Immobilized pH Gradient Strips Research Articles

Two dimensional gel electrophoresis using narrow pH 3-5.6 immobilised pH gradient strips identifies potential novel disease biomarkers in plasma or serum

Two dimensional gel electrophoresis using narrow pH 3-5.6 immobilised pH gradient strips identifies potential novel disease biomarkers in plasma or serum

Identification and Characterization of an Oocyte Factor Required for Porcine Nuclear Reprogramming

Nuclear reprogramming of somatic cells can be induced by oocyte factors. Despite numerous attempts, the factors responsible for successful nuclear reprogramming remain elusive. In the present study, we found that porcine oocytes with the first polar body collected at 42 h of in vitro maturation had a stronger ability to support early development of cloned embryos than porcine oocytes with the first polar body collected at 33 h of in vitro maturation. To explore the key reprogramming factors responsible for the difference, we compared proteome signatures of the two groups of oocytes. 18 differentially expressed proteins between these two groups of oocytes were discovered by mass spectrometry (MS). Among these proteins, we especially focused on vimentin (VIM). A certain amount of VIM protein was stored in oocytes and accumulated during oocyte maturation, and maternal VIM was specifically incorporated into transferred somatic nuclei during nuclear reprogramming. When maternal VIM function was inhibited by anti-VIM antibody, the rate of cloned embryos developing to blastocysts was significantly lower than that of IgG antibody-injected embryos and non-injected embryos (12.24 versus 22.57 and 21.10%; p < 0.05), but the development of in vitro fertilization and parthenogenetic activation embryos was not affected. Furthermore, we found that DNA double strand breaks dramatically increased and that the p53 pathway was activated in cloned embryos when VIM function was inhibited. This study demonstrates that maternal VIM, as a genomic protector, is crucial for nuclear reprogramming in porcine cloned embryos.

IPG strip-based peptide fractionation for shotgun proteomics.

Efficient fractionation of peptides is an essential prerequisite for comprehensive analysis of complex protein mixtures by shotgun mass spectrometry. The separation of peptides by isoelectric focusing is particularly attractive due to its orthogonality to reverse-phase HPLC. Here, we present a protocol for in-gel peptide isoelectric focusing using immobilized pH gradient strips. The method shows high resolving power for up to 1 mg of sample and is highly reproducible.

Soybean Proteomics for Unraveling Abiotic Stress Response Mechanism

Plant response to abiotic stresses depends upon the fast activation of molecular cascades involving stress perception, signal transduction, changes in gene and protein expression and post-translational modification of stress-induced proteins. Legumes are extremely sensitive to flooding, drought, salinity and heavy metal stresses, and soybean is not an exception of that. Invention of immobilized pH gradient strips followed by advancement in mass spectrometry has made proteomics a fast, sensitive and reliable technique for separation, identification and characterization of stress-induced proteins. As the functional translated portion of the genome plays an essential role in plant stress response, proteomic studies provide us a finer picture of protein networks and metabolic pathways primarily involved in stress tolerance mechanism. Identifying master regulator proteins that play key roles in the abiotic stress response pathway is fundamental in providing opportunities for developing genetically engineered stress-tolerant crop plants. This review highlights recent contributions in the field of soybean biology to comprehend the complex mechanism of abiotic stress acclimation. Furthermore, strengths and weaknesses of different proteomic methodologies of extracting complete proteome and challenges and future prospects of soybean proteome study both at organ and whole plant levels are discussed in detail to get new insights into the plant abiotic stress response mechanism.

