Proteomic analysis of human very low-density lipoprotein by two-dimensional gel electrophoresis and MALDI-TOF/TOF

Biochemical studies of lipoproteins have shed light on their composition, highly contributing to the comprehension of their function. Due to the complexity of their structure, however, an in-depth structural analysis, in terms of components and PTMs, may still unravel important players in physiological and pathological processes of lipid metabolism. In this study, we performed a protein map of very low-density lipoprotein (VLDL) using a 2-DE MALDI-TOF/TOF proteomic approach. Several VLDL-associated apolipoproteins were identified, including five isoforms of apoE, three isoforms of apoC-IV, and one isoform each of apoC-III, apoM, apoA-I, and apoA-IV. Notably, we also identified seven isoforms of apoL-I and two isoforms of prenylcysteine lyase as new VLDL-associated proteins. Furthermore, we were able to identify PTM of apoE, which was found to be differently O-glycosylated at Thr212 residue, and PTM of apoL-I which we described, for the first time, to be phosphorylated at Ser296. While the physiological relevance of our finding remains to be assessed, we believe that our results will be useful as reference for future studies of VLDL structure in specific physiopathological conditions.     

A sodium dodecyl sulfate--polyacrylamide gel electrophoresis system that separates peptides and proteins in the molecular weight range of 2500 to 90,000

A discontinuous sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis system is described which provides superior resolution of polypeptides with molecular weights from approximately 2500 to 90,000. The system utilizes a relatively low-mobility acetate ion in the stacking gel and high-mobility strong anions, sulfate and chloride, as leading and trailing ions in the separating gel. The entire system is run at pH 7.8. The separating gel contains 8 M urea, and can be used at acrylamide concentrations from 5 to 18%, all with 5% crosslinker concentrations. Using a number of protein standards, the calibration curves obtained with this system are linear over the molecular weight range from 2500 to 90,000, regardless of acrylamide concentration. These studies indicate that by providing good resolution of small peptides, this system greatly extends the utility of one-dimensional peptide mapping techniques.     

Evaluation of isoelectric focusing running conditions during two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis: variation of gel patterns with changing conditions and optimized isoelectric focusing conditions

Five major isoelectric focusing (IEF) parameters--volt-hours; concentrations of acrylamide, NaOH, and H3PO4; and equilibration time--were systematically varied to determine the effect of each on two-dimensional IEF/sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel patterns and to optimize IEF conditions. Alterations in each parameter affected the gel pattern, frequently causing uncertainty in the identification of spots between conditions. The results emphasize the need for internal analytical consistency, and indicate that gel pattern comparisons between laboratories can be complicated if different IEF conditions are employed. The systematic evaluation indicated that optimized patterns were obtained when increased concentrations of NaOH and H3PO4 (to 50 and 25 mM, respectively) and run durations of 10,000 V-h or longer were used.     

Allergome: the characterization of allergens based on a 2D gel electrophoresis approach

Type I hypersensitivity reactions are in constant progression in industrialized countries. The physiopathologic mechanism of these diseases implicates the production of specific immunoglobulin (Ig)E to allergenic molecules, their binding to the Fcε receptor on the surface of mast cells and basophils, and the release of inflammatory mediators when allergens are introduced into the body and crosslink with the IgE bound to the cell surface. An allergen is defined as a molecule that induces the production of, and binds to, IgE. The identification of the allergenic molecules is an important goal to improve diagnosis and treatment of allergy. This characterization aims to extract proteins from the allergenic source, to analyze IgE specificity by immunoblotting and to identify the proteins that bind IgE.     

