High-resolution Two-dimensional Polyacrylamide Gel Electrophoresis Research Articles

Soluble Alpha-APP (sAPPalpha) Regulates CDK5 Expression and Activity in Neurons

A growing body of evidence suggests a role for soluble alpha-amyloid precursor protein (sAPPalpha) in pathomechanisms of Alzheimer disease (AD). This cleavage product of APP was identified to have neurotrophic properties. However, it remained enigmatic what proteins, targeted by sAPPalpha, might be involved in such neuroprotective actions. Here, we used high-resolution two-dimensional polyacrylamide gel electrophoresis to analyze proteome changes downstream of sAPPalpha in neurons. We present evidence that sAPPalpha regulates expression and activity of CDK5, a kinase that plays an important role in AD pathology. We also identified the cytoprotective chaperone ORP150 to be induced by sAPPalpha as part of this protective response. Finally, we present functional evidence that the sAPPalpha receptor SORLA is essential to mediate such molecular functions of sAPPalpha in neurons.

Ascaris lumbricoides and Ascaris suum: Comparative proteomic studies using 2-DE coupled with mass spectrometry

Roundworms of the genus Ascaris comprise species of medical (A. lumbricoides) and veterinary (A. suum) importance. Despite the economic importance, public health impact, and worldwide prevalence both parasites still have a controversial taxonomy and their proteomes are poorly described in databases. In the present study, high-resolution two-dimensional polyacrylamide gel electrophoresis (2-DE) coupled with MALDI-TOF/TOF MS was used to characterize and contrast proteomic profiles of adult female A. lumbricoides and A. suum. Approximately, 3-fold higher protein spots were obtained in the present study compared to traditional method, and more than 630 and 750 protein spots were reproducibly detected among the 6 gels examined (3 gels for each of A. lumbricoides and A. suum). The peptide mass finger printings (PMFs) were acquired successfully for 17 from 22 differential 2-DE spots, corresponding to 12 unique proteins. Among them, 2 Asu-enriched and 3 Alu-specific proteins were found in A. lumbricoides; and 6 Asu-enriched and 1 Alu-specific proteins were found in A. suum. Functional analysis revealed low level of difference among differentially expressed proteins of the two nematode species, while the 1 species-specific protein of A. suum was different from that of A. lumbricoides. The proteomic patterns of A. lumbricoides and A. suum provide proteomic evidence that these two taxa are very closely related. This work indicates that the combined 2-DE and MALDI-TOF/TOF MS approaches employed in the current study allow the identification of proteins whose expression is different between Ascaris species.

Selection and Identification of Acute Proteins with Proteomic Techniques in Rhinencephalon of Cyprinus Carpio After Wounded Acutely

A combined available approach of low osmosis and freeze-soluble with liquid nitrogen was used to disrupt the rhinencephalon tissue of Cyprinus carpio (CC). A centrifugal method with low speed was used to extract proteins in rhinencephalon tissue of CC (CCRT), and high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) map with 1200 protein spots was obtained. In this study, several techniques were used for extracting and separating proteome in CCRT. Three lysis solutions were prepared to disrupt the tissue in the fish, and used to extract proteome, water-soluble proteins and fat-soluble proteins, respectively. Both water-soluble proteins and fat-soluble protein from the CCRT were effectively separated by an optimized 2D-PAGE method with high resolution. With treating by 10% acetic acid, six acute proteins in CCRT were selected by proteomic techniques, and further identified by peptide mass fingerprinting (PMF) and database research, respectively. Three of these differential proteins can be primarily defined to be 70 kD heat-shock protein, DNA ligase IV, and β 5 tubulin. It was proposed that these proteins may be used as markers to study the repairing pathway and mechanism in brain once wounded.

Studies on middle and posterior silk glands of silkworm ( Bombyx mori) using two-dimensional electrophoresis and mass spectrometry

Silk glands, present in the larval stage of the silkworm, produce threads of silky material to form the cocoon and are mainly composed of three parts: the anterior, the middle, and the posterior silk glands, each playing different roles in silk secretion. High-resolution two-dimensional polyacrylamide gel electrophoresis and computer-assisted analysis were used to investigate quantitative and qualitative differences between the middle and posterior silk glands. Silver staining revealed over 600 spots for each sample, mostly distributed from 15 to 100 kDa with pH 4–7. Computer-assisted image analysis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and post-source decay technology suggested that there were significant differences in spot distribution and expression between the middle and posterior silk glands. In addition, 98 spots from the posterior silk gland were excised and further investigated following trypsin digestion. The results suggested that more than 20% of the 88 proteins identified were related to heat-shock proteins and chaperones. Redox system and DNA replication proteins involved in silk protein synthesis were also detected in the posterior silk gland. Interestingly, two novel serpin proteins were identified in the middle silk gland, and to a lesser extent in the posterior gland, which were presumed to be involved in regulation of proteolytic activity and protection of silk proteins from degradation.