Comparative study of six methods of protein extraction for two-dimensional gel electrophoresis of proteomic profiling in poplar stems

Azri, W., Ennajah, A. and Jing, M. 2013. Comparative study of six methods of protein extraction for two-dimensional gel electrophoresis of proteomic profiling in poplar stems. Can. J. Plant Sci. 93: 895-901. Protein extraction is a crucial step in two-dimensional gel electrophoresis (2-DE) analysis of proteins, since it can have significant impact on both the quantity and the quality of protein detection. The present study is a comparison between six previously published protocols of protein extraction (A, B, C, D, E, and F) aiming to determine a suitable method to extract total proteins from poplar stems, a recalcitrant plant tissue. The obtained results revealed that method F (optimized method B), combining detergents (CHAPS, Triton X-100, and low sodium dodecyl sulfate amounts) and chaotropes (thiourea and urea), gave the best solution for the problem of protein solubilization. Method F enabled the detection of more than 300 spots reproducible on the 2-DE gel with pH 4-7 immobilized pH gradient strips ...

Comparative study of six methods of protein extraction for two-dimensional gel electrophoresis of proteomic profiling in poplar stems

Azri, W., Ennajah, A. and Jing, M. 2013. Comparative study of six methods of protein extraction for two-dimensional gel electrophoresis of proteomic profiling in poplar stems. Can. J. Plant Sci. 93: 895–901. Protein extraction is a crucial step in two-dimensional gel electrophoresis (2-DE) analysis of proteins, since it can have significant impact on both the quantity and the quality of protein detection. The present study is a comparison between six previously published protocols of protein extraction (A, B, C, D, E, and F) aiming to determine a suitable method to extract total proteins from poplar stems, a recalcitrant plant tissue. The obtained results revealed that method F (optimized method B), combining detergents (CHAPS, Triton X-100, and low sodium dodecyl sulfate amounts) and chaotropes (thiourea and urea), gave the best solution for the problem of protein solubilization. Method F enabled the detection of more than 300 spots reproducible on the 2-DE gel with pH 4–7 immobilized pH gradient strips and 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, using silver staining. Our results suggest that Method F is expected to have excellent applications in proteomic studies of poplar stems.

Developing a reproducible method for the high-resolution separation of peritoneal dialysate proteins on 2-D gels

PurposeDespite recent progress in the proteomic analysis of peritoneal dialysate effluent (PDE), there remains unresolved problems in the development of an optimal sample preparation method. Experimental designWe examined five protocols for concentrating PDE proteins and the effects of immobilized pH gradient (IPG) strips with different pH ranges and sample loading techniques. In addition, we examined three kits for depleting high abundance proteins by SDS–PAGE and two-dimensional gel electrophoresis (2-DE). ResultsPDE proteins precipitated with 75% acetonitrile (ACN) showed the greatest number of protein spots by 2-DE, with over 800 distinct spots. Higher-resolution images were obtained using IPG strips with a pH range of 4–7. The ProteoPrep immunoaffinity albumin and IgG depletion kit removed high abundance proteins with higher efficiency and more compatibility with isoelectric focusing (IEF). Removing high abundance proteins also increased the resolution and improved the intensity of low abundance proteins. Conclusion and clinical relevanceHigh-resolution 2-DE images of PDE proteins were obtained by concentrating samples with 75% ACN, using pH 4–7 IPG strips, and depleting high abundance proteins. This optimized method will enable future studies to discover predictive biomarkers of disease in patients on dialysis.

A Novel Prefractionation Method Combining Protein and Peptide Isoelectric Focusing in Immobilized pH Gradient Strips

To increase sensitivity and analytical depth in shotgun proteomics, prefractionation of complex samples is often used. Here we describe a novel prefractionation method, Sandwich high resolution isoelectric focusing, which combines both protein and peptide isoelectric focusing. In the first step, intact proteins are separated on the basis of isoelectric point (pI) using traditional immobilized pH gradient (IPG) strips. Segments in the IPG-strip containing proteins of interest are subsequently cut out and applied to in-strip digestion, without subsequent peptide elution. In the second peptide isoelectric focusing step, the strip segments are used as loading bridges. The peptides are thereby directly applied to the peptide isoelectric focusing, without an intermediate elution step, and separated on narrow range IPG strips to reduce the complexity on the peptide level. In the final step, the peptides are eluted into 96-well plates and analyzed with mass spectrometry. In a proof of principle experiment, using this method to zoom in on pI regions of interest in human plasma, we identify over 800 proteins, with concentrations spanning over 6 orders of magnitude.