镉胁迫下三个萝卜栽培种蛋白质变化的双向电泳比较研究

在0.5mmol·L^-1 Cd²⁺处理时,小五缨(XWY)的发芽率下降至92%,并且随着Cd²⁺浓度的增加,发芽率逐渐降低,在1和5mmol·L^-1 Cd²⁺浓度处理时,发芽率分别降至83%和67%。象牙白(XYB)则在5mmol·L^-1 Cd²⁺浓度时,发芽率下降。卫青(WQ)在0.05mmol·L^-1 Cd²⁺浓度时,发芽率已降至83%, 1和5mmol·L^-1时则下降至58%。幼苗生长也明显受Cd²⁺处理影响,在0.05mmol·L^-1 Cd²⁺处理时, 3种栽培萝卜的幼苗生长均受到明显影响,并且随Cd²⁺浓度的增加,生长受抑加重。从发芽率和幼苗生长两种实验结果看,卫青对Cd²⁺最为敏感。双向电泳结果表明, Cd²⁺处理后3种栽培萝卜幼苗中蛋白质组分有明显变化。小五缨中, 0.1 mmol·L^-1Cd²⁺处理后,有5个蛋白质点消失, 15个新的蛋白质点被诱导产生。象牙白中, 2个蛋白质点消失, 1个蛋白质点含量减少, 13个新的蛋白质点被诱导产生。卫青中, 12个新的蛋白质点诱导产生,但没有发现蛋白质点消失现象。Cd²⁺处理后, 3种栽培萝卜中,蛋白质合成的变化与幼苗生长受抑存在明显相关性,这一实验结果对于探讨萝卜Cd²⁺害的生化机理是有重要意义的。     

Sub-proteome differential display: single gel comparison by 2D electrophoresis and mass spectrometry

Two-dimensional (2D) gel electrophoresis and mass spectrometry (MS) have been used in comparative proteomics but inherent problems of the 2D electrophoresis technique lead to difficulties when comparing two samples. We describe a method (sub-proteome differential display) for comparing the proteins from two sources simultaneously. Proteins from one source are mixed with radiolabelled proteins from a second source in a ratio of 100:1. These combined proteomes are fractionated simultaneously using column chromatographic methods, followed by analysis of the pre-fractionated proteomes (designated sub-proteomes) using 2D gel electrophoresis. Silver staining and (35)S autoradiography of a single gel allows precise discrimination between members of each sub-proteome, using commonly available computer software. This is followed by MS identification of individual proteins. We have demonstrated the utility of the technology by identifying the product of a transfected gene and several proteins expressed differentially between two renal carcinoma proteomes. The procedure has the capacity to enrich proteins prior to 2D electrophoresis and provides a simple, inexpensive approach to compare proteomes. The single gel approach eliminates differences that might arise if separate proteome fractionations or 2D gels are employed.     

Protein expression profiles in Saccharomyces cerevisiae during apoptosis induced by H2O2

We identified the proteins involved during apoptosis induced by H2O2 in Saccharomyces cerevisiae, and analyzed the global protein pattern by 2-DE. We analyzed classical parameters of apoptosis such as chromatin condensation, DNA fragmentation, and morphology changes of cells. Exposure of yeast cells to nonphysiological doses of peroxides decreases the expression (or increases degradation) of enzymes involved in protection against oxidative stress. This leads the yeast cells to a reduction of their antioxidant defense and makes the cells more prone to apoptosis. In our data the down expression of peroxiredoxin II and GST I, could induce a perturbation of mitochondrial function with an alteration of permeability of the membrane leading to the mitochondria-mediated apoptosis. Moreover, we identified a new spot of a classical glycolytic enzyme: the glyceraldehyde 3-phosphate dehydrogenase during apoptosis. It is known that GAPDH is an extremely abundant glycolytic enzyme with multiple functions and that its overexpression is evident during apoptosis induced by a variety of stimuli. Our results confirm that it is a major intracellular messenger mediating apoptotic death and that this new spot of GAPDH could be an intracellular sensor of oxidative stress during apoptosis induced by H2O2 in S. cerevisiae.     

Bacterial genome mapping by two-dimensional pulsed-field gel electrophoresis (2D-PFGE)

Macrorestriction mapping is often the first step toward a thorough physical and genetic characterization of a bacterial genome. The problem of deducing the order of partially or completely digested macrorestriction fragments to yield a physical genome map may readily be solved by applying twodimensional pulsed-field gel electrophoresis (2D-PFGE) techniques. These powerful methods are quick and technically easy to perform; specifically, they are independent of DNA probes and should therefore be applicable to any bacterial species irrespective of its prior genetic characterization. In this article, detailed step-by-step protocols are given to set up, run, and evaluate 2D pulsed-field gels. Two basic methods are described: partial/complete 2D gels of one restriction enzyme and complete/complete 2D gels of two different restriction enzymes. Other topics include preparation of bacterial genomic DNA, screening for suitable rare-cutting restriction enzymes and determination of optimal running conditions. Accompanied by many notes, these protocols are meant to offer the novice a sound and rapid access to these important methods.     

