One-dimensional Sodium Dodecyl Sulfate-polyacrylamide Gel Electrophoresis Research Articles

Proteomic Profiling of Maternal Serum for Early Risk Analysis of Preterm Birth

Background: The absence of absolute clinical indicators and suitable biomarkers hinders the timely diagnosis of women at risk of preterm birth. It influences roughly 12% of births. At delivery and clinical presentation, preterm births are generally inspected based on the gestational period. Different disturbed pathways are associated with the signs of at-risk pregnancies. Objective: The main purpose of this study is to analyze and explore the serum proteome of early deliveries and help health care professionals to improve the understanding of the progression of preterm birth. Methods: In the present study, 200 pregnant females of 20-30 years of age were selected. We collected samples of second and third-trimester pregnant females, out of which 40 females delivered preterm. We further divided them into three groups, i.e., extremely preterm group, very preterm, and controls. Overall comparison of serum profiles of all the three groups expressing fourteen proteins ranging between 200-10kDa was made. Serum proteins were isolated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and photographed by totalLab quant software. Groups were evaluated using the ANOVA Tukey’s Post Hoc analysis. Results: Proteins of 69kDa and 15kDa expressed a significant decrease when compared with control subjects. In contrast, the proteins of 23kDa expressed a significant increase, while the proteins of 77kDa, 45kDa, and 25kDa demonstrated no considerable variation. Conclusion: The serum proteins showing significant difference as compared to the control group will serve as predictive biomarkers for at-risk pregnancies. The present study is expected to considerably improve the understanding of the disease pathogenesis along with improved diagnostic and therapeutic approaches leading to better management of pregnancy and reducing the risk of preterm birth.

Screening of selenium containing proteins in the Tris-buffer soluble fraction of African catfish (Clarias gariepinus) fillets by laser ablation-ICP-MS after SDS-PAGE and electroblotting onto membranes

The anti-carcinogenic properties of selenium against certain types of cancer when present in organic forms justify the increasing interest in development of selenium fortified food. In this particular study, African catfish (Clarias gariepinus) were fed with a Se-enriched diet in order to enhance the selenium concentration in the fish fillet up to 0.85 ± 0.06 mg Se kg−1 (0.26 ± 0.02 mg Se kg−1 in control). Selenium distribution in proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was studied for better understanding of the metabolism of this element in the organism. Soluble proteins (Tris buffer) were investigated by conventional application of a glass homogenizer in comparison to application of ultrasound probe sonication, and the latter was convincing due to higher protein extraction efficiency and the ease and speed of application. Laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) was successfully applied for screening of selenium in proteins in fillets of African catfish separated by one-dimensional SDS-PAGE and after electroblotting onto nitrocellulose membranes, with a total Se amount below 0.4 ng loaded into a well. Selenium was detected in more than 11 protein spots. In order to improve the protein separation of one-dimensional SDS-PAGE, pre-fractionation of the Tris buffer soluble protein extract by size exclusion was carried out. Tryptic digestion of the Se-containing bands was performed for protein identification by nano-HPLC coupled to electrospray-MS/MS. A database search revealed several proteins which are located in muscle tissue. However, the very low Se concentration circumvents the detection of selenium containing amino acids in the tryptic peptides by electrospray-MS/MS.

Protein Expression in Vibrio parahaemolyticus 690 Subjected to Sublethal Heat and Ethanol Shock Treatments

Cells of Vibrio parahaemolyticus 690 were subjected either to heat shock at 42°C for 45 min or to ethanol shock in the presence of 5% ethanol for 60 min. The protein profiles of the unstressed and stressed V. parahaemolyticus cells were compared. Additionally, the induction of DnaK- and GroEL-like proteins in the unstressed and stressed cells of V. parahaemolyticus was also examined. Analysis with one-dimensional sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) indicated that three proteins with molecular masses of 93, 77, and 58 kDa were induced by both heat shock and ethanol shock. The protein patterns revealed by two-dimensional electrophoresis were more detailed than those revealed by one-dimensional SDS-PAGE. It was found that heat shock and ethanol shock affected the expression of a total of 28 proteins. Among them, four proteins with molecular masses of 94, 32.1, 26.7, and 25.7 kDa were enhanced by both heat shock and ethanol shock. Furthermore, immunoblot analysis showed the presence of a GroEL-like protein with a molecular mass of 61 kDa in the test organism, with the heat-shocked and ethanol-shocked cells producing a GroEL-like protein in a larger quantity than the unstressed cells. However, DnaK-like protein was not detectable in either the unstressed or the stressed cells.

