Functional characterization of two-dimensional gel-separated proteins using sequential staining

Proteins separated by two-dimensional (2-D) gel electrophoresis can be visualized using various protein staining methods. This is followed by downstream procedures, such as image analysis, gel spot cutting, protein digestion, and mass spectrometry (MS), to characterize protein expression profiles within cells, tissues, organisms, or body fluids. Characterizing specific post-translational modifications on proteins using MS of peptide fragments is difficult and labor-intensive. Recently, specific staining methods have been developed and merged into the 2-D gel platform so that not only general protein patterns but also patterns of phosphorylated and glycosylated proteins can be obtained. We used the new Pro-Q Diamond phosphoprotein dye technology for the fluorescent detection of phosphoproteins directly in 2-D gels of mouse leukocyte proteins, and Pro-Q Emerald 488 glycoprotein dye to detect glycoproteins. These two fluorescent stains are compatible with general protein stains, such as SYPRO Ruby stain. We devised a sequential procedure using Pro-Q Diamond (phosphoprotein), followed by Pro-Q Emerald 488 (glycoprotein), followed by SYPRO Ruby stain (general protein stain), and finally silver stain for total protein profile. This multiple staining of the proteins in a single gel provided parallel determination of protein expression and preliminary characterization of post-translational modifications of proteins in individual spots on 2-D gels. Although this method does not provide the same degree of certainty as traditional MS methods of characterizing post-translational modifications, it is much simpler, faster, and does not require sophisticated equipment and expertise in MS.     

Iterative data analysis is the key for exhaustive analysis of peptide mass fingerprints from proteins separated by two-dimensional electrophoresis

Peptide mass fingerprinting (PMF) is a powerful tool for identification of proteins separated by two-dimensional electrophoresis (2-DE). With the increase in sensitivity of peptide mass determination it becomes obvious that even spots looking well separated on a 2-DE gel may consist of several proteins. As a result the number of mass peaks in PMFs increased dramatically leaving many unassigned after a first database search. A number of these are caused by experiment-specific contaminants or by neighbor spots, as well as by additional proteins or post-translational modifications. To understand the complete protein composition of a spot we suggest an iterative procedure based on large numbers of PMFs, exemplified by PMFs of 480 Helicobacter pylori protein spots. Three key iterations were applied: (1) Elimination of contaminant mass peaks determined by MS-Screener (a software developed for this purpose) followed by reanalysis; (2) neighbor spot mass peak determination by cluster analysis, elimination from the peak list and repeated search; (3) re-evaluation of contaminant peaks. The quality of the identification was improved and spots previously unidentified were assigned to proteins. Eight additional spots were identified with this procedure, increasing the total number of identified spots to 455.     

Quantitative evaluation of proteins in one- and two-dimensional polyacrylamide gels using a fluorescent stain

The characteristics of protein detection and quantitation with SYPRO Ruby protein gel stain in one- and two-dimensional polyacrylamide gels were evaluated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of three different purified recombinant proteins showed that the limits of detection were comparable to the limits of detection with ammoniacal silver staining and were protein-specific, ranging from 0.5 to 5 ng. The linearity of the relationship between protein level and SYPRO Ruby staining intensity also depended on the individual protein, with observed linear dynamic ranges of 200-, 500-, and, 1000-fold for proteins analyzed by SDS-PAGE. SYPRO Ruby protein gel stain was also evaluated in two-dimensional electrophoretic (2-DE) analysis of Escherichia coli proteins. The experiment involved analysis of replicates of the same sample as well as dilution of the sample from 0.5 to 50 nug total protein across gels. In addition to validating the 2-DE system itself, the experiment was used to evaluate three different image analysis programs: Z3 (Compugen), Progenesis (Nonlinear Dynamics), and PDQuest (Bio-Rad). In each program, we analyzed the 2-DE images with respect to sensitivity and reproducibility of overall protein spot detection, as well as linearity of response for 20 representative proteins of different molecular weights and pI. Across all three programs, coefficients of variation (CV) in total number of spots detected among replicate gels ranged from 4 to 11%. For the 20 representative proteins, spot quantitation was also comparable with CVs for gel-to-gel reproducibility ranging from 3 to 33%. Using Progenesis and PDQuest, a 1000-fold linear dynamic range of SYPRO Ruby was demonstrated with a single known protein. These two programs were more suitable than Z3 for examining individual protein spot quantity across a series of gels and gave comparable results.     