Establishment of two-dimensional electrophoresis system of caudal gland

To establish an effective separation system of 2-DE for the proteome of caudal gland, and provide foundation for revealing the mechanisms of histological development and pharmacological activities. The total proteins of caudal gland were extracted by TCA/acetone precipitation, phenol extraction/methanol-ammonium acetate precipitation and trizol-base method respectively and separated by immobilized pH gradient (IPG) strips prior to SDS-PAGE. Loading protein sample size and isoelectric focusing conditions were optimized. The gels were stained with Coomassie brilliant blue, scanned and then analyzed using PDQuest 8.0 analysis software. The total proteins of caudal gland extracted by trizol-base method were the highest quality and could meet the needs of 2-DE. With 300 microg of proteins loaded on 7 cm pH 3-10 IPG strip followed by isoelectric focusing program II ,a satisfying 2-DE profiles were obtained. The total number of disticted protein spots was 209 with the optimized system. A well-resolved 2-DE patterns of caudal gland were obtained by this optimized system. This method could be applied to prepare other similar tissue sample and 2-DE studies.

Proteomic analysis of the endophytic fungus Undifilum oxytropis

The filamentous Ascomycete fungus Undifilum oxytropis is an endophyte of locoweed plants of the genera Oxytropis that produces a toxic alkaloid swainsonine. Swainsonine, an alpha-mannosidase inhibitor causes a general toxicosis and neurological problems (locoism) when consumed by grazing animals. Swainsonine is also being assessed for its anti-cancer properties. While the ecology of U. oxytropis has been studied, little is known about the genetics and proteomics of any swainsonine-producing fungus. To help understand the proteins in U. oxytropis , the proteome of U. oxytropis was analyzed using 2-dimensional electrophoresis (2-DE). Proteins from U. oxytropis mycelia were extracted and separated by in-gel isoelectric focusing (IEF). The entire immobilized pH gradient (IPG) strip was cut into a set of gel sections and each gel section was digested with trypsin and then identified using liquid chromatography tandem mass spectrometry (LC-MS/MS). 2-DE maps were also developed for U. oxytropis to define its proteome. In the isoelectric point (pI) range of 3-11 and 10-250 kDa ranges, more than 450 spots were detected in 2-DE silver-stained gels, and 52 proteins were identified by LC-MS/MS. Most of the identified proteins were involved in energy production, oxidoreductase activity, carbohydrate metabolic process, amino acid and cellular ketone metabolic process. A large group of identified proteins were related to stress proteins and heat shock proteins. This work presents the first two-dimensional reference map of this alkaloid-producing fungus. Details of the proteome serve as a baseline for further study of this swainsonine-producing fungus and are essential for a reverse genetic analysis of the fungus. Keywords: Undifilum oxytropis fungus, two-dimensional gel electrophoresis, proteome reference map, liquid chromatography tandem mass spectrometry, swainsonine

Proteomic analysis of infiltrating ductal carcinoma tissues by coupled 2-D DIGE/MS/MS analysis

