2D Polyacrylamide Gel Electrophoresis Research Articles

DIGE Analysis of Animal Tissues.

Two-dimensional difference gel electrophoresis (2D-DIGE) is an acrylamide gel electrophoresis-based technique for protein separation and quantification in complex mixtures. The technique addresses some of the drawbacks of conventional 2D polyacrylamide gel electrophoresis (2D-PAGE), offering improved sensitivity, more limited experimental variation, and accurate within-gel matching. 2D-DIGE is based on direct labeling of proteins with isobaric fluorescent dyes (known as CyDyes: Cy2, Cy3, and Cy5) prior to isoelectric focusing (IEF). Here, up to two samples and a reference pool (internal standard) can be mixed and loaded onto IEF for first dimension prior to SDS (sodium dodecyl sulfate)-PAGE separation in the second dimension. After the electrophoretic run, the gel is imaged at the specific excitation wavelength for each dye, in sequence, and gel scans are recorded separately. For each individual protein spot, intensities recorded at the different wavelengths are integrated and the ratio between volumes normalized to that of the internal standard. This provides an immediate appreciation of protein amount variations under the different conditions tested. In addition, proteins of interest can still be excised and identified with conventional mass spectrometric techniques and further analyzed by other biochemical methods. In this chapter, we describe application of this methodology to separation and quantitation of protein mixtures from porcine muscle exudate, collected following centrifugation of muscle specimens (centrifugal drip) for the characterization of quality parameters of importance in meat industry.

Effect of MG‐132 on myofibrillogenesis and the ubiquitination of GAPDH in quail myotubes

In the three-step myofibrillogenesis model, mature myofibrils are formed through two intermediate structures: premyofibrils and nascent myofibrils. We have recently reported that several inhibitors of the Ubiquitin Proteosome System, for example, MG-132, and DBeQ, reversibly block progression of nascent myofibrils to mature myofibrils. In this investigation, we studied the effects of MG132 and DBeQ on the expression of various myofibrillar proteins including actin, myosin light and heavy chains, tropomyosin, myomesin, and myosin binding protein-C in cultured embryonic quail myotubes by western blotting using two loading controls-α-tubulin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Surprisingly, we found that MG-132 affected the level of expression of GAPDH but DBeQ did not. Reverse transcription polymerase chain reaction (RT-PCR) and quantitative reverse transcription-PCR (qRT-PCR) showed no significant effect of MG-132 on GAPDH transcription. Two-dimensional (2D) western blot analyses with extracts of control and MG-132-treated cells using anti-ubiquitin antibody indicated that MG132-treated myotubes show a stronger emitter-coupled logic signal. However, Spot% and Spot volume calculations for all spots from both western blot film signals and matched Coomassie-stained 2D polyacrylamide gel electrophoresis showed that the intensity of staining in a spot of ~39 kDa protein is 3.5-fold lower in the gel of MG-132-treated extracts. Mass spectrometry analyses identified the ~39 kDa protein as quail GAPDH. Immunohistochemical analysis of fixed MG-132-treated myotubes with anti-GAPDH antibody showed extensive clump formation, which may be analogous to granule formation by stress response factors in MG132-treated cells. This is the first report on in vivo ubiquitination of GAPDH. This may be essential for the moonlighting (Jeffery, 1999) activity of GAPDH for tailoring stress in myotubes.

An Effective Method of Isolating Honey Proteins.

Honey is a natural sweetener composed mostly of sugars, but it contains also pollen grains, proteins, free amino acids, and minerals. The amounts and proportions of these components depend on the honey type and bee species. Despite the low content of honey protein, they are becoming a popular study object, and have recently been used as markers of the authenticity and quality of honey. Currently, the most popular methods of protein isolation from honey are dialysis against distilled water, lyophilization of dialysate, or various precipitation protocols. In this work, we propose a new method based on saturated phenol. We tested it on three popular polish honey types and we proved its compatibility with both 1D and 2D polyacrylamide gel electrophoresis (PAGE) and MS (mass spectrometry) techniques. The elaborated technique is also potentially less expensive and less time-consuming than other previously described methods, while being equally effective.