Proteome analysis of activated murine B-lymphocytes

Proteins extracted from murine B-lymphocytes after in vitro stimulation by lipopolysaccharide were separated by two-dimensional (2-D) polyacrylamide gel electrophoresis and analyzed by matrix assisted laser desorption/ionization mass spectrometry. Structural information on the protein entities from 153 spots was obtained. Since many of these spots occur as members of spot families, a smaller number --98 genes-- was found to be coding for the identified spots. The elucidated proteins belong to groups of functional categories; we found 26 enzymes, 36 regulatory proteins, 15 chaperones, 15 structural proteins, 4 immunoglobulins, 1 ribosomal and 1 histone protein. A comparison between expected and observed molecular masses yields a good correlation for the majority of the compared spot entities. This set of proteins now identified in the context of a lymphocyte 2-D gel pattern should advance further studies on lymphocyte functions.     

Capillary column high-performance liquid chromatographic-electrospray ionization triple-stage quadrupole mass spectrometric analysis of proteins separated by two-dimensional polyacrylamide gel electrophoresis application to cerebellar protein mapping

A method is presented for the structural characterization of proteins separated by two-dimensional poly-acrylamide gel electrophoresis (2D-PAGE). The method includes separation of a protein mixture by 2D-PAGE, recovery of proteins from the gel spots revealed by copper staining and analysis of the proteins by triple-stage quadrupole mass spectrometry using an electrospray ionization interface (ESI-TSQMS). Prior to the mass spectrometric analysis, the extracted proteins were passed through a small reversed-phase column (10 × 4.0 mm I.D.) to remove salts and gel-derived contaminants and then introduced into the mass spectrometer through a reversed-phase capillary column with 0.25 mm I.D. Application of the method to the analysis of rat cerebellar proteins suggests that the molecular mass could be accurately determined with sub-picomole amounts of protein samples derived from one or two 2D gels. The method was also useful for peptide mapping and determination of amino acid sequences of proteins micro-prepared from the 2D gel. Because 2D-PAGE has an excellent resolving power in protein separation and because capillary LC-ESI-TSQMS provides structural information with very small amounts of samples, the combined system of 2D-PAGE and capillary LC-ESI-TSQMS described here should allow wide applications to molecular studies of genes and proteins, such as identifications of protein spots on 2D gels, confirmation of gene/protein sequences and analysis of post-translational modification of proteins present naturally in tissue/cell extracts or expressed by recombinant DNA techniques.     

Identification of proteins in human cerebrospinal fluid using liquid-phase isoelectric focusing as a prefractionation step followed by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionisation mass spectrometry

Cerebrospinal fluid (CSF) is in close proximity to the brain and changes in the protein composition of CSF may be indicative of altered brain protein expression in neurodegenerative disorders. Analysis of brain-specific proteins in CSF is complicated by the fact that most CSF proteins are derived from the plasma and tend to obscure less abundant proteins. By adopting a prefractionation step prior to two-dimensional gel electrophoresis (2-DE), less abundant proteins are enriched and can be detected in complex proteomes such as CSF. We have developed a method in which liquid-phase isoelectric focusing (IEF) is used to prefractionate individual CSF samples; selected IEF fractions are then analysed on SYPRO-Ruby-stained 2-D gels, with final protein identification by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS). To optimise the focusing of the protein spots on the 2-D gel, the ampholyte concentration in liquid-phase IEF was minimised and the focusing time in the first dimension was increased. When comparing 2-D gels from individual prefractionated and unfractionated CSF samples it is evident that individual protein spots are larger and contain more protein after prefractionation of CSF. Generally, more protein spots were also detected in the 2-D gels from prefractionated CSF compared with direct 2-DE separations of CSF. Several proteins, including cystatin C, IgM-kappa, hemopexin, acetyl-coenzyme A carboxylase-alpha, and alpha-1-acid glycoprotein, were identified in prefractionated CSF but not in unfractionated CSF. Low abundant forms of posttranslationally modified proteins, e.g. alpha-1-acid glycoprotein and alpha-2-HS glycoprotein, can be enriched, thus better resolved and detected on the 2-D gel. Liquid-phase IEF, as a prefractionation step prior to 2-DE, reduce sample complexity, facilitate detection of less abundant protein components, increases the protein loads and the protein amount in each gel spot for MALDI-MS analysis.     

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.     

Deamidation as a widespread phenomenon in two-dimensional polyacrylamide gel electrophoresis of human blood plasma proteins

The human plasma protein patterns obtained by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is a good model system for post-translational modifications because of the existence of several "ladders" of protein spots [Anderson, N. L., Anderson, N. G., Electrophoresis 1991, 12, 883-906], so-called "trains" of spots. Our investigation of several proteins, amongst others beta2-microglobulin and the haptoglobin chains, found the differences in isoelectric points (p/) to be due to deamidation of asparagines. After enzymatic cleavage with endopeptidases in the 2-D polyacrylamide gel, the asparagine and deamidated asparagine containing peptides were separated and quantified by reversed-phase HPLC. In order to separate these peptides, a neutral pH system was established and, as a result, the differences in hydrophobicity of asparagine-containing and deamidated asparagine-containing peptides increased. But how do deamidated asparagines contribute to the observed spot pattern? One spot in the 2-D gel consists of a mixture of protein species with the same number of deamidated asparagines but on different sequence position sites. The difference between the spots in the "ladder" is a growing number of negative charges introduced in the protein by an increasing number of deamidated asparagines. As a consequence, the mass difference between two spots is exactly 1 Da, which is shown in this paper for intact protein masses and the corresponding deamidated peptides.     