Proteomic Profiling Identifies an UV-Induced Activation of Cofilin-1 and Destrin in Human Epidermis

The human skin is the only line of defense against UV radiation. A series of responses to protect the skin are induced by UV radiation. In this study, a proteomic approach was used to study these responses. We have performed high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis of (solar simulated) UV-exposed reconstructed skin equivalents as well as native skin. Differentially expressed proteins were processed for mass spectrometric analysis, when consistent differences were observed in all individual human skin equivalents. In addition to proteins known to be involved in UV responses (HSP27, MnSOD, and PDX-2), we identified two novel proteins that were downregulated following UV exposure. Further analysis revealed that these proteins were the phosphorylated forms of the actin cytoskeleton modulators cofilin-1 and destrin. The de-phosphorylation of cofilin-1 was confirmed using western blotting of UV-exposed skin equivalents and ex vivo skin protein extracts. In conclusion, our study indicates the potency of a proteomic approach to study UV-induced changes in a tissue culture system mimicking human skin as well as excised human skin.

Proteome analysis of the silkworm (Bombyx mori. L) colleterial gland during different development stages

The silkworm, Bombyx mori, colleterial gland developed very slowly until 2 days before emergence, then markedly enlarged due to the accumulation of a glue-like substances (mainly including 85% water and 11% proteins). However, the No glue (Ng) mutant female moth secreted only very little glue-like substance and laid loose eggs naturally. High-resolution two-dimensional polyacrylamide gel electrophoresis, followed by computer-assisted analysis, was used to screen the secretory region of colleterial gland protein patterns during different development stages to find quantitative and qualitative difference in protein expression during the pupae and moth stages. More than 700 protein spots were resolved in different developmental stages from the secretory region of the glands and most of the proteins were distributed in the mass range from 30 to 70 kD with pH 4-8. Through comparison and analysis, it was found that 3 proteins were only expressed in the later pupae stage (one or two days before emergence) and moth stage. Furthermore, these proteins were not expressed in the Ng mutant especially actin. There was a great variation of some protein expression volume during the development. Protein spots that changed more than 1.5-fold in expression level (relative to day 9), including 6 spots that were down-regulated and 2 spots that were up-regulated in expression were excised for identification by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Results indicated that actins that participated or regulated the exocytosis of colleterial gland and other differentially expressed proteins might be related to colleterial gland development or the secretion of a glue-like substance.

The protein profile of acetazolamide-treated sera in mice bearing Lewis neoplasm

The aim of the present research is to analyze the proteome of neoplasm serum before and after treated with acetazolamide (20, 40, 80 mg kg −1 d −1 for 3 days p.o.). The Lewis lung carcinoma mice were used and carried out a comprehensive proteomic analysis by using the technologies of high-resolution two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and mass spectrometry (MS). The results showed that the acetazolamide could dramatically reduce the lung metastasis and primary tumor growth. Its most potent inhibition rate on lung metastases was reach to 77.7% at the dose of 80 mg kg −1 d −1. The two dimension electrophoresis and software analysis reveal 393 protein spots in control gel, 385 protein spots were detected in treated gel and matched 209 protein spots with control gel, indicating that intensive changes had occurred during the process of treatment. Two obviously different spots were cut off from gel and for the peptide mass fingerprinting. Data base searching showed the two proteins' peptide much more mach with Histone H2B fragment and Ubc-like protein CROC1 fragment. The results suggest that acetazolamide has a strong anti-tumor and anti-metastasis effect on Lewis-lung-carcinoma. The mechanism may be related to its regulation on plenty of proteins, in particular, on upregulation of H2B and CROC-1 expression of postreplicational DNA repair related protein in serum.