Investigation of polyacrylamide hydrogel-based molecularly imprinted polymers using protein gel electrophoresis

In conjunction with polyacrylamide gel electrophoresis (PAGE), molecular imprinting methods have been applied to produce a multilayer mini-slab in order to evaluate how selectively and specifically a hydrogel-based molecularly imprinted polymer (MIP) binds bovine haemoglobin (BHb, ~64.5 kDa). A three-layer mini-slab comprising an upper and lower layer and a MIP, or a non-imprinted control polymer dispersion middle layer has been investigated. The discriminating MIP layer, also based on polyacrylamide, was able to specifically bind BHb molecules in preference to a protein similar in molecular weight such as bovine serum albumin (BSA, ~66 kDa). Protein staining allowed us to visualise the protein retention strength of the MIP layer under the influence of an electric field. This method could be applied to other proteins with implications in effective protein capture, disease diagnostics, and protein analysis.     

Comparative analysis of protein aggregates by blue native electrophoresis and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a three-dimensional geometry gel

We describe the comparative analysis of protein aggregates by combining blue native electrophoresis and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 3-D geometry gel for simultaneous processing of many samples. The first native electrophoresis step, separating the aggregates, is carried out for a series of samples in parallel lanes within a slab gel. This gel is then placed on the top surface of a cylindrical, 3-D geometry gel for the second denaturing electrophoresis step, separating the proteins composing the aggregates. The samples migrate parallel to the vertical axis of the gel cylinder. Data are acquired online by photodetection of laser-induced fluorescence during electrophoresis. For this purpose, the samples are fluorescently labeled within the slab gel after the first separation step. A 3-D geometry gel separates the equivalent of many conventional SDS slab gels represented by vertical layers in the 3-D gel body. In this way, many samples are analyzed in the same gel under identical conditions, improving comparability and resolution and making the process considerably more efficient. This novel technique allowed the identification of several aggregate classes of recombinant proteins expressed in bacteria. We observed that proteins preferentially bind to homolog polypeptides, but also seem to form a trapping mesh co-aggregating with other proteins. The aggregation pattern revealed by this technique supplements data obtained from standard two-dimensional gel electrophoresis analysis. We expect interesting applications, for instance in aggregate monitoring of clinical samples. It should be feasible to quickly gain a diagnostic picture during amyloid-related neurodegenerative disease development or to observe drug effects on protein aggregation.     

Identification of stool proteins in C57BL/6J mice by two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry

Gastrointestinal disease is a major cause of mortality in humans and animals, and the detection of disease-associated protein in stool is an established diagnostic method in this context. Yet, no data currently exists about the protein composition of mammalian faeces. Using a newly developed two-dimensional (2D) gel method, 28 of the most abundant proteins in murine faeces were identified. Mammalian faeces contains protein from multiple species (from the individual, from gastrointestinal bacteria, from food, etc.). Yet, it was found that the majority of mouse stool proteins were of mouse origin, with a minority of proteins being derived from food (in particular soybean glycinin and conglycinin) and bacteria (flagellin). Most mouse proteins were proteases and saccharidases derived from the exocrine pancreas. In addition, two unexpected mouse proteins were identified: one was a newly described mucin-like protein from intestinal goblet cells (FcγBP); the other was the secreted form of carbonic anhydrase (type VI) from salivary gland. The data suggest that 2D analysis of faecal protein is likely to provide meaningful information about the physiological stage of the gastrointestinal tract. Compared with studies based on biopsies, faecal protein analysis may reduce the number of laboratory animals, and might also allow quicker bridging from animal studies to humans, where biopsy material is more difficult to obtain and is less relevant for general practice use.     

Protein expression analysis ofChlamydia pneumoniae persistence by combined surface-enhanced laser desorption ionization time-of-flight mass spectrometry and two-dimensional polyacrylamide gel electrophoresis

