analysis

Protein identification using automated data-dependent tandem mass spectrometry (MS/MS) is now a standard procedure. However, in many cases data-dependent acquisition becomes redundant acquisition as many different peptides from the same protein are fragmented, whilst only a few are needed for unambiguous identification. To increase the quality of information but decrease the amount of information, a nonredundant MS (nrMS) strategy has been developed. With nrMS, data analysis is an integral part of the overall MS acquisition and analysis, and not an endpoint as typically performed. In this nrMS workflow a matrix assisted laser desorption/ionization-time of flight-time of flight (MALDI-TOF/TOF) instrument is used. MS and restricted MS/MS data are searched and identified proteins are used to generate an "exclusion list", after in silico digestion. Peptide fragmentation is then restricted to only the most intense ions not present in the exclusion list. This process is repeated until all peaks are accounted for or the sample is consumed. Compared to nanoLC-MS/MS, nrMS yielded similar results for the analysis of six pooled two-dimensional electrophoresis (2-DE) spots. In comparison to standard data-dependent MALDI-MS/MS for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel band analysis, nrMS dramatically increased the number of identified proteins. It was also found that this new workflow significantly increased sequence coverage by identifying unexpected peptides, which can result from post-translational modifications.     

Proteomic analysis in clear cell renal cell carcinoma: identification of differentially expressed protein by 2-D DIGE

Renal cell carcinoma (RCC), the most common neoplasm affecting the adult kidney, is characterised by heterogeneity of histological subtypes, drug resistance, and absence of molecular markers. Two-dimensional difference gel electrophoresis (2-D DIGE) technology in combination with mass spectrometry (MS) was applied to detect differentially expressed proteins in 20 pairs of RCC tissues and matched adjacent normal kidney cortex (ANK), in order to search for RCC markers. After gel analysis by DeCyder 6.5 and EDA software, differentially expressed protein spots were excised from Deep Purple stained preparative 2DE gel. A total of 100 proteins were identified by MS out of 2500 spots, 23 and 77 of these were, respectively, over- and down-expressed in RCC. The Principal Component Analysis applied to gels and protein spots exactly separated the two sample classes in two groups: RCC and ANK. Moreover, some spots, including ANXA2, PPIA, FABP7 and LEG1, resulted highly differential. The DIGE data were also confirmed by immunoblotting analysis for these proteins. In conclusion, we suggest that applying 2-D DIGE to RCC may provide the basis for a better molecular characterization and for the discovery of candidate biomarkers.     

Two-dimensional gel electrophoresis-based analysis provides global insights into the cotton ovule and fiber proteomes

Proteomic analysis of upland cotton was performed to profile the global detectable proteomes of ovules and fibers using two-dimensional electrophoresis (2DE). A total of 1,203 independent protein spots were collected from representative 2DE gels, which were digested with trypsin and identified by matrix-assisted laser desorption and ionization-time-offlight/ time-of-flight (MALDI-TOF/TOF) mass spectrometry. The mass spectrometry or tandem mass spectrometry (MS or MS/MS) data were then searched against a local database constructed from Gossypium hirsutum genome sequences, resulting in successful identification of 975 protein spots (411 for ovules and 564 for fibers). Functional annotation analysis of the 975 identified proteins revealed that ovule-specific proteins were mainly enriched in functions related to fatty acid elongation, sulfur amino acid metabolism and post-replication repair, while fiber-specific proteins were enriched in functions related to root hair elongation, galactose metabolism and D-xylose metabolic processes. Further annotation analysis of the most abundant protein spots showed that 28.96% of the total proteins in the ovule were mainly located in the Golgi apparatus, endoplasmic reticulum, mitochondrion and ribosome, whereas in fibers, 27.02% of the total proteins were located in the cytoskeleton, nuclear envelope and cell wall. Quantitative real-time polymerase chain reaction (qRT-PCR) analyses of the ovule-specific protein spots P61, P93 and P198 and fiber-specific protein spots 230, 477 and 511 were performed to validate the proteomics data. Protein-protein interaction network analyses revealed very different network cluster patterns between ovules and fibers. This work provides the largest protein identification dataset of 2DE-detectable proteins in cotton ovules and fibers and indicates potentially important roles of tissue-specific proteins, thus providing insights into the cotton ovule and fiber proteomes on a global scale.     