Protein Expression Profile of Twenty-Week-Old Diabetic db/db and Non-Diabetic Mice Livers: A Proteomic and Bioinformatic Analysis.

Type 2 diabetes mellitus is characterized by insulin resistance in the liver. Insulin is not only involved in carbohydrate metabolism, it also regulates protein synthesis. This work describes the expression of proteins in the liver of a diabetic mouse and identifies the metabolic pathways involved. Twenty-week-old diabetic db/db mice were hepatectomized, after which proteins were separated by 2D-Polyacrylamide Gel Electrophoresis (2D-PAGE). Spots varying in intensity were analyzed using mass spectrometry, and biological function was assigned by the Database for Annotation, Visualization and Integrated Discovery (DAVID) software. A differential expression of 26 proteins was identified; among these were arginase-1, pyruvate carboxylase, peroxiredoxin-1, regucalcin, and sorbitol dehydrogenase. Bioinformatics analysis indicated that many of these proteins are mitochondrial and participate in metabolic pathways, such as the citrate cycle, the fructose and mannose metabolism, and glycolysis or gluconeogenesis. In addition, these proteins are related to oxidation–reduction reactions and molecular function of vitamin binding and amino acid metabolism. In conclusion, the proteomic profile of the liver of diabetic mouse db/db exhibited mainly alterations in the metabolism of carbohydrates and nitrogen. These differences illustrate the heterogeneity of diabetes in its different stages and under different conditions and highlights the need to improve treatments for this disease.

Ubiquitin-Conjugating Enzyme E2 L3 is Downregulated by the Chikungunya Virus nsP2 Protease.

Chikungunya virus (CHIKV) is a mosquito transmitted alphavirus that causes chikungunya fever in humans. The CHIKV non-structural protein 2 (nsP2) is a multifunctional protein that additionally modulates the host cell to dampen the innate immune response and inhibit other cellular processes. To further investigate the interactions of nsP2 with host cells, the protease domain of CHIKV nsP2 (nsP2-pro) is transfected into Hela cells, and differential protein expression is detected by 2D polyacrylamide gel electrophoresis. A total of 21 differentially regulated (six upregulated, 15 downregulated) spots are observed, of which five are identified by mass spectrometry. The downregulation of one of the identified proteins, ubiquitin-conjugating enzyme E2 L3 (UBE2L3) is confirmed by western blotting of both nsP2-pro transfection and CHIKV natural infection, and the downregulation of UBE2L3 is additionally shown to require an enzymatically active nsP2 protease domain. Transfection of full length UBE2L3 into HEK293T/17 cells prior to CHIKV infection reduce levels of infection and E protein expression but do not alter RNA genome levels. These results suggest that UBE2L3 is a cellular target of the CHIKV nsP2 protease, and this possibly mediates the pathogenesis of chikungunya fever.

DIGE Analysis of Animal Tissues.

Two-dimensional difference gel electrophoresis (2D-DIGE) is an acrylamide gel electrophoresis-based technique for protein separation and quantification in complex mixtures. The technique addresses some of the drawbacks of conventional 2D polyacrylamide gel electrophoresis (2D-PAGE), offering improved sensitivity, more limited experimental variation and accurate within-gel matching. DIGE is based on direct labeling of proteins with isobaric fluorescent dyes (known as CyDyes: Cy2, Cy3, and Cy5) prior to isoelectric focusing (IEF). Here, up to two samples and a reference pool (internal standard) can be mixed and loaded onto IEF for first dimension prior to SDS-PAGE separation in the second dimension. After the electrophoretic run, the gel is imaged at the specific excitation wavelength for each dye, in sequence, and gel scans are recorded separately. For each individual protein spot, intensities recorded at the different wavelengths are integrated and the ratio between volumes normalized to that of the internal standard. This provides an immediate appreciation of protein amount variations under the different conditions tested. In addition, proteins of interest can still be excised and identified with conventional mass spectrometry techniques and further analyzed by other biochemical methods. In this chapter, we describe the application of this methodology to separation and quantitation of proteins mixtures from porcine muscle exudate, collected following centrifugation of muscle specimens (centrifugal drip) for the characterization of quality parameters of importance in the meat industry.