The aim of this study was to examine the protein expression profiles of persistentChlamydia pneumoniae by two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Although 2D PAGE is still the method of choice for separating and detecting components of complex protein mixtures, it has several distinct disadvantages; i.e., being labor-intensive and having a bias toward proteins within the dynamic range of the gel condition. Hence, SELDI-TOF-MS technology was used to complement 2D PAGE.C. pneumoniae-infected HEp2 cells were treated with or without IFN-γ, and protein expression profiles were determined at 48 h postinfection (hpi). Unfractionated monolayers were also used for protein profiling by SELDI-TOF, using two different chip surface types: weak cation exchanger and hydrophobic surface. Under IFN-γ-induced persistence,C. pneumoniae expresses an altered protein expression profile. Twenty chlamydial proteins showed differential regulatory patterns by SELDI-TOF-MS, two of which, HSP-70 cofactor, and a hypothetical protein, were identified by 2D PAGE and mass spectrometry. Two additional proteins, phosphatidylserine decarboxylase and 30S ribosomal protein S17, were exclusively identified by SELDI TOF-MS analysis, as these were not present in sufficient quantity for detection by 2D PAGE. We propose that a combination of 2D-PAGE and SELDI-TOF-MS may complement the disadvantages of each technique alone and may provide a rapid and precise screening technique.     

Rapid identification of pickle yeasts by fluorescent PCR and microtemperature-gradient gel electrophoresis

Monitoring the yeast populations within pickle soaking fluid is imperative for ensuring optimum taste, but these analyses have proven time-consuming and expensive, limiting their industrial application. Here, yeasts were identified in the soaking fluid from Japanese radish pickles using fluorescent PCR amplification of the variable D1/D2 region of the 26S rDNA, followed by analysis with microtemperature-gradient gel electrophoresis (micro-TGGE). This smaller version of the normal TGGE apparatus is capable of analyzing samples 10- to 20-fold faster without sacrificing data quality. Each primer set was labeled with a different fluorescent dye, allowing easy isolation of the various PCR products and identification of the bands corresponding to the various yeasts. The results indicate that fluorescent PCR and micro-TGGE may be a useful new method for rapid, easy monitoring of yeast flora in various food industries. This new method can be used on a daily basis to provide overviews of yeast flora during pickle production, allowing producers to quickly grasp pickle readiness at a single glance.     

Data standards for proteomics: mitochondrial two-dimensional polyacrylamide gel electrophoresis data as a model system

Proteomics has emerged as a major discipline that led to a re-examination of the need for consensus and a nationally sanctioned set of proteomics technology standards. Such standards for databases and data reporting may be applied to two-dimensional polyacrylamide gel electrophoresis (2D PAGE) technology as a pilot project for assessing global and national needs in proteomics, and the role of the National Institute of Standards and Technology (NIST) and other similar standards and measurement organizations. The experience of harmonizing the heterogeneous data included in the Protein Data Bank (PDB) provides a paradigm for technology in an area where significant heterogeneity in technical detail and data storage has evolved. Here we propose an approach toward standardizing mitochondrial 2D PAGE data in support of a globally relevant proteomics consensus.     

聚丙烯酰胺凝胶电泳配合荧光增白剂显色检测木霉几丁质酶同工酶谱

为提高木霉几丁质酶检测方法的准确性和灵敏度,建立一种快速检测几丁质酶同工酶的方法。采用活性凝胶电泳、变性凝胶电泳、原位显色凝胶电泳结合荧光增白剂（Calcofluor white M2R）显色从绿色木霉LTR-2发酵产物中检测几丁质酶同工酶。活性凝胶电泳在粗酶液浓缩5倍时显示两条活性谱带,变性凝胶电泳在浓缩10倍时显示一条活性谱带,原位显色凝胶电泳在浓缩20倍时显示两条不清晰的活性谱带,SDS-PAGE显示这两条活性谱带的分子量分别为65kDa和42kDa。结果表明活性聚丙烯酰胺凝胶电泳和Calcofluor white M2R显色相结合的方法在几丁质酶上样量为0.47U时具有较好的分辨能力,是检测木霉几丁质酶同工酶的有效的方法。     

Construction of a two-dimensional gel electrophoresis protein database for the Nicotiana tabacum cv. Bright Yellow-2 cell suspension culture