Proteome analysis of rice tissues by two-dimensional electrophoresis: an approach to the investigation of gibberellin regulated proteins

Protein databases constructed using high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) were used to explore the proteome expressed in various rice tissues. Proteins from leaf sheath, root, and cultured suspension cells were systematically analyzed using 2D-PAGE, mass spectrometry and Edman sequencing, followed by database searching. In all, 79 of the 431 spots detected by 2D-PAGE in the leaf sheath, 73 of the 508 spots in the root and 140 of the 962 spots in the cultured suspension cells could be identified. Protein lists were constructed for each tissue and used to investigate the effects of gibberellin (GA) treatment. In the leaf sheath, root and cultured suspension cells, 8, 21, and 14 of the identified proteins, respectively, were regulated by GA. These proteins included polypeptides involved in general metabolism, energy production, transcriptional regulation and signal transduction in the leaf sheath; in metabolism and defense in the root; and in metabolism, energy production, cell growth, defense and signal transduction in the cultured suspension cells. These results indicate that the proteome databases assembled in these studies will be useful for the rapid assessment of changes in protein content in specific tissues, and that proteins regulated by GA may play a significant role in tissue growth.

Analysis of Protein Expression along the Normal to Tension Wood Gradient in Eucalyptus gunnii

AbstractThis paper examines the contribution of various xylem proteins to wood formation inEucalyptus gunnii. Proteins were extracted from differentiating xylem harvested from a crooked tree, separated by high-resolution two-dimensional polyacrylamide gel electrophoresis, visualised by silver nitrate staining and analysed with a computer-assisted system for single protein spot quantification. Growth strain measurements allowed xylem samples to be classified quantitatively from tension wood to normal wood. Regression of lignin content on growth strain showed that a decrease in lignin content corresponded to decreasing growth strain values,i.e., presence of tension wood. Out of the 140 studied protein spots, 12 were significantly associated with growth strain: 7 being less abundant in tension wood and 5 being more abundant in tension wood. A clustered-correlation analysis was performed to study protein expression simultaneously along the gradient of gravistimulated stressed xylem tissue. Proteins were found to form “expression clusters”.

Identification of new proteins in follicular fluid of mature human follicles.

Proteins present in human follicular fluid (HFF) have been poorly characterized to date. The purpose of our study was to analyse the protein content and identify new proteins originating from fluid of mature human follicles. A total of six females from infertile couples referred for in vitro fertilization (IVF) were stimulated and 44 follicular fluid samples from mature follicles yielding an oocyte were collected 34-36 h after human chorionic gonadotropin administration. HFF samples were processed for high-resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Comparative analysis of the 2-D gels revealed up to 600 spots, of which four were selected because of variations in their expression level. Using direct sequencing procedures (Edman degradation) or matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS), these four spots were identified as three new proteins: thioredoxin peroxydase 1 (TDPX1), transthyretin (TTR) and retinol-binding protein (RBP). The proteins identified here may emerge as potential candidates for specific functions during folliculogenesis and may prove useful as biomedical markers for follicle and/or oocyte maturation.

Identification of α1-Antitrypsin Variants in Plasma with the Use of Proteomic Technology

Proteomic technology permits the investigation of genetic metabolic diseases at the level of protein expression. Changes in the expression, polypeptide structure, and posttranslational modification of individual proteins can be detected in complex mixtures of proteins. We used high-resolution two-dimensional polyacrylamide gel electrophoresis to separate isoforms of plasma proteins and detect abnormalities of mass and/or charge. We confirmed the identity of the separated proteins by in-gel digestion with proteases and N-glycanases and then analyzed the released peptides and glycans by matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry. Complete characterization of the polypeptide sequences and glycosylation of alpha(1)-antitrypsin isoforms was achieved in plasma from controls and from patients with three different known alpha(1)-antitrypsin deficiencies and congenital disorder of glycosylation type Ia. This study shows that proteomic techniques are a powerful and sensitive means of detecting changes in the amino acid sequence and abnormal posttranslational modifications of specific proteins in a complex biologic matrix.

Proteolytic cleavage of staphylococcal exoproteins analyzed by two-dimensional gel electrophoresis.

Extracellular proteases of Staphylococcus aureus are emerging as potential virulence factors that are relevant to the pathogenicity of staphylococcal infections. These proteases may also be involved in the proteolytic cleavage of other exoproteins released from this organism. To define the target exoproteins and their sites of cleavage by proteases, high-resolution two-dimensional polyacrylamide gel electrophoresis followed by N-terminal amino acid sequencing of exoprotein spots was performed. Two to three hundred exoprotein spots were detected at the early-stationary phase of cultures of S. aureus NCTC8325, and then at the late-stationary stage most of these high molecular protein spots became invisible due to further proteolytic degradation. As the result of N-terminal analysis, lipase, triacylglycerol lipase, orf619 protein and orf388 protein were detected as multiple spots at the early-stationary phase. We found that these exoproteins were cleaved at 3, 7, 4 and 4 different sites, respectively, by proteases. According to the M.W. and pI of each peptide spot obtained from the gel and their matches with calculated values in addition to their N-terminal sequences, we showed that the positions of putative peptides resulted from proteolytic cleavage of these proteins.