There is a growing interest in protein expression profiling aiming to identify novel diagnostic markers in breast cancer. Proteomic approaches such as two-dimensional differential gel electrophoresis coupled with tandem mass spectrometry analysis (2-D DIGE/MS/MS) have been used successfully for the identification of candidate biomarkers for screening, diagnosis, prognosis and monitoring of treatment response in various types of cancer. Identifying previously unknown proteins of potential clinical relevance will ultimately help in reaching effective ways to manage the disease. We analyzed breast cancer tissues from five tumor and five normal tissue samples from ten breast cancer subjects with infiltrating ductal carcinoma (IDC) by 2-D DIGE using two types of immobilized pH gradient (IPG) strips: pH 3-10 and pH 4-7. From all the spots detected, differentially expressed (p < 0.05 and ratio > 2) were 50 spots. Of these, 39 proteins were successfully identified by MS, representing 29 different proteins. Ten proteins were overexpressed in the tumor samples. The 2-D DIGE/MS/MS analysis revealed an increase in the expression levels in tumor samples of several proteins not previously associated with breast cancer, such as: macrophage-capping protein (CAPG), phosphomannomutase 2 (PMM2), ATPase ASN1, methylthioribose-1-phosphate isomerase (MRI1), peptidyl-prolyl cis-trans isomerase FKBP4, cellular retinoic acid-binding protein 2 (CRABP2), lamin B1 and keratin, type II cytoskeletal 8 (KRT8). Ingenuity Pathway Analysis (IPA) revealed highly significant (p = 10(-26)) interactions between the identified proteins and their association with cancer. These proteins are involved in many diverse pathways and have established roles in cellular metabolism. It remains the goal of future work to test the suitability of the identified proteins in samples of larger and independent patient groups.

The effect of pre-existing maternal obesity on the placental proteome: two-dimensional difference gel electrophoresis coupled with mass spectrometry

Our aim was to study the protein expression profiles of placenta obtained from lean and obese pregnant women with normal glucose tolerance at the time of term Caesarean section. We used two-dimensional difference gel electrophoresis (2D-DIGE), utilising narrow-range immobilised pH gradient strips that encompassed the broad pH range of 4-5 and 5-6, followed by MALDI-TOF mass spectrometry of selected protein spots. Western blot and quantitative RT-PCR (qRT-PCR) analyses were performed to validate representative findings from the 2D-DIGE analysis. Eight proteins were altered (six down-regulated and two up-regulated on obese placentas). Annexin A5 (ANXA5), ATP synthase subunit beta, mitochondria (ATPB), brain acid soluble protein 1 (BASP1), ferritin light chain (FTL), heterogeneous nuclear ribonucleoprotein C (HNRPC) and vimentin (VIME) were all lower in obese patients. Alpha-1-antitrypsin (A1AT) and stress-70 protein, mitochondrial (GRP75) were higher in obese patients. Western blot analysis of ANXA5, ATPB, FTL, VIME, A1AT and GRP75 confirmed the findings from the 2D-DIGE analysis. For brain acid soluble protein 1 and HNRPC, qRT-PCR analysis also confirmed the findings from the 2D-DIGE analysis. Immunohistochemical analysis was also used to determine the localisation of the proteins in human placenta. In conclusion, proteomic analysis of placenta reveals differential expression of several proteins in patients with pre-existing obesity. These proteins are implicated in a variety of cellular functions such as regulation of growth, cytoskeletal structure, oxidative stress, inflammation, coagulation and apoptosis. These disturbances may have significant implications for fetal growth and development.

Comparative Root Protein Profiles of Korean Ginseng (Panax ginseng) and Indian Ginseng (Withania somnifera)

Ginsenosides and withanolides are the secondary metabolites from Panax ginseng and Withania somnifera, respectively. These compounds have similar biological properties. Two-dimensional electrophoresis (2-DE) analysis was utilized to reveal the protein profile in the roots of both plants, with the aim of clarifying similarly- and differentially-expressed proteins. Total proteins of Korea ginseng (P. ginseng) and Indian ginseng (W. somnifera) roots were separated by 2-DE using a pH 4-7 immobilized pH gradient strip in the first dimension and 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis in the second dimension. The protein spots were visualized by silver staining. Twenty-one P. ginseng proteins and 35 W. somnifera proteins were chosen for identification by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry; of these, functions were ascribed to 14 and 22 of the P. ginseng and W. somnifera proteins, respectively. Functions mainly included general cell metabolism, defense and secondary metabolism. ATPase and alcohol dehydrogenase proteins were expressed in both plants. The results of this study, to our knowledge, are the first to provide a reference 2-DE map for the W. somnifera root proteome, and will aid in the understanding of the expression and functions of proteins in the roots of Korean ginseng and Indian ginseng.

