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.     

The role of bioinformatics in two-dimensional gel electrophoresis

Over the last two decades, two-dimensional electrophoresis (2-DE) gel has established itself as the de facto approach to separating proteins from cell and tissue samples. Due to the sheer volume of data and its experimental geometric and expression uncertainties, quantitative analysis of these data with image processing and modelling has become an actively pursued research topic. The results of these analyses include accurate protein quantification, isoelectric point and relative molecular mass estimation, and the detection of differential expression between samples run on different gels. Systematic errors such as current leakage and regional expression inhomogeneities are corrected for, followed by each protein spot in the gel being segmented and modelled for quantification. To assess differential expression of protein spots in different samples run on a series of two-dimensional gels, a number of image registration techniques for correcting geometric distortion have been proposed. This paper provides a comprehensive review of the computation techniques used in the analysis of 2-DE gels, together with a discussion of current and future trends in large scale analysis. We examine the pitfalls of existing techniques and highlight some of the key areas that need to be developed in the coming years, especially those related to statistical approaches based on multiple gel runs and image mining techniques through the use of parallel processing based on cluster computing and the grid technology.     

Protein spot detection and quantification in 2-DE gel images using machine-learning methods

Two-dimensional gel electrophoresis (2-DE) is the most established protein separation method used in expression proteomics. Despite the existence of sophisticated software tools, 2-DE gel image analysis still remains a serious bottleneck. The low accuracies of commercial software packages and the extensive manual calibration that they often require for acceptable results show that we are far from achieving the goal of a fully automated and reliable, high-throughput gel processing system. We present a novel spot detection and quantification methodology which draws heavily from unsupervised machine-learning methods. Using the proposed hierarchical machine learning-based segmentation methodology reduces both the number of faint spots missed (improves sensitivity) and the number of extraneous spots introduced (improves precision). The detection and quantification performance has been thoroughly evaluated and is shown to compare favorably (higher F-measure) to a commercially available software package (PDQuest). The whole image analysis pipeline that we have developed is fully automated and can be used for high-throughput proteomics analysis since it does not require any manual intervention for recalibration every time a new 2-DE gel image is to be analyzed. Furthermore, it can be easily parallelized for high performance and also applied without any modification to prealigned group average gels.     

用于双向凝胶电泳分析的奶牛乳清蛋白样品制备方法的比较

为建立适用于双向凝胶电泳分析的奶牛乳清蛋白的制备方法,分别比较了直接裂解法、三氯乙酸-丙酮法,Trizol法和2-D clean up kit法对奶牛乳清蛋白提取效率和双向凝胶电泳图谱的影响.用2-D Quant Kit试剂盒测定蛋白浓度,分别用十二烷基磺酸钠 聚丙烯酰胺凝胶电泳和双向凝胶电泳进行奶牛乳清蛋白的分离.蛋白定量结果表明,2-D clean up kit法产率最高,直接裂解法、三氯乙酸-丙酮法次之,trizol法产率最低;十二烷基磺酸钠-聚丙烯酰胺凝胶电泳结果表明,2- D clean up kit法提取的蛋白质量最高;双向电泳图谱分析表明,2-D clean up kit法得到的蛋白图谱与另外3种方法相比,检测到的蛋白点最多,图谱背景清晰,分辨率最高.结果提示,2-D clean-up法相对最适合于双向凝胶电泳分析奶牛乳清蛋白样品的制备,尤其对一些低丰度高分子量蛋白的分离效果较为明显.     

Comparing SILAC and two-dimensional gel electrophoresis image analysis for profiling urokinase plasminogen activator signaling in ovarian cancer cells

Two-dimensional gel electrophoresis (2-DE) image analysis is conventionally used for comparative proteomics. However, there are a number of technical difficulties associated with 2-DE protein separation that limit the depth of proteome coverage, and the image analysis steps are typically labor-intensive and low-throughput. Recently, mass spectrometry-based quantitation strategies have been described as alternative differential proteome analysis techniques. In this study, we investigated changes in protein expression using an ovarian cancer cell line, OVMZ6, 24 h post-stimulation with the relatively weak agonist, urokinase-type plasminogen activator (uPA). Quantitative protein profiles were obtained by MALDI-TOF/TOF from stable isotope-labeled cells in culture (SILAC), and these results were compared to the quantitative ratios obtained using 2-DE gel image analysis. MALDI-TOF/TOF mass spectrometry showed that differential quantitation using SILAC was highly reproducible (approximately 8% coefficient of variation (CV)), and this variance was considerably lower than that achieved using automated 2-DE image analysis strategies (CV approximately 25%). Both techniques revealed subtle alterations in cellular protein expression following uPA stimulation. However, due to the lower variances associated with the SILAC technique, smaller changes in expression of uPA-inducible proteins could be found with greater certainty.     