Using two-dimensional gel electrophoresis (2-DE) and electrospray-tandem mass spectrometry (ESI-MS/MS), we have started the proteome analysis of the cell line Nicotiana tabacum cv. Bright Yellow-2 (tobacco BY-2). The BY-2 cell suspension culture is widely used as a model system to study the growth and development of plant cells. We present a protocol describing the sample preparation and 2-DE, enabling us to separate and display more than 1000 proteins from this cell culture. A reference gel was generated, using immobilized pH gradient isoelectric focusing in a linear gradient from pH 3 to 10 and 12% Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Although the tobacco genome is not sequenced yet, a range of protein spots from this reference map was identified by means of a semi-automated liquid chromatography-ESI-quadrupole time of flight-tandem MS (LC-ESI-QTOF-MS-MS) setup and cross-species matching. These data were integrated in a database, which can be accessed at http://tby2-www.uia.ac.be/tby2/. On the on-line reference map, the identified protein spots are hyperlinked to individual protein entries. Each protein entry contains all identification information, as well as links to relevant entries in other on-line databases. Comprehensive search functions are implemented. Especially for an unsequenced but widespread model organism like tobacco BY-2, such a reference database is a convenient source for protein information that brings protein identification within reach without the need for extensive MS. This publicly accessible database provides a solid basis for tobacco BY-2 proteomics in the future.     

Protein markers of ischemic insult in brain endothelial cells identified using 2D gel electrophoresis and ICAT-based quantitative proteomics

The blood-brain barrier (BBB) is formed by endothelial cells of cerebral microvessels sealed by tight junctions. Ischemic brain injury is known to initiate a series of biochemical and molecular processes that lead to the disruption of the BBB, development of vascular inflammation, and subsequent neurovascular remodeling including angiogenesis. Molecular effectors of these changes are multiple and are regulated in a dynamic fashion. The current study was designed to analyze changes in cellular and secreted proteins in rat brain endothelial cells (BEC) exposed to ischemic insult in vitro using two complementary quantitative proteomic approaches: two-dimensional gel electrophoresis (2DE) and isotope-coded affinity tag (ICAT)-based proteomics. We show a comprehensive qualitative and quantitative comparison between the two proteomic methods applied to the same experimental system with respect to their reproducibility, specificity, and the type of proteins identified. In total, >160 proteins showed differential expression in response to the ischemic insult, with 38 identified by 2DE and 138 by ICAT. Only 15 proteins were commonly identified. ICAT showed superior reproducibility over 2DE and was more suitable for detecting small, large, basic, hydrophobic, and secreted proteins than 2DE. However, positive identification of proteins by MS/MS was more reliably done using a 2DE-based method compared to ICAT. Changes in proteins involved in nucleic acid, protein, and carbohydrate metabolism, signal transduction, cell structure, adhesion and motility, immunity and defense, cell cycle, and apoptosis were observed. The functional significance of observed protein changes was evaluated through a multifaceted protein classification and validation process, which included literature mining and comparative evaluation of protein changes in analogous in vitro and in vivo ischemia models. The comparative analyses of protein changes between the in vitro and in vivo models demonstrated a significant correlative relationship, emphasizing the 'translational' value of in vitro endothelial models in neurovascular research.     

Two-dimensional gel electrophoresis image registration using block-matching techniques and deformation models

Block-matching techniques have been widely used in the task of estimating displacement in medical images, and they represent the best approach in scenes with deformable structures such as tissues, fluids, and gels. In this article, a new iterative block-matching technique—based on successive deformation, search, fitting, filtering, and interpolation stages—is proposed to measure elastic displacements in two-dimensional polyacrylamide gel electrophoresis (2D–PAGE) images. The proposed technique uses different deformation models in the task of correlating proteins in real 2D electrophoresis gel images, obtaining an accuracy of 96.6% and improving the results obtained with other techniques. This technique represents a general solution, being easy to adapt to different 2D deformable cases and providing an experimental reference for block-matching algorithms.     

Flicker image comparison of 2-D gel images for putative protein identification using the 2DWG meta-database

With the availability of two-dimensional (2-D) gel electrophoresis databases that have many characterized proteins, it may be possible to compare a researcher’s gel images with those in relevant databases. This may lead to the putative identification of unknown protein spots in a researcher’s gel with those characterized in a given database, saving the researcher time and money by suggesting monoclonal antibodies to try in confirming these identifications. We have developed two tools to help with this comparison: (1) Flicker, http://www.lecb.ncifcrf.gov/flicker/, a Java applet program running in the researcher’s Web browser, to visually compare their gels against gels on the Internet; and (2) the 2DWG meta-database, http://www.lecb.ncifcrf.gov/2dwgDB/, a searchable database of locations of 2-D electrophoretic gel images found on the Internet. Recent additions to Flicker allow users to click on a protein spot in a gel that is linked to a federated 2D gel database, such as SWISS-2DPAGE, and have it retrieve a report from that Web database for that protein.