Heparin-binding proteins of canine seminal plasma

Heparin-binding proteins (HBP) from seminal plasma have been expected to participate in modulation of the acrosomal reaction, and have been correlated with fertility in some species. However, they have not been described in the dog. The aim of this study was to document the HBPs of canine seminal plasma. Six pooled samples of seminal plasma from three crossbred dogs were used. The HBPs were isolated by heparin affinity chromatography and the fractions recovered were pooled. One-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out on 12 and 18% vertical minigels. The stained gels were scanned and the molecular weight (kDa) values for each band within a lane were calculated by image analysis software. The electrophoresis analysis of the pooled eluded fractions identified 19 bands, with molecular weights varying from 61.5 to 5.2 kDa. Previous studies, using one-dimensional SDS-PAGE, identified two bands (67 and 58.6 kDa), which were positively correlated with some semen parameters (sperm motility, sperm vigor, percentage of morphologically normal sperm and plasma membrane integrity). The 61.5 kDa band detected in the present study apparently corresponded to the 58.6 kDa band identified previously. Canine seminal plasma contained HBP; since HBP modulate the acrosome reaction in other species, they may have the same function in the dog. Further studies are necessary to better characterize this protein and determine if it is associated with fertility in the dog.

In situ alkylation with acrylamide for identification of cysteinyl residues in proteins during one- and two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis

Cysteinyl residues in proteins were alkylated with acrylamide during sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) to yield a thioether derivative, cys-S-beta-propionamide (PAM cys). The process was termed in situ alkylation with acrylamide. Using this method, the recovery of PAM-cys peptides from bovine serum albumin (BSA) was 88.6% at 10 picomol in one-dimensional (1-D) SDS-PAGE and 97.1% at 50 picomol in two-dimensional (2-D) SDS-PAGE. The coverage of tryptic peptide of BSA in 1-D and 2-D SDS-PAGE was 83.7% and 81.1%, respectively. The advantages of in situ alkylation with acrylamide were the following: (i) cysteinyl peptides were effectively derived in a single PAM cys and then proteins were precisely identified using databases; (ii) marked reduction of salts compared with post alkylation, e.g., using carboxymethylamide (CAM), resulting in higher signal intensity and wider coverage of cysteinyl peptides from PAM cys, compared with those of CAM derivatives, in mass spectrometry peptide mapping; and (iii) shorter duration by excluding the processes of post alkylation and desalting before peptide mapping.

Role of germinal vesicle on protein synthesis in rat oocyte during in vitro maturation.

To investigate the role of the germinal vesicle (GV) on in vitro maturation (IVM) of rat oocytes, we examined protein synthesis during IVM by comparing polypeptide patterns in control and enucleated oocytes using one and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Separation of polypeptides extracted from the cytoplasm of GV by one-dimensional SDS-PAGE revealed that a 55 kDa polypeptide was present only in the GVs of rat oocytes. At 0, 12, 24, 36, and 44 hr after PMSG injection, prior to the initiation of maturation, enucleated oocytes synthesized the same major polypeptides as cumulus intact (CI) oocytes. During meiotic maturation, no major changes were detected in protein synthesis from prophase (GV stage) to prometaphase I (0-6 hr IVM). However, after entry into prometaphase I (7 hr IVM), striking changes were seen; a 24 kDa polypeptide disappeared and expression of a 34 kDa polypeptide became stronger. This pattern lasted until metaphase II. We detected no major differences in the pattern of protein synthesis between CI and enucleated oocytes using two-dimensional PAGE. These results indicate that protein synthesis in the maturing rat oocyte is controlled by cytoplasmic regulators rather than intrinsic nuclear components.