Proteomics of Culture Filtrate of Prevalent Mycobacterium tuberculosis Strains: 2D-PAGE Map and MALDI-TOF/MS Analysis

Although diverse efforts have been done to identify biomarkers for control of tuberculosis using laboratory strain Mycobacterium tuberculosis H37Rv, the disease still poses a threat to mankind. There are many emerging M. tuberculosis strains, and proteomic profiling of these strains might be important to find out potential targets for diagnosis and/or prevention of tuberculosis. We evaluated the comparative proteomic profiling of culture filtrate (CF) proteins from prevalent M. tuberculosis strains (Central Asian or Delhi type; CAS1_Del, East African-Indian; EAI-3 and Beijing family) by 2D polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization–time-of-flight mass spectrometry. As a result, we could identify 12 CF proteins (Rv0066c, Rv1310, Rv3375, Rv1415, Rv0567, Rv1886c, Rv3803c, Rv3804c, Rv2031c, Rv1038c, Rv2809, and Rv1911c), which were consistently increased in all prevalent M. tuberculosis strains, and interestingly, two CF proteins (Rv2809, Rv1911c) were identified with unknown functions. Consistent increased intensity of these proteins suggests their critical role for survival of prevalent M. tuberculosis isolates, and some of these proteins may also have potential as diagnostic and vaccine candidates for tuberculosis, which needs to be further explored by immunological analysis.

Increased AAA-TOB3 correlates with lymph node metastasis and advanced stage of lung adenocarcinoma.

This study was to investigate the differential mitochondrial protein expressions in human lung adenocarcinoma and provide preliminary data for further exploration of the carcinogenic mechanism. Total proteins of A549 and 16HBE mitochondria were extracted through 2D polyacrylamide gel electrophoresis (2-DE). The differential mitochondria proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and were further confirmed by Western blot, immunoelectron microscopy and immunohistochemistry (IHC) in A549 cells as well as lung adenocarcinoma tissues. A total of 41 differentially expressed protein spots were found in A549 mitochondria. Of them, 15 proteins were highly expressed and 26 proteins were lowly expressed in the mitochondria of A549 (by more than 1.5 times). Among the 15 more highly expressed proteins, AAA-TOB3 (by more than 3 times) was highly expressed in the mitochondria of A549 compared with the 16HBE, by LC-MS/MS identification. High electron density and clear circular colloidal gold-marked AAA-TOB3 particles were observed in the A549 cells via immunoelectron microscopy. Besides, AAA-TOB3 was confirmed to be elevated in lung adenocarcinoma by Western blot and IHC. Moreover, increased AAA-TOB3 correlated with lymph node metastasis and advanced stage of lung adenocarcinoma (p<0.05). AAA-TOB3 was highly expressed in lung adenocarcinoma, and the up-regulation of AAA-TOB3 correlated with lymph node metastasis and advanced stage of lung adenocarcinoma, which suggested that it could serve as a potential molecular marker for lung adenocarcinoma.

Analysis of differentially expressed proteins in Muscovy duck embryo fibroblasts infected with virulent and attenuated Muscovy duck reovirus by two-dimensional polyacrylamide gel electrophoresis.