An improved sample preparation method for analyzing mycobacterial proteins in two-dimensional gels

Two-dimensional gel electrophoresis (2-DE) is currently a widely used analytical method for resolving complex mixtures of proteins. Sample preparation has a marked influence on 2-DE pattern. To reduce impurities and to increase the low-abundance proteins, protein precipitation is often used for the preparation of samples before 2-DE. In this study, we revealed that addition of SDS prior to TCA precipitation of mycobacterial cell extract proteins increases the resolution of the 2-D gel pattern.     

Multiresolution image registration for two-dimensional gel electrophoresis

In proteomic research, two-dimensional electrophoresis (2-D) is an important tool for investigating differential patterns of qualitative and quantitative protein expression. The strength of the technique is due to its unrivalled power of being able to separate simultaneously thousands of proteins. The key to the comparison of 2-D protein profiles, however, lies in the use of a fast and robust image matching process which is essential to the subsequent quantification procedure. To satisfy the growing demand for a robust and fully automatic method of matching 2-D gel protein separation profiles, we describe in this paper a novel registration technique based on image intensity distribution rather than selected features. The method uses a multiresolution representation of the gel profiles and exploits the fact that coarse approximations to the optimal matching can be extracted efficiently from low-resolution images. This permits the removal of misalignments at different scales in a systematic manner and the strength of the new method has been confirmed by a double blind trial of 111 2-D gel pairs. The proposed method requires neither landmarks nor an a priori image alignment, and takes about five seconds for processing a typical gel pair on a standard personal computer.     

Establishment of a near-standard two-dimensional human urine proteomic map

A proteomic map for human urine on two-dimensional (2-D) gels has been developed. Initial studies demonstrated that the urine proteins prepared by conventional methods showed interference and poor reproducibility in 2-D electrophoresis (2-DE). To address this issue, urine samples were dialyzed to remove any interfering molecules. The dialysis of urine proteins and the concentration by lyophilization without fractionation significantly improved the reproducibility and resolution and likely represents the total urine proteins on a 2-D gel. In addition, removing albumin from urine using Affi-Gel Blue helped to identify the low-abundant proteins. Using the developed method, we prepared proteins from urine collected from healthy females and males. The large inter- and intra-subject variation in protein profiles on 2-D gels made it difficult to establish a normal human urine proteomic 2-D map. To resolve this problem, urinary proteins were prepared from the pooled urine collected from 20 healthy females and males, respectively. The established male and female urine proteomes separated on 2-D gels were almost identical except for some potential sex-dependent protein spots. We have annotated 113 different proteins on the 2-D gel by peptide mass fingerprinting (PMF). We propose that the established total urine proteome can be used for 2-DE analysis, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and identification of novel disease-specific biomarkers.     

Proteomic analysis of primary esophageal squamous cell carcinoma reveals downregulation of a cell adhesion protein, periplakin

Recent advances in two-dimensional electrophoresis (2-DE) such as fluorescent 2-D differential gel electrophoresis (2-D DIGE) has made it possible to detect and quantitate the critical changes involved in disease pathogenesis. We have previously identified novel proteins with altered expression in primary colorectal cancer using agarose 2-DE that has a higher loading capacity than immobilized pH gradient gel. The aim of this study is to identify novel proteins with altered expression in primary esophageal cancer using the powerful method of agarose 2-DE and agarose 2-D DIGE. Excised tissues from 12 patients of primary esophageal cancer were obtained. Proteins with altered expression between cancer and adjacent non-cancer tissues were analyzed by agarose 2-D DIGE and identified by mass spectrometry. Thirty-three proteins out of 74 spots with altered expression in tumors were identified. Among them, a 195-kDa protein, periplakin, was significantly downregulated in esophageal cancer, which was confirmed by immunoblotting. Immunohistochemistry showed that periplakin was mainly localized at cell-cell boundaries in normal epithelium and dysplastic lesions, while it disappeared from cell boundaries, shifted to cytoplasm, in early cancers and scarcely expressed in advanced cancers. These results suggest that periplakin could be a useful marker for detection of early esophageal cancer and evaluation of tumor progression.     

Identification and characterization of the Sulfolobus solfataricus P2 proteome

Via combined separation approaches, a total of 1399 proteins were identified, representing 47% of the Sulfolobus solfataricus P2 theoretical proteome. This includes 1323 proteins from the soluble fraction, 44 from the insoluble fraction and 32 from the extra-cellular or secreted fraction. We used conventional 2-dimensional gel electrophoresis (2-DE) for the soluble fraction, and shotgun proteomics for all three cell fractions (soluble, insoluble, and secreted). Two gel-based fractionation methods were explored for shotgun proteomics, namely: (i) protein separation utilizing 1-dimensional gel electrophoresis (1-DE) followed by peptide fractionation by iso-electric focusing (IEF), and (ii) protein and peptide fractionation both employing IEF. Results indicate that a 1D-IEF fractionation workflow with three replicate mass spectrometric analyses gave the best overall result for soluble protein identification. A greater than 50% increment in protein identification was achieved with three injections using LC-ESI-MS/MS. Protein and peptide fractionation efficiency; together with the filtration criteria are also discussed.     