Development of an in-house protocol for the OFFGEL fractionation of plant proteins

Protein sample prefractionation using off-gel technology coupled to mass spectrometry-based proteomics has proven very efficient in identifying novel proteins of low abundance in various biological systems with the exception of plant tissues. In this study, we have prefractionated protein samples from wheat leaves and apoplastic fluid using the Agilent 3100 OFFGEL fractionator along 3-10 pH range into 24 fractions. Two methods were compared: 1/ the Agilent method based on the use of a proprietary starter kit which comprises all the necessary consumables, and 2/ an in-house method in which focusing solutions were made using electrophoresis grade chemicals, and immobilised pH gradient (IPG) strips and electrode pads were acquired elsewhere. Method comparisons and quality of focusing were assessed using pH distributions, one- and two-dimensional patterns. Both methods produced excellent electrophoretic patterns, with generally more better-resolved bands/spots observed with the in-house method. This study will allow other laboratories using OFFGEL device to consider in-house solutions.

Optimization of Two-Dimensional Gel Electrophoresis for Kenaf Leaf Proteins

Abstract To establish a suitable and effective protocol of protein extraction for two-dimensional gel electrophoresis (2-DE) analysis in kenaf leaf tissues, three extraction methods (trichloroacetic acid/acetone, urea/thiourea, and phenol extraction methods) were applied to the extraction of kenaf leaf protein. The results were compared in regard to protein extraction efficiency, sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and 2-DE gels. Furthermore, the 2-DE system was optimized for four aspects: the pH range of IPG (immobilized pH gradient) stripes, sampling methods, sample volumes, and concentration of polyacrylamide gels. The data presented showed that the phenol extraction method is the best method to perform 2-DE analysis of kenaf leaf protein. The protein extracted from phenol extraction method reached the purity of (26.40±0.859)%, showed (25.67±1.53) protein bands in one dimension SDS-PAGE gels, and (1 374±54.44) protein spots on 2-DE gels. The research also indicates that kenaf leaf protein spots were distributed mainly within the pH range of 4-8. More clear background with a better distribution effect and many protein spots could be obtained on 2-DE gels under the conditions of active rehydration loading, 24 cm IPG strips (linear pH gradient of 4-7), 1.4 mg samples, and 12% SDS-PAGE gels.

Evaluation of Protocols Used in 2-D Electrophoresis for Proteome Analysis of Young Rice Caryopsis

In order to obtain a high-resolution electrophorogram of rice young panicle proteome, we evaluated various protocols commonly used in two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE) of proteins, including gel staining protocol, pH range of immobilized pH gradient (IPG) strips and sample loading quantity. Results showed that a silver staining protocol using sensitized solution containing glacial acetic acid, sodium acetate and sodium thiosulfate (reported by Heukeshoven and Dernick in 1988) and a Coomassie Brilliant Blue staining method using solution containing G-250, ammonium sulfate and phosphoric acid (reported by Pink et al. in 2010) demonstrated the superior staining effect. In addition, we also showed that higher resolution was achieved when IPG gel strip with pH range of 5-8 was used, compared to that with pH range of 4-7. Finally, the optimal loading quantity was determined as 130µg using the 17cm-long nonlinear IPG strip with pH 5-8 in combination with the silver nitrate staining protocol. The evaluated results would be helpful in proteome analysis of young rice caryopsis.

Proteome Profile of ZebrafishDanio rerioOlfactory Bulb Based on Two-Dimensional Gel Electrophoresis Matrix-Assisted Laser Desorption/Ionization MS/MS Analysis