Muscovy duck reovirus (MDRV) belongs to the Orthoreovirus genus of the Reoviridae family, which is a significant poultry pathogen leading to high morbidity and mortality in ducklings. However, the pathogenesis of the virus is not well understood. In the present study, two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE) combined with LC-MS-MS was used to identify differentially expressed proteins between Muscovy duck embryo fibroblasts (MDEF) infected with virulent (MV9710 strain) and attenuated (CA strain) MDRV and non-infected MDEFs. A total of 115 abundant protein spots were identified. Of these, 59 of differentially expressed proteins were detected, with functions in metabolism and utilization of carbohydrates and nucleotides, anti-stress, and regulation of immune and cellular process. GO analysis of the identified proteins showed that they belonged to the classes molecular function (141 proteins), cellular component (62 proteins), and biological process (146 proteins). The results were validated by qRT-PCR, which suggests that the analysis method of 2D PAGE combined with LC-MS-MS used in this study is reliable. This study lays a foundation for further investigation of the biology of MDRV infection in MDEF.

Identification of pathogenic factors potentially involved in Staphylococcus aureus keratitis using proteomics

Staphylococcus is a leading cause of microbial keratitis, characterized by destruction of the cornea by bacterial exoproteins and host-associated factors. The aim of this study was to compare extracellular and cell-associated proteins produced by two different isolates of S. aureus, a virulent clinical isolate (Staph 38) and a laboratory strain (Staphylococcus aureus 8325-4) of weaker virulence in the mouse keratitis model. Proteins were analyzed using 2D polyacrylamide gel electrophoresis and identified by subsequent mass spectrometry. Activity of staphylococcal adhesins was assessed by allowing strains to bind to various proteins adsorbed onto polymethylmethacrylate squares. Thirteen proteins in the extracellular fraction and eight proteins in the cell-associated fractions after bacterial growth were produced in increased amounts in the clinical isolate Staph 38. Four of these proteins were S. aureus virulence factor adhesins, fibronectin binding protein A, staphopain, glyceraldehyde-3-phosphate dehydrogenase 2 and extracellular adherence protein. The clinical isolate Staph 38 adhered to a greater extent to all mammalian proteins tested, indicating the potential of the adhesins to be active on its surface. Other proteins with increased expression in Staph 38 included potential moonlighting proteins and proteins involved in transcription or translation. This is the first demonstration of the proteome of S. aureus isolates from keratitis. These results indicate that the virulent clinical isolate produces more potentially important virulence factors compared to the less virulent laboratory strain and these may be associated with the ability of a S. aureus strain to cause more severe keratitis.

Purification of nasulysin-1: A new toxin from Porthidium nasutum snake venom that specifically induces apoptosis in leukemia cell model through caspase-3 and apoptosis-inducing factor activation

Nasulysin-1, a new zinc-metalloproteinase from the snake venom of the hognose pit viper Porthidium nasutum, was purified to homogeneity using molecular exclusion chromatography and high performance liquid chromatography on a reverse phase column. The molecular mass of the purified enzyme was 25,900 kDa and pI 4.1, as determined by 1D and 2D polyacrylamide gel electrophoresis. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis of the N-terminal amino acid sequence (1FSPRYIELVVVADHGMFKKYNSNLNTIR28; 1TASLANLEVWSK12; 1DLLPR6) of the purified nasulysin-1, shows close structural homology with other snake venom metalloproteinases isolated from different snake venoms. The purified nasulysin-1 showed specific apoptosis-inducing activity in Jurkat and K562 cells, a T-cell acute lymphocytic leukemia (ALL) and chronic myeloid leukemia (AML) cell model, respectively, without affecting the viability of human lymphocyte cells. After 48 h treatment, nasulysin-1 (20 μg/mL) induced loss of the mitochondrial membrane potential (ΔΨm), activated the apoptosis-inducing factor (AIF), activated the protease caspase-3, and induced chromatin condensation and DNA fragmentation, all hallmarks of apoptosis. These results strongly suggest that nasulysin-1 selectively induces apoptosis to eliminate leukemia cells. Thus, these data warrant further investigation into the use of the metalloproteinase protein, nasulysin-1 as a potential therapeutic agent for treating leukemia.