Yeast proteome map (update 2006)

To improve the potential of two-dimensional gel electrophoresis for proteomic investigations in yeast we have undertaken the systematic identification of Saccharomyces cerevisiae proteins separated on 2-D gels. We report here the identification of 187 novel protein spots. They were identified by two methods, mass spectrometry and gene inactivation. These identifications extend the number of protein spots identified on our yeast 2-D proteome map to 602, i.e. nearly half the detectable spots of the proteome map. These spots correspond to 417 different proteins. The reference map and the list of identified proteins can be accessed on the Yeast Protein Map server (www.ibgc.u-bordeaux2.fr/YPM).     

Mass spectrometrical identification of brain proteins including highly insoluble and transmembrane proteins

Conventional two-dimensional electrophoresis (2DE) is the main technique used for protein profiling of tissues and cells, however separation of strongly acidic, basic or highly insoluble proteins is still limited. A series of methods have been proposed to cope with this problem and the use of discontinuous gel electrophoresis in an acidic buffer system using the cationic detergent benzyldimethyl-n-hexadecylammonium chloride (16-BAC) with subsequent SDS-PAGE followed by mass spectrometry showed that results from 2DE can be complemented by this approach. It was the aim of this study to separate and identify proteins from whole mouse brain that were not demonstrated by 2DE. For this purpose samples were homogenised, soluble proteins were removed by ultracentrifugation and the water-insoluble pellet was resuspended in a mixture containing urea, 16-BAC, glycerol, pyronine Y and dithiothreitol. Electrophoresis was run in the presence of 16-BAC, the strip from the gel containing separated proteins was cut out and was re-run on SDS-PAGE. Protein spots were analyzed by MALDI-TOF-TOF mass spectrometry. One hundred and six individual proteins represented by 187 spots were unambiguously identified consisting of 42 proteins with predicted pI values of pI>8.0, 25 with a 6.0相似文献   

Mass spectrometric identification of proteins in complex post-genomic projects. Soluble proteins of the metabolically versatile, denitrifying 'Aromatoleum' sp. strain EbN1

The rapidly developing proteomics technologies help to advance the global understanding of physiological and cellular processes. The lifestyle of a study organism determines the type and complexity of a given proteomic project. The complexity of this study is characterized by a broad collection of pathway-specific subproteomes, reflecting the metabolic versatility as well as the regulatory potential of the aromatic-degrading, denitrifying bacterium 'Aromatoleum' sp. strain EbN1. Differences in protein profiles were determined using a gel-based approach. Protein identification was based on a progressive application of MALDI-TOF-MS, MALDI-TOF-MS/MS and LC-ESI-MS/MS. This progression was result-driven and automated by software control. The identification rate was increased by the assembly of a project-specific list of background signals that was used for internal calibration of the MS spectra, and by the combination of two search engines using a dedicated MetaScoring algorithm. In total, intelligent bioinformatics could increase the identification yield from 53 to 70% of the analyzed 5,050 gel spots; a total of 556 different proteins were identified. MS identification was highly reproducible: most proteins were identified more than twice from parallel 2DE gels with an average sequence coverage of >50% and rather restrictive score thresholds (Mascot >or=95, ProFound >or=2.2, MetaScore >or=97). The MS technologies and bioinformatics tools that were implemented and integrated to handle this complex proteomic project are presented. In addition, we describe the basic principles and current developments of the applied technologies and provide an overview over the current state of microbial proteome research.     

Proteomics in Drosophila melanogaster: first 2D database of larval hemolymph proteins

A proteomic approach was used for the identification of larval hemolymph proteins of Drosophila melanogaster. We report the initial establishment of a two-dimensional gel electrophoresis reference map for hemolymph proteins of third instar larvae of D. melanogaster. We used immobilized pH gradients of pH 4-7 (linear) and a 12-14% linear gradient polyacrylamide gel. The protein spots were silver-stained and analyzed by nanoLC-Q-Tof MS/MS (on-line nanoscale liquid chromatography quadrupole time of flight tandem mass spectrometry) or by Matrix assisted laser desorption time of flight MS (MALDI-TOF MS). Querying the SWISSPROT database with the mass spectrometric data yielded the identity of the proteins in the spots. The presented proteome map lists those protein spots identified to date. This map will be updated continuously and will serve as a reference database for investigators, studying changes at the protein level in different physiological conditions.     