Separation of human erythrocyte membrane associated proteins with one-dimensional and two-dimensional gel electrophoresis followed by identification with matrix-assisted laser desorption/ionization-time of flight mass spectrometry

A classical proteomic analysis was used to establish a reference map of proteins associated with healthy human erythrocyte ghosts. Following osmotic lysis and differential centrifugation, ghost proteins were separated by either one-dimensional gel electrophoresis (1-DE) or two-dimensional gel electrophoresis (2-DE). Selected protein bands or spots were excised and trypsinized before mass spectrometric analyses and data mining was performed using the SWISS-PROT and NCBI nonredundant databases. A total of 102 protein spots from a 2-D gel were successfully identified. These corresponded to 59 distinct polypeptides with the remaining 43 being isoforms. As for the 1-D gel, 44 polypeptides were identified, of which 19 were also found on the 2-D gel. Most of the 19 common polypeptides were membrane cytoskeletal proteins that are often referred to as the "band" proteins. The remaining 25 polypeptides that were found exclusively on 1-D gels were proteins with high hydrophobicity (e.g., sorbitol dehydrogenase and glucose transporter) and high molecular mass (e.g., Kell blood group glycoprotein and Janus-kinase 2). A higher number of signaling proteins was also identified on 1-D gels compared to 2-D gels. These included Ras, cAMP dependent protein kinase and TGF-beta receptor type 1 precursor.     

A reference map of a human pituitary adenoma proteome

In order to compare the proteomes from different cell types of pituitary adenomas for our long-term goal to clarify the molecular mechanisms that participate in the formation of pituitary adenoma, and to detect any tumor-related marker for an "early-stage" diagnosis, the two-dimensional gel electrophoresis (2-DE) reference map of a pituitary adenoma tissue proteome is described here. A vertical, two-dimensional (2-D) polyacrylamide gel electrophoresis system and PDQuest image analysis software have been used to provide a high level of between-gel reproducibility and to accurately array each protein expressed in a pituitary adenoma tissue. Mass spectrometry (matrix-assisted laser desorption/ionization-time of flight MALDI-TOF and liquid chromatography-electrospray ionization-quadrupole-ion trap LC-ESI-Q-IT) and protein databases were used to characterize each protein in the 2-D gel. The results demonstrate that a good reproducibility of the 2-D gel pattern was attained. The position deviation of matched spots among four 2-D gels was 1.95 +/- 0.45 mm in the isoelectric focusing direction, and 1.70 +/- 0.53 mm in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis direction. A total of ca. 1000 protein spots were separated by 2-DE, and 135 protein spots that represent 111 proteins were characterized with mass spectrometry (96 spots for MALDI-TOF, 39 spots for LC-ESI-Q-IT). The characterized proteins include pituitary hormones, cellular signals, enzymes, cellular-defense proteins, cell-structure proteins, transport proteins, etc. Those proteins were located in the cytoplasmic, cellular membrane, mitochondrial, endoplasmic reticulum, nuclear, ribonucleosome, extracellular fractions, or were secreted in plasma, etc. Those identified proteins contribute to a functional profile of the pituitary adenoma proteome. These data will be used to expand the proteome database of the human pituitary, which can be accessed in the website http://www.utmem.edu /proteomics.     

Efficient prefractionation of low-abundance proteins in human plasma and construction of a two-dimensional map

Human plasma is the most clinically valuable specimen, containing not only a dynamic concentration range of protein components, but also several groups of high-abundance proteins that seriously interfere with the detection of low-abundance potential biomarker proteins. To establish a high-throughput method for efficient depletion of high-abundance proteins and subsequent fractionation, prior to molecular analysis of proteins, we explored how coupled immunoaffinity columns, commercially available as multiple affinity removal columns (MARC) and free flow electrophoresis (FFE), could apply to the HUPO plasma proteome project. Here we report identification of proteins and construction of a human plasma 2-DE map devoid of six major abundance proteins (albumin, transferrin, IgG, IgA, haptoglobin, and antitrypsin) using MARC. The proteins were identified by PMF, matching with various internal 2-DE maps, resulting in a total of 144 nonredundant proteins that were identified from 398 spots. Tissue plasminogen activator, usually present at 10-60 ng/mL plasma, was also identified, indicative of a potentially low-abundance biomarker. Comparison of representative 2-D gel images of three ethnic groups (Caucasian, Asian-American, African-American) plasma exhibited minor differences in certain proteins between races and sample pretreatment. To establish a throughput fractionation of plasma samples by FFE, either MARC flow-through fractions or untreated samples of Korean serum were subjected to FFE. After separation of samples on FFE, an aliquot of each fraction was analyzed by 1-D gel, in which MARC separation was a prerequisite for FFE work. Thus, a working scheme of MARC --> FFE --> 1-D PAGE --> 2-D-nanoLC-MS/MS may be considered as a widely applicable standard platform technology for fractionation of complex samples like plasma.     