Changes in proteome profiles of rat liver microsomes induced by silicon dioxide nanoparticles

The effect of daily intragastric administration of an aqueous dispersion of silicon nanoparticles (NPs) (the dose range from 1.0 mg/kg to 100 mg/kg body weight for 28 days) to rats on the proteomic profile of liver microsomes has been investigated by 2D-electrophoresis followed by subsequent mass spectrometry identification. The liver microsomal fraction was isolated by differential centrifugation and its protein composition was analyzed by 2D-polyacrylamide gel electrophoresis. Identification of protein spots was carried out using MALDI-TOF mass spectrometric analysis. The mass spectrometry analysis revealed the protein GRP78 (78 kD glucose-regulated protein precursor), belonging to the family of heat shock proteins. This protein present in animals of the control group was not detected in NP-treated rats of group 2 (1 mg/kg body weight/day) and group 3 (10 mg/kg body weight/day). This protein predominantly localized in the liver cell endoplasmic reticulum and plasma membrane has the chaperone biological activity. Possible mechanisms of the effects of engineered nanoparticles on biosynthetic processes in the body are discussed.

Ascorbic acid at pre-anthesis modulate the thermotolerance level of wheat (Triticum aestivum) pollen under heat stress

The reproductive stage of wheat is highly sensitive to the heat stress, especially pollen viability. Here, we report the alterations in the expression of heat shock proteins and antioxidant enzymes in wheat pollen of thermotolerant (C-306) and susceptible (HD2329) cultivars under the heat shock (42 °C, 2 h) with or without ascorbic acid (400 mM) treatment. A significant fold change in expression of catalase (CAT) and ascorbate peroxidase (APX) was observed in the pollens of HD2329 in response to 400 mM ascorbic acid (AsA) before heat stress (T1). The SOD transcript was observed significantly high in C-306 pollens compared to HD2329 in response to T1. The transcripts of high and low molecular weight HSPs (HSP70, HSP90, HSP17 and HSP26) were very high in pollens of C-306 as compared to HD2329. Semi-quantitative RT-PCR showed marked variations in the transcript of HSPs and antioxidant enzymes in pollens of C-306 and HD2329 under T0 and T1 treatments. 1D and 2D polyacrylamide gel electrophoresis of pollens showed more differentially expressed proteins in C-306 than in HD2329 in response to T1. A significant increase in the accumulation of osmolyte (proline), H2O2 and endogenous AsA levels were observed in the pollens of C-306 than in HD2329, in response to T0 and T1 treatments. Immunoblot analysis using monoclonal antibodies of HSP70 and HSP90 showed abundance of HSP70 proteins in the pollens of C-306 and HSP90 proteins in pollens of HD2329. Scanning electron microscopy of pollen under the heat stress showed disintegrated and dehydrated exine layer and alteration in pollen structure from spheroid to ovoid in both the cultivars. The pollen viability was found to be less in HD2329 than in C-306 in response to T1 treatment. A significant increase in the total antioxidant capacity was observed in pollens of C-306 as compared to HD2329 in response to the treatments. Findings indicate that pre-anthesis treatment of 400 mM AsA before HS (42 °C, 2 h) enhances the thermotolerance capacity of wheat pollens, as assessed through biochemical markers; further investigations on similar elicitors may yield useful information on mitigating the effect of the terminal heat on reproductive system of wheat.

Changes in the serum proteome of canine lymphoma identified by electrophoresis and mass spectrometry

The serum proteome of canine lymphoma was characterised by one dimensional (1D) serum protein electrophoresis (SPE) on agarose gels, two dimensional (2D) polyacrylamide gel electrophoresis (PAGE) and tandem mass spectrometry (MS). Results were compared with serum proteome data collected previously from the sera of healthy dogs. Twenty-one dogs with high grade multicentric lymphoma had significantly elevated quantities of α2 globulins on 1D SPE. Further separation of the serum proteins was performed on three dogs using a 2D PAGE system. Thirty-six different proteins were identified in 38 bands submitted for MS. Most of the proteins were the same as those previously identified in the sera of healthy dogs. Haptoglobin was identified in the sera of all three dogs with lymphoma and could account for the increased levels of α2 globulins. α2 Macroglobulin, α-antichymotrypsin and inter-α-trypsin inhibitor were also present in dogs with lymphoma. Clusterin, an anti-apoptotic protein, was identified in the serum of one dog with lymphoma. Kininogen, which is present in the sera of healthy dogs, was absent in all three dogs with lymphoma. The 2D electrophoresis technique identified alterations in the serum proteome of dogs with lymphoma and supported previous findings that canine lymphoma has an inflammatory component.