Protein identification methods in proteomics.

A combination of high-resolution two-dimensional (2-D) polyacrylamide gel electrophoresis, highly sensitive biological mass spectrometry, and the rapidly growing protein and DNA databases has paved the way for high-throughput proteomics. This review concentrates on protein identification. We first discuss the use of protein electroblotting and Edman sequencing as tools for de novo sequencing and protein identification. In the second part, we highlight matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) as one of the main contemporary analytical methods for linking gel-separated proteins to entries in sequence databases. In this context we describe the two main MALDI-MS-based identification methods: (i) peptide mass fingerprinting, and (ii) post-source decay (PSD) analysis. In the last part, we briefly emphasize the importance of sample preparation for obtaining highly sensitive and high-quality MALDI-MS spectra.

Protein identification methods in proteomics

A combination of high-resolution two-dimensional (2-D) polyacrylamide gel electrophoresis, highly sensitive biological mass spectrometry, and the rapidly growing protein and DNA databases has paved the way for high-throughput proteomics. This review concentrates on protein identification. We first discuss the use of protein electroblotting and Edman sequencing as tools for de novo sequencing and protein identification. In the second part, we highlight matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) as one of the main contemporary analytical methods for linking gel-separated proteins to entries in sequence databases. In this context we describe the two main MALDI-MS-based identification methods: (i) peptide mass fingerprinting, and (ii) post-source decay (PSD) analysis. In the last part, we briefly emphasize the importance of sample preparation for obtaining highly sensitive and high-quality MALDI-MS spectra.

2‐D Proteome analysis protocols, by Andrew J. Link, Ed. 1999. Totowa, New Jersey: Humana Press. 704 pp. $89.50 (hardcover)

For the past 24 years, high-resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) has been the technique of choice for analyzing the protein composition of cells, tissues, and fluids, as well as for studying changes in global patterns of gene expression elicited by a wide array of effectors. The technique, which was originally described independently by O'Farrell and Klose, offers the highest resolution of protein separation and, therefore, plays a central role in “proteomics,” an area of functional genomics that combines a plethora of techniques to resolve (2-D PAGE), identify (peptide sequencing by Edman degradation, mass spectrometry, Western immunoblotting), quantify (scanners, phosphorimagers), and characterize proteins, as well as to store (image analysis and 2-D PAGE databases), communicate, and interface protein data with that emerging from the various genome mapping and sequencing projects. Proteomics, together with genomics, cDNA arrays, phage antibody libraries, and transgenics and knockouts, today provide an impressive array of technologies for studying gene expression in both health and disease.

The different expression of proteins recognized by monoclonal anti-heat shock protein 70 (hsp70) antibody in human colonic diseases.

Analysis of the expression of hsp70 (70 kDa heat-shock proteins) in normal and pathological tissues might prove their potential diagnostic and prognostic values. In the present study, we combined high-resolution two-dimensional polyacrylamide gel electrophoresis with immunoblotting to study hsp70 expression in normal, preneoplastic and neoplastic colonic mucosa. By monoclonal anti-hsp70 antibody, recognizing both the constitutive and inducible forms of hsp70, we have detected six charge isoforms localized in the pH 5.1-5.3 range and in the molecular mass range of 68-69 kDa in normal colonic mucosa. Immunostaining of hsp70 in polypous and malignant tissues revealed qualitative as well as quantitative changes in the expression of more acidic isoforms of hsp70 in comparison with normal tissue. Furthermore, the different basic isoforms of hsp70 were detected in chronically inflamed colonic mucosa from patients suffering from ulcerative colitis (UC) or Crohn's disease (CD). Besides the standard hsp70 protein pattern, additional proteins with molecular masses of about 39, 40, 74 and 75 kDa were variably immunostained in normal and pathological specimens. These observations suggest that hsp70 expression may be closely linked to disease etiology and/or pathophysiology.

Identification and characterization of wheat grain albumin/globulin allergens.