Zebrafish (Danio rerio), is one of the most extensively studied chordate model animal in the field of neuroscience and development biology. Understanding the proteome profile of various tissues and organs of Danio rerio has become increasingly important for inching it as an alternate translational model animal. This study aimed in understanding the proteome profile of D. rerio brain's olfactory bulb exclusively. The proteome pattern of olfactory bulb of D. rerio was determined using the two-dimensional gel electrophoresis approach involving both full range and three various narrow pH range immobilized pH gradient strips, followed by mass spectrometry and tandem mass spectrometry analysis. 221 various proteins were characterized based on matrix-assisted laser desorption/ionization mass spectrometry and tandem mass spectrometry analysis as olfactory bulb-specific proteins. The identified proteins represented various pI, mass, functions, and localization. The different proteins acknowledged in this study were found to be associated with diverse metabolic pathways, enzymatic activities, and neurological functions. Glycolysis, gluconeogenesis, and oxidative phosphorylation are the major metabolic pathways found associated with the identified proteins. The identified protein catalogue was also found to be participating in various network pathways such as GABAergic neurophysiological neurotransmission, remodeling of cytoskeleton, neurofilaments, and cell adhesion. This study provides the D. rerio olfactory bulb two-dimensional gel electrophoresis proteome map and the details of 221 olfactory bulb-specific proteins.

Proteomic Analysis of Human Saliva From Lung Cancer Patients Using Two-Dimensional Difference Gel Electrophoresis and Mass Spectrometry

Lung cancer is often asymptomatic or causes only nonspecific symptoms in its early stages. Early detection represents one of the most promising approaches to reduce the growing lung cancer burden. Human saliva is an attractive diagnostic fluid because its collection is less invasive than that of tissue or blood. Profiling of proteins in saliva over the course of disease progression could reveal potential biomarkers indicative of oral or systematic diseases, which may be used extensively in future medical diagnostics. There were 72 subjects enrolled in this study for saliva sample collection according to the approved protocol. Two-dimensional difference gel electrophoresis combined with MS was the platform for salivary proteome separation, quantification, and identification from two pooled samples. Candidate proteomic biomarkers were verified and prevalidated by using immunoassay methods. There were 16 candidate protein biomarkers discovered by two-dimensional difference gel electrophoresis and MS. Three proteins were further verified in the discovery sample set, prevalidation sample set, and lung cancer cell lines. The discriminatory power of these candidate biomarkers in lung cancer patients and healthy control subjects can reach 88.5% sensitivity and 92.3% specificity with AUC = 0.90. This preliminary data report demonstrates that proteomic biomarkers are present in human saliva when people develop lung cancer. The discriminatory power of these candidate biomarkers indicate that a simple saliva test might be established for lung cancer clinical screening and detection.

Proteomic profiling of hempseed proteins from Cheungsam

Proteomic profiling of hempseed proteins from a non-drug type of industrial hemp (Cannabis sativa L.), Cheungsam, was conducted using two-dimensional gel electrophoresis and mass spectrometry. A total of 1102 protein spots were resolved on pH 3–10 immobilized pH gradient strips, and 168 unique protein spots were identified. The proteins were categorized based on function, including involvement in energy regulation (23%), metabolism (18%), stress response (16%), unclassified (12%), cytoskeleton (11%), binding function (5%), and protein synthesis (3%). These proteins might have important biological functions in hempseed, such as germination, storage, or development.

Two dimensional gel electrophoresis using narrow pH 3-5.6 immobilised pH gradient strips identifies potential novel disease biomarkers in plasma or serum

Two-dimensional gel electrophoresis (2-DE) is a protein separation technique often used to separate plasma or serum proteins in an attempt to identify novel biomarkers. This protocol describes how to run 2-DE gels using narrow pH 3-5.6 immobilised pH gradient strips to separate 2 mg of serum proteins. pH 3-6 ampholytes are used to enhance the solubility of proteins in this pH range before the serum proteins are separated in the first dimension by isoelectric point (isoelectric focusing) followed by molecular weight (SDS-PAGE). This approach using the pH 3-5.6 range differs from pH ranges more commonly used for serum or plasma biomarker discovery which span three or more pH units (e.g. pH 3-10 and 4-7), and has the advantage that the pH range lies outside the range of three highly abundant proteins and therefore improves separation and representation of low abundance features. The protocol described takes approximately 8 days.