Characterisation of the normal canine serum proteome using a novel electrophoretic technique combined with mass spectrometry

One dimensional (1D) serum protein electrophoresis (SPE) on agarose gels is a frequently used diagnostic tool for canine diseases; however, little is known regarding the precise composition of the different protein fractions in normal or diseased animals. In this study, to analyse the canine serum proteome in more detail, conventional 1D SPE was combined with second dimension (2D) polyacrylamide gel electrophoresis (PAGE), followed by tandem mass spectrometry (MS). One dimensional SPE was performed on the sera of 17 healthy dogs to establish normal reference ranges for the albumin and globulin sub-fractions. Two representative serum samples from healthy dogs were further separated using a novel method of 2D PAGE, leading to the generation of 26 distinct bands across the six main sub-fractions, which were subjected to MS analysis. Thirty-two proteins were identified, most of which were found in both dogs. Twenty proteins belonged specifically to the species Canis lupus familiaris, with the remaining 12 proteins belonging to other mammalian species, likely reflecting incomplete sequencing knowledge of canine proteins. Two dimensional electrophoresis and MS allowed identification of canine serum albumin precursor, serpin peptidase inhibitor, kininogen-1, vitamin D binding protein, haemopexin, complement C4 and a variety of immunoglobulin class molecules, along with localisation of these proteins within serum protein subfractions.

ICP-MS-assisted identification of selenium-containing proteins in 2D gels using a new capillary HPLC–ICP MS interface and Orbitrap tandem mass spectrometry

An improved instrumental approach is discussed for the identification of Se-containing proteins separated by 2D polyacrylamide gel electrophoresis. A protein, detected in a gel by laser ablation ICP-MS 2D 78Se imaging, was excised from another gel obtained in parallel under identical conditions, and digested with trypsin. Selenium-peptides were detected by capillary HPLC–ICP MS using a new robust commercial interface and an ICP mass spectrometer fitted with a frequency-matching RF generator facilitating work at high concentrations of acetonitrile in the mobile phase. The identification of selenopeptides was carried out by means of hybrid linear quadrupole trap-Orbital trap MS offering a higher intrascan dynamic range and better mass accuracy than the hitherto used Q-TOF (time-of-flight) mass spectrometers. An example of the identification of Se-containing glyceraldehyde-3-phosphate dehydrogenase-3 in yeast is discussed.

Correlation of expression levels of ANXA2, PGAM1, and CALR with glioma grade and prognosis

Biomarkers for the diagnosis and prognosis of gliomas are lacking. To elucidate new diagnostic and prognostic targets, a routine method is used to evaluate differences between the protein profile of normal and tumor cells. The object of the current study was to investigate novel differentially expressed proteins and their roles in gliomas. Differences in the protein profile were compared using 2D polyacrylamide gel electrophoresis using C6 glioma cells and rat astrocytes. The mRNA and protein expression of ANXA2, PGAM1, and CALR were analyzed in glioma tissues and normal brain tissues. The expression of ANXA2 in the U87 glioma cell line was interrupted using short interfering RNA duplexes, and the role of ANXA2 in the migration and invasiveness of glioma cells was assessed. The expression of ANXA2, PGAM1, and CALR was examined further by immunohistochemical analysis using 130 glioma samples obtained in patients, and their prognostic roles in gliomas were evaluated using Kaplan-Meier and Cox regression analyses. Significantly higher expression levels of ANXA2 and PGAM1 and a lower level of CALR were found in glioma samples than in the normal brain samples. ANXA2, PGAM1, and CALR expression correlated with the grade and survival of patients with gliomas. Multivariate analysis further revealed that ANXA2 was an independent prognostic marker for glioma. After ANXA2 expression was suppressed using short interfering RNA, U87 cells had decreased migratory and invasive capabilities in vitro. Protein expression alterations in ANXA2, PGAM1, and CALR were found in gliomas, and ANXA2 provided a novel prognostic value.