Identification of proteins in slow continuous ultrafiltrate by reversed-phase chromatography and proteomics

Continuous modes of renal replacement therapy (CRRT) are increasingly being utilized in the intensive care unit. The removal of cytokines and other inflammatory proteins during ultrafiltration may be responsible for some of the beneficial effects of CRRT. We used proteomic tools to identify proteins found in the ultrafiltrate from a patient with acute renal failure. Identification of these proteins could help elucidate the mechanism(s) of improved outcome with continuous renal replacement therapy. Protein was loaded on a reversed-phase C4 column and eluted with stepwise isocratic flows starting with 0%, 5%, 10%, 25%, and 50% of acetonitrile. Effluent was collected, pooled, desalted, and separated by two-dimensional gel electrophoresis (2DE). Reversed-phase separation improved the resolution and the number of spots seen on the gels. Protein spots were digested with trypsin and spotted onto MALDI plates. Proteins were identified by either peptide mass fingerprinting using a MALDI-TOF mass spectrometer or by peptide sequencing using a MALDI-TOF/TOF tandem mass spectrometer. From 196 spots cut, 47 were identified, representing multiple charge forms of 10 different proteins. Proteins identified were albumin, apolipoprotein A-IV, beta-2-microglobulin, lithostathine, mannose-binding lectin associated serine protease 2 associated protein, plasma retinol-binding protein, transferrin, transthyretin, vitamin D-binding protein and Zn alpha-2 glycoprotein. Continuous renal replacement therapy is frequently used in acutely ill patients with renal failure. Removal of proteins occurs during this process. The physiological significance of this protein removal is unclear. Identification of these proteins will lead to better understanding of the role of protein removal in continuous renal replacement therapy.     

Proteomics analysis of major royal jelly protein changes under different storage conditions

Protein changes in fresh royal jelly (RJ) were compared when stored at -20, 4 degrees C, and room temperature (RT) for 12 months. Protein was partially identified using combinations of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF/MS), gel filtration chromatography, nanoLC MS/MS, and a protein engine identification tool applied to the honeybee genome. Significantly more protein spots were found in fresh (85 spots) and -20 degrees C (81 spots) stored RJ than in samples stored at 4 degrees C (73 spots) and at RT (70 spots) for 1 year. Most identified spots, 56, 57, 51, 46, corresponding to RJ sample of the fresh, -20 degrees C, 4 degrees C, and RT, were assigned to major royal jelly proteins (MRJPs). Marked differences were found in the heterogeneity of the MRJPs, in particular, MRJP3. The quantity of MRJP1 decreased significantly following the temperature trend in all images, but MRJP 2 and -3 did not increase or decrease following the temperature trend, thus, suggesting that MRJP 1-3 are sensitive to temperature. However, MRJP4, 5, glucose oxidase (GOD), peroxiredoxin (PRDX), and glutathione S-transferase (GST) S1 were clearly absent in all images in samples held at RT for 1 year. This indicates that they are the proteins most sensitive to storage temperature and protein markers for freshness of RJ. Combining chromatography and nanoLC MS/MS results, we tentatively conclude that MRJP5 is a reliable freshness marker and that the best way to maintain quality of RJ is under freezing conditions.     

In-gel digestion for mass spectrometric characterization of proteins and proteomes

In-gel digestion of proteins isolated by gel electrophoresis is a cornerstone of mass spectrometry (MS)-driven proteomics. The 10-year-old recipe by Shevchenko et al. has been optimized to increase the speed and sensitivity of analysis. The protocol is for the in-gel digestion of both silver and Coomassie-stained protein spots or bands and can be followed by MALDI-MS or LC-MS/MS analysis to identify proteins at sensitivities better than a few femtomoles of protein starting material.     

Horseradish esterases: detection,purification and identification

Our goal is to characterize esterases from horseradish tissues and assign their physiological roles. In the present study we focused on isolation, purification and identification of esterases from different horseradish tissues: plantlets and two tumor tissue lines. Horizontal IEF system enabled separation of six esterase isoforms with quite different pI values as well as with pronounced differences in expression levels among analyzed tissues. Esterases were extracted, fractionated by means of cation exchange chromatography, and analyzed by planar gel electrophoresis (SDS–PAGE) and isoelectrical focusing (IEF), UV/Vis spectroscopy, MALDI mass spectrometry (MS) and MALDI-MS/MS. Several chromatographic strategies were applied for esterase purification and characterization. Two subsequent cation exchange chromatographic steps based on SP-Sepharose FF material, followed by in-solution digestion combined with MALDI-MS and MS/MS proved to be the best strategy for identification of two esterase proteins, namely Pectinesterase/pectinesterase inhibitor 18 and GDSL esterase/lipase ESM1.     