Comparative Proteome Analysis of Human Temporal Cortex Lobes by Two-Dimensional Electrophoresis and Identification of Selected Common Proteins

Human brain proteins were isolated from left and right temporal cortex lobes at the age of 73, 23, 84 years and separated by two-dimensional gel electrophoresis (2-DE). 2-DE was carried out with an immobilized pH gradient strip in the first dimension and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. Over 800 polypeptide spots were resolved with a silver-staining protocol by computerized 2-D gel analsis. Seven of the polypeptide spots were evidently distinguishable between human left and right temporal lobes. Four of the polypeptide spots were larger and three were smaller in human right temporal lobe. One of these three protein spots that have descendent expression in human right temporal lobe was identified as carbonyl reductase (NADPH) 1 by MALDI-TOF MS. Thirty-three common spots were identified by ESI-MS/MALDI-TOF MS/Edman sequencing and a protein database search. These identified proteins include some important enzymes and regulating proteins.     

Application of laser capture microdissection combined with two-dimensional electrophoresis for the discovery of differentially regulated proteins in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is the most lethal of all the common malignancies and markers for early detection or targets for treatment of this disease are urgently required. The disease is characterised by a strong stromal response, with cancer cells usually representing a relatively small proportion of the cells in the tumor mass. We therefore performed laser capture microdissection (LCM) to enrich for both normal and malignant pancreatic ductal epithelial cells. Proteins extracted from these cells were then separated by two-dimensional gel electrophoresis (2-DE). The limited amounts of protein in the LCM procured samples necessitated the detection of 2-DE resolved proteins by silver staining. Consequently, loading equivalent amounts of protein onto gels was essential. However, we found that conventional means of measuring total protein in the samples were not sufficiently accurate. We therefore adopted a strategy in which the samples were first separated by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis, stained with silver stain and subjected to densitometry. Evaluation of the staining intensity was then used to normalise the samples. We found that the protein profiles from undissected normal pancreas and LCM-acquired non-malignant ductal epithelial cells from the same tissue block were different, underpinning the value of LCM in our analysis. The comparisons of protein profiles from nonmalignant and malignant ductal epithelial cells revealed nine protein spots that were consistently differentially regulated. Five of these proteins showed increased expression in tumor cells while four showed diminished expression in these cells. One of the proteins displaying enhanced expression in tumor cells was identified as the calcium-binding protein, S100A6. To determine the incidence of S100A6 overexpression in pancreatic cancer, we carried out immunohistochemical analysis on sections from a pancreas cancer tissue array containing 174 duplicate normal and malignant pancreatic tissue samples, from 46 pancreas cancer patients. Normal pancreatic ductal epithelia were either devoid of detectable S100A6 or showed weak expression only. Moderately or poorly differentiated tumors, by contrast, showed a higher incidence and a higher level of S100A6 expression. These observations indicate that the combination of LCM with 2-DE provides an effective strategy to discover proteins that are differentially expressed in PDAC.     

Different techniques for urinary protein analysis of normal and lung cancer patients

Many components in urine are useful in clinical diagnosis and urinary proteins are known as important components to define many diseases such as proteinuria, kidney, bladder and urinary tract diseases. In this study, we focused on the comparison of different sample preparation methods for isolating urinary proteins prior to protein analysis of pooled healthy and lung cancer patient samples. Selective method was used for preliminary investigation of some putative urinary protein markers. Urine samples were passed first through a gel filtration column (PD-10 desalting column) to remove high salts and subsequently concentrated. Remaining interferences were removed by ultrafiltration or four precipitation methods. The analysis of urinary proteins by high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed many similarities in profiles among preparation methods and a few profiles were different between normal and lung cancer patients. In contrast, the results of two-dimensional gel electrophoresis (2-DE) showed more distinctly different protein patterns. Our finding showed that the sequential preparation of urinary proteins by gel filtration and ultrafiltration could retain most urinary proteins which demonstrated the highest protein spots on 2-D gels and able to identify preliminary urinary protein markers related to cancer. Although sequential preparation of urine samples by gel filtration and protein precipitation resulted in low amounts of proteins on 2-D gels, high Mr proteins were easily detected. Therefore, there are alternative choices for urine sample preparation for studying the urinary proteome and identifying urinary protein markers important for further preclinical diagnostic and therapeutic applications.