Identification of differentially expressed proteins in wheat after benzothiadiazole treatment

The systemic acquired resistance (SAR) mechanism is stimulated by biological and chemical agents in response to pathogen infection as a part of the innate immunity response system of plants. The externally applied synthetic chemical, benzo-(1,2,3)-thiadiazole-7-carbothioic acid-S-methyl ester (BTH), is also known to induce a SAR response in plants. Studies identifying genes induced or suppressed by BTH are limited. Only few genes responding to BTH are determined. The focus of this preliminary study is to identify the gene products affected by BTH in wheat. A 2D-polyacrylamide gel electrophoresis (PAGE) analysis was carried out with BTH treated Triticum aestivum cv. Gerek-79 and mock treated samples. Following 2D-PAGE image analysis, the selected differentially expressed protein spots were identified by nanoLC-ESI-MS/MS. Among the 26 protein spots distinguished, five were found to be increased upon BTH treatment, another set of seven spots were absent in the control sample.Thus, they were apparent only in the gel of the BTH treated sample (+BTH), whereas five protein spots disappeared in the gel of the BTH treated plants (-BTH). Up-regulation of some proteins such as OEE2 (oxygen evolving enhancer protein) and COR (cold-responsive) LEA (late embryogenesis abundant)/RAB (responsive to abscisic acid, ABA) and down-regulation of some proteins such as RuBisCo LSU (large subunit), fructose 1, 6-biphosphate aldolase (AldP), methyl binding domain protein 6 (MBD6), and 3-isopropylmalate dehydrogenase are shown for the first time in BTH treatment of wheat.

Proteomic Analysis of Plasma Proteins in Diabetic Rats by 2D Electrophoresis and MALDI-TOF-MS

Despite tremendous advances in our understanding of the molecular basis of diabetes mellitus, substantial gaps still remain in our understanding of disease pathogenesis and in the development of effective strategies for early diagnosis and treatment. The proteomic approach has offered many opportunities and challenges in identifying new marker proteins and therapeutic targets, i.e., using 2D-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry. The differential protein expressions were analyzed in alloxan-induced diabetic rats treated with Cynodon dactylon leaf extract. The plant extract was administered for 15 days that resulted in a significant increase in plasma insulin and C-peptide levels. We have also identified four differentially expressed proteins from rat plasma. These four diabetes-associated proteins were broadly classified into three groups as per their function: (1) lipid metabolism-associated protein (Apo A-IV), (2) antioxidant activity-related proteins [preprohaptoglobin and heat shock proteins B8 (HspB8)], and (3) muscle function-related protein (TPM3). Apo A-IV, HspB8, and preprohaptoglobin may play a key role in the recovery of diabetes mellitus and also prevent the diabetes-associated complications such as prevention of oxidative stress due to free radical and free hemoglobin. These results show the value of proteomic approach in identifying the potential markers that may eventually serve as diagnostic markers or therapeutic targets.

2DSpotDB: A Database for the Annotated Two-dimensional Polyacrylamide Gel Electrophoresis of Pathogen Proteins

The biological interpretation of two-dimensional (2D) gel electrophoresis experiments is a key step toward understanding the functions of biological systems. We here present a web-based integrated database, called 2DSpotDB, for the management of proteome data derived from several pathogens. The 2DSpotDB was established as a part of the management of a pathogen proteome project at the Korea National Institute of Health. The goals of the 2DSpotDB implementation are to store and define important pathogen genes, retrieve information obtained by 2D polyacrylamide gel electrophoresis and mass spectrometry, and create an integrated system to provide pathogen proteome information for biological scientists. This database currently contains 14 gels and information on 387 protein spots, among which 329 proteins were identified and annotated.