Two-dimensional electrophoresis aided by personal computer analysis for screening of mutant proteins in inherited diseases

A rapid and reproducible method of two-dimensional electrophoresis was developed for screening of abnormal proteins expressed in fibroblasts from patients with inherited diseases. After silver staining, the electrophoresis gel was subjected to semiautomatic digitizer-personal computer analysis: scanning with an image sensor video camera connected to a digitizer, followed by quantitative determination and statistical analysis with a personal computer. The protein spots analyzed by this method showed quantitative variations of various degrees, particularly in 2 of 247 spots examined. Seven spots were not always detected in control and pathological cells in this study. Slight variations in molecular weight were observed in 3 different spots.     

Novel procedure for the identification of proteins by mass fingerprinting combining two-dimensional electrophoresis with fluorescent SYPRO red staining

The fluorescent sensitive SYPRO Red dye was successfully employed to stain proteins in two-dimensional gels for protein identification by peptide mass fingerprinting. Proteins which are not chemically modified during the SYPRO Red staining process are well digested enzymatically in the gel and hence the resulting peptides can be efficiently eluted and analysed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). A SYPRO Red two-dimensional gel of a complex protein extract from Candida albicans was analysed by MALDI-TOF MS. The validity of SYPRO Red staining was demonstrated by identifying, via peptide mass fingerprinting, 10 different C. albicans proteins from a total of 31 selected protein spots. The peptide mass signal intensity, the number of matched peptides and the percentage of coverage of protein sequences from SYPRO Red-stained proteins were similar to or greater than those obtained in parallel with the modified silver protein gel staining. This work demonstrates that fluorescent SYPRO Red staining is compatible with the identification of proteins separated on polyacrylamide gel and that it can be used as an alternative to silver staining. As far as we know, this is the first report in which C. albicans proteins separated using 2-D gels have been identified by peptide mass fingerprinting. The improved technique described here should be very useful for carrying out proteomic studies.     

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.     

Rat liver cytosolic protein changes after ethanol exposure studied by two-dimensional electrophoresis

Rats were fed liquid food containing ethanol in concentrations ranging from 1-5% for 13 weeks. Livers were removed for histopathology and the liver cytosolic protein fraction was prepared and used for two-dimensional gel electrophoresis (2D-PAGE). Polypeptides were visualized by silver staining. Scanning was made for estimation of the relative abundance of protein in each polypeptide spot in the gels and for comparison between rats. Visual inspection and scanning of gels with the stained polypeptide spots obtained after equilibrium isoelectric focusing and non-equilibrium pH gradient electrophoresis revealed that: 1) within the control rat and ethanol-treated rat livers the numbers of polypeptide spots detected using isoelectric focusing in the first dimension were approximately 500 and for non-equilibrium pH gradient electrophoresis 400; 2) in the control group the variation in the estimated amount of protein in each spot was remarkably small; 3) pronounced differences in the relative abundance of protein in several of the spots was observed in the ethanol-exposed rats as compared to controls. Dose-response relations and possible causes for the effects of ethanol are discussed.     

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。     

Two-dimensional electrophoresis analysis of proteomics based on image feature and mathematical morphology

Protein analysis techniques, including 2-D electro- phoresis, image analysis, biological mass spectrometry, database search, etc., are fundamental technologies of proteomics, a front area in biochemistry and life sci- ences[1―3]. Among them image analysi…     

Protein profiling of rat cerebella during development

Protein profiles of developing rat cerebella were analyzed by means of two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). The analysis of adult rat cerebellum gave rise to a protein map comprising approximately 3000 spots detectable by silver staining following high resolution 2-DE with a pH range of 3-10 and a mass range of 8-100 kDa. To obtain landmarks for comparison of developmental profiles of cerebellar proteins, 100 spots were subjected to peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and 67 spots were assigned on the map. Analysis of profiles of the developing cerebella revealed significant changes in the expression of proteins during development. In most cases the expression levels of proteins increased as the cerebellum matured, while the expression of 42 spots appeared specific or remarkably abundant in the immature cerebellum. Peptide mass fingerprinting of these spots allowed us to identify 29 proteins, which include, in addition to proteins of unknown function, many proteins known to have roles in the development of the central nervous system. These results suggest that the proteomic approach is valuable for mass identification of proteins involved in cerebellar morphogenesis.     