Bakers' asthma, an immediate-type allergic response to the inhalation of cereal flours, is an important occupational disease among workers of the baking and milling industries, and the salt-soluble proteins of wheat and rye flour dust are considered the most relevant allergens. In order to identify and characterize the major IgE-binding proteins, the polypeptide composition of the albumin/globulin protein fraction obtained from different cultivars was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and high-resolution two-dimensional polyacrylamide gel electrophoresis with immobilized pH gradients in the first dimension (IPG-Dalt), followed by immunoblotting with sera from asthmatic bakers. Relevant allergens were isolated by micropreparative IPG-Dalt and blotting onto polyvinylidenedifluoride membranes and identified by amino acid composition analysis or N-terminal amino acid sequence analysis. SDS-PAGE, IPG-Dalt, and immunoblotting demonstrated that the sera of the bakers allergic to flour contained IgE antibodies which bound to numerous albumin/globulin polypeptides in the 70, 55, 35, 26-28, and 14-18 kDa areas. More detailed investigations using IPG-Dalt revealed cultivar-specific differences in IgE-binding. It was also demonstrated that the majority of the allergens were not single polypeptide spots, but consisted of up to ten isoforms of similar molecular mass but different isoelectric points. Amino acid composition analysis and N-terminal amino acid sequence analysis, which were performed for nine allergens located in the 14-18, 26-28, and 35 kDa areas, revealed homologies to amylase/protease inhibitors, acyl-CoA oxidase and fructose-bisphosphate-aldolase from wheat, barley, maize, and rice, respectively.

From genome to proteome: protein map of Haemophilus influenzae.

High-resolution two-dimensional (2-D) polyacrylamide gel electrophoresis allows the separation of complex biological mixtures (i.e., several hundred proteins from a bacterial cell lysate) in a single experiment. In this report proteins from Haemophilus influenzae were separated by 2-D gels and analyzed by peptide mass fingerprinting and/or amino acid analysis. By comparing the peptide mass profiles and the amino acid composition with the Haemophilus influenzae database, 119 protein spots were identified. The combination of amino acid analysis and peptide mass fingerprinting is a powerful tool for a rapid and economical identification of a large number of proteins resolved by 2-D gels. Studies on gene regulation and changes of protein expression upon drug treatment require quick and serial analysis techniques to efficiently identify potential new drug targets.

Rapid detection of the main human plasma glycoproteins by two-dimensional polyacrylamide gel electrophoresis lectin affinoblotting.

Glycoprotein modifications in the glycan moiety can occur in diseases such as cancers, inflammatory processes and alcoholism. We combined high-resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) with lectin affinoblotting in order to establish the normal human plasma glycoprotein map. Human plasma proteins were separated by mini 2-D PAGE (7 x 9 cm), transferred onto polyvinylidene difluoride membranes and incubated with biotinylated lectins. We focused our study on lectins binding sialic acid and galactose residues. Known plasma glycoproteins such as alpha 1-antitrypsin, alpha 1-antichymotrypsin, alpha 2-HS glycoprotein, alpha 1-acid glycoprotein, haptoglobin beta-chain and transferrin were easily detected in ng amounts. This protocol was adequate to establish a normal plasma glycoprotein map and will allow the study of glycoproteins in diseases.

Secretagogue regulation of pancreatic acinar cell protein phosphorylation shown by large-scale 2D-PAGE

High-resolution large-scale two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) combined with computer-assisted image analysis was used to construct a database of secretagogue/second messenger-induced phosphoprotein modifications in intact rat pancreatic acinar cells. Isolated acini were labeled with 32Pi, exposed to hormones and other test agents, and subjected to large-scale 2D-PAGE and autoradiography. This procedure resolved 500 phosphoproteins in pancreatic acinar whole cell lysates, approximately 90% of which were localized in the soluble fraction of centrifuged samples. Soluble proteins were further characterized as to heat and acid stability. Cholecystokinin (CCK), carbachol, and bombesin altered the phosphorylation state of about 27 proteins with both increases and decreases observed. Subsets of proteins were phosphorylated in response to phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), calcium ionophore A-23187, and adenosine 3',5'-cyclic monophosphate (cAMP) analogue 8-bromo-cAMP. One of these proteins was identified as the myristoylated, alanine-rich, C-kinase substrate (MARCKS) protein by immunoprecipitation. The time course and dose response of phosphorylation changes due to CCK showed considerable variation between proteins, although a temporal hierarchy of phosphorylation events was clearly exhibited. Particularly striking was the rapid dephosphorylation within 30 s of a 19-kDa soluble protein to a minimum of 20 +/- 1% of control. Increased phosphorylation of the MARCKS and other TPA-regulated proteins suggests that CCK, carbachol, bombesin, and the CCK partial agonist, JMV-180, all activate protein kinase C in intact acini.