Large-scale identification of proteins in human salivary proteome by liquid chromatography/mass spectrometry and two-dimensional gel electrophoresis-mass spectrometry

Human saliva contains a large number of proteins and peptides (salivary proteome) that help maintain homeostasis in the oral cavity. Global analysis of human salivary proteome is important for understanding oral health and disease pathogenesis. In this study, large-scale identification of salivary proteins was demonstrated by using shotgun proteomics and two-dimensinal gel electrophoresis-mass spectrometry (2-DE-MS). For the shotgun approach, whole saliva proteins were prefractionated according to molecular weight. The smallest fraction, presumably containing salivary peptides, was directly separated by capillary liquid chromatography (LC). However, the large protein fractions were digested into peptides for subsequent LC separation. Separated peptides were analyzed by on-line electrospray tandem mass spectrometry (MS/MS) using a quadrupole-time of flight mass spectrometer, and the obtained spectra were automatically processed to search human protein sequence database for protein identification. Additionally, 2-DE was used to map out the proteins in whole saliva. Protein spots 105 in number were excised and in-gel digested; and the resulting peptide fragments were measured by matrix-assisted laser desorption/ionization-mass spectrometry and sequenced by LC-MS/MS for protein identification. In total, we cataloged 309 proteins from human whole saliva by using these two proteomic approaches.     

Two dimensional electrophoresis of the exo-proteome produced from community acquired methicillin resistant Staphylococcus aureus belonging to clonal complex 80

Two-dimensional electrophoresis (2DE) combined with mass spectrometry was used to characterize the exo-proteome secreted by two strains (ER13 and ER21) representing community acquired methicillin resistant Staphylococcus aureus (CA-MRSA) belonging to clonal complex 80 (CC80). Common spots were detected between the 2 gels using the Progenesis SameSpots software. Two hundred and fifty-one and 312 spots from the exo-proteome of ER13 and ER21 were resolved, respectively. 2DE overlap comparison showed that 59 spots were shared. LC–MS/MS analysis identified 57 proteins from these spots comprising about 21% extracellular, 48% cytoplasmic, 2% cytoplasmic membrane, 2% cell wall, and 26% with unknown localization. The identified proteins were classified with respect to their Gene Ontology (GO) annotation as ～24% virulence determinants and toxins, ～17% involved in carbohydrate metabolism, ～14% involved in environmental stress, and ～12% associated with cell division. The identification of the enterotoxin B from the exo-products of both strains used in our study, as belonging to CC80 was interesting.     

应用蛋白质组学的方法筛选胰腺干细胞标记物

探索利用蛋白质组学的方法筛选胰腺干细胞的标记物.通过大鼠胰腺大部分切除(Px)的方法建立胰腺增生模型,采用双向电泳(2DE)分离比较Px术后第3天大鼠增生胰腺组织与假手术(Sx)非增生胰腺组织蛋白质的表达谱差异,用基质辅助激光解吸离子化飞行时间质谱(MALDI-TOF-MS)和肽质量指纹图谱(PMF)的方法对差异蛋白进行鉴定,进一步经生物信息学检索,筛选与胚胎发育和细胞分化相关的蛋白质分子.蛋白质印迹分析蛋白质HSP47、Vimentin在Px大鼠胰腺组织中的表达,以验证2DE的结果.2DE显示Sx和Px大鼠胰腺组织平均蛋白质点数分别为(1369±31)和(1315±28)个,其中共有91个蛋白质点出现1.5倍以上的表达差异.所有差异蛋白质点PMF鉴定出53种蛋白质,其中Vimentin,cytokeratin8(CK8),lymphocytecytosolicprotein1(L-plastin),heterogeneousnuclearribonucleoproteinA2/B1(hnRNPA2/B1)和L-arginine:glycineamidinotransferase(AGAT)与胚胎发育和细胞分化有关,这些蛋白质可能是胰腺干细胞潜在的候选标记物.总之,蛋白质组学的方法利用其高通量的特点能有效发现潜在的胰腺干细胞生物学标记物.