Analytical approach to the metallomic of Nile tilapia (Oreochromis niloticus) liver tissue by SRXRF and FAAS after 2D-PAGE separation: Preliminary results

An investigation was made into calcium, iron and zinc in protein spots in samples of Nile tilapia (Oreochromis niloticus) liver tissue obtained after protein separation by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and subsequent qualitative and quantitative evaluation by synchrotron radiation X-ray fluorescence (SRXRF) and Flame Atomic Absorption Spectrometry (FAAS). An analysis of the fluorescence spectra indicated the presence of calcium, iron and zinc in 12, 6 and 8 liver protein spots, respectively. The metal ions found were distributed mainly in proteins with a molar mass of less than 40.00 kDa and more than 12.00 kDa, with pI in the range of 4.70-9.40. The only exception was a spot presenting protein with a molar mass of 10.10 kDa. In addition to calcium, iron and zinc, sulfur and phosphorus - which are non-metals that may be part of the protein structure, were also detected. After microwave-assisted acid mineralization of the proteins spots, a FAAS estimation of the concentration of calcium, iron and zinc ions bound to these proteins indicated a range of 1.08-5.80 mg g⁻¹, 2.02-8.03 mg g⁻¹ and 1.60-8.55 mg g⁻¹, respectively.     

The scaled volume as an image analysis variable for detecting changes in protein expression levels by silver stain.

A new variable for measuring the relative intensities of silver stained protein spots on two-dimensional gels is described. The scaled volume (SV) more accurately measures the intensity of protein spots and accounts for differences frequently encountered when trying to compare two gels than other variables such as relative volume ratio, optical density, or relative optical density. The SV scales the signal of interest by the noise (gel background) with secondary signals removed (spots not of interest, e.g., technical artifacts). The SV of spot intensities offers a better dynamic response to protein amount for the model proteins studied here. Depending on the quantity of protein loaded onto gels, we have observed a coefficient of variation range of 0.2 to 1.3. We also observe that the SV silver stain response follows a characteristic exponential profile for different proteins.     

应用蛋白质组学方法分析猪晶状体不同区域上皮细胞中蛋白质的表达差异

应用蛋白质组学方法分析比较猪晶状体中央和周边上皮细胞的蛋白质表达差异。将32个正常猪晶状体前囊膜所附着于的上皮细胞分为中央和周边两部分，经二维凝胶电泳分离和凝胶考马斯亮兰染色，质谱（MALDI—TOF—MS）鉴定差异蛋白质斑点，并对鉴定的蛋白质进行分类。结果显示来自中央与周边区域的猪晶体上皮细胞的蛋白在二维凝胶上分别有801和886个蛋白质斑点，鉴定出差异表达蛋白84个：差异表达的蛋白质在功能上有一定趋向性，主要涉及代谢、细胞骨架、信号转导／细胞周期／转录因子等。     

Esophageal carcinoma-associated proteins detected by two-dimensional polyacrylamide gel electrophoresis

Patterns of proteins of five surgically resected esophageal carcinomas were studied by two-dimensional polyacrylamide gel electrophoresis with silver staining. The samples of normal esophageal mucosa and esophageal carcinoma from the same patient were compared. Each gel had ca. 300 protein spots and had a similar pattern of proteins. Four spots were observed in all of the esophageal carcinomas that were not present in any of the normal mucosae. The molecular weights and isoelectric points were 46,000 and 5.3, 46,000 and 5.2, 36,000 and 4.7 and 33,000 and 5.1, respectively. One spot was observed in all of the normal mucosae but not in any of the esophageal carcinomas. Its molecular weight and isoelectric point were 27,000 and 5.3, respectively.     

Metabolically stable isotope labeling prior to electrophoretic protein separation reveals differences in fractional synthesis rates between mitochondrial aldehyde dehydrogenase isoforms

Living mice were subjected to whole body labeling by intravenous infusion of [(13)C]glucose as the sole carbon source. After 10 h infusion the mice were sacrificed, and liver proteins were separated by two-dimensional polyacrylamide gel electrophoresis. Five spots were found to contain mitochondrial aldehyde dehydrogenase (ALDH2) by matrix assisted time of flight mass spectrometry protein identification. By measuring the isotopologue mass distributions of peptide ions, and modeling the (13)C content of the precursor amino acid pool, the fractional synthesis rate of ALDH2 molecules synthesized during the labeling period was determined. One of the five spots was observed to have a five-fold higher fraction of (13)C-containing newly synthesized ALDH2 than the spot with the highest ALDH2 content, and contained more than 60% of newly synthesized ALDH2 although it accounted for less than 20% of the total ALDH2 detected. The total range in the fraction of (13)C-containing proteins between different ALDH2 spots approached 50-fold. The ability to quantitatively characterize different protein isoforms of biological origin for ALDH2 and other proteins from living animals provides new avenues for the exploration of protein function.