Chapter 16 Two-Dimensional Separation of Axonemal Proteins

The procedure described in this chapter is meant to resolve the molecular components of Chlamydomonas axonemes. It can be applied to all kinds of axonemes for various purposes—namely, the analysis of axonemal fractions, the identification of defective gene products, the recognition of cytoskeletal proteins, and, finally, the definition of “molecular signatures” that are formed by specific substructures. These signatures consist of characteristic two-dimensional patterns of spots. The development of two-dimensional electrophoretic procedures capable of resolving nearly all axonemal proteins provided the opportunity to identify the molecular components of all major axonemal substructures. Axoneme is composed of hundreds of distinct polypeptides that have different concentrations and are post-translationally modified. The procedure discussed consists of two successive gel electrophoreses of polypeptides that, first, are resolved in a pH gradient and, then, are separated by conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). During the first step the polypeptides retain their own electric charge but do not reach their isoelectric point. The procedure works very well for separating proteins that are smaller than 200,000 Da. Larger proteins need analysis by one-dimensional SDS-PAGE. The electrophoreses are performed in two homemade, vertical apparatus that hold 16-cm-wide, 1Pcm-long, 0.3-cm-thick glass plates and 35.5-cm-wide, 26.5-cm-long, 0.4-cm-thick glass plates, respectively. The dimensions of the apparatus for the second electrophoresis are designed so that two samples can be run side-by-side in an identical acrylamide gradient. The procedure is described in detail. Following the second electrophoresis, detection of polypeptides is performed either by silver staining of wet gels or autoradiography of dried gels. In nonequilibrium electrophoresis of polypeptides in a gradient of pH, all polypeptides under analysis remain in the gel. Polypeptides that could irreversibly precipitate at their isoelectric point in a focusing gel remain soluble and can be separated in the gel used for the second electrophoresis.

Response of embryos of the sea urchin Strongylocentrotus purpuratus to aqueous petroleum waste includes the expression of a high molecular weight glycoprotein

The present study investigated the sublethal effects of the largest aqueous waste associated with offshore oil development, produced water (PW), on sea urchin embryo development. PW inhibited normal gastrulation in purple sea urchin (Strongylocentrotus purpuratus) embryos exposed at the hatching stage. The exposed embryos did not form complete archenterons, and secondary mesenchyme cells exhibited an unusual behavior with respect to the wall of the blastocoelic cavity. In addition, an abundance of extracellular matrix was observed in the blastocoelic cavities in the embryos exposed to higher concentrations of PW. An inhibition of the development of embryos through the pluteus stage was observed as a result of PW exposure. This was manifested by inhibition of normal spicule formation with concomitant abnormal pluteus morphology. To determine if a biochemical stress response occurs as a result of PW exposure, embryos at the early gastrula stage were exposed to PW for 2 h, followed by an additional 1 h in 3H-leucine and PW. These embryos expressed a 253 kDa protein as observed by one-dimensional SDS (sodium dodecyl sulfate) polyacrylamide gel electrophoresis and autoradiography. This protein could occasionally be observed in gels stained for protein, and was glycosylated as demonstrated by staining with periodic acid Schiff (PAS). The 70 to 73 kDa heat-shock proteins previously described in sea urchin embryos (and other organisms) as a result of stress were not synthesized as a result of PW exposure. Heat shock did not induce synthesis of the 253 kDa glycoprotein. Combined PW and heat-shock exposure elicited both the heat-shock response (73 kDa protein) as well as expression of the 253 kDa glycoprotein. These data demonstrate the use of expression of endogenous macromolecular markers to distinguish exposures of embryos to different perturbations. Arsenic (as sodium arsenite), a metal constituent of PW, also induced the 253 kDa glycoprotein in early gastrula-stage embryos. We suggest that this glycoprotein may be a marker for PW (and a metal constituent) exposure in sea urchin embryos, and may be related to morphological abnormalities.

Bcy1, the regulatory subunit of cAMP-dependent protein kinase in yeast, is differentially modified in response to the physiological status of the cell.

The regulatory subunit of cAMP-dependent protein kinase in yeast, encoded by the BCY1 gene, is known to be required under certain conditions such as growth on nonfermentable carbon sources and entry into stationary phase. We have identified novel isoforms of Bcy1 in cells under these conditions. The isoforms are distinguishable by their migration on one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 2-dimensional nonequilibrium pH gradient gel electrophoresis. The isoforms observed by one-dimensional SDS-PAGE bind cAMP, as determined by [32P]8-azido-cAMP labeling (diagnostic of Bcy1 protein). Proteins isolated from cells grown to stationary phase in rich medium exhibit five antibody-reactive bands, by one-dimensional SDS-PAGE immunoblot analysis, with apparent molecular masses of 50, 52, 55, 59 and 61 kDa. Total Bcy1 protein increases at least 8-fold between exponential and stationary phase. Analysis of proteins from a variety of yeast mutants indicated that 1) many of the observed modifications of Bcy1 are dependent upon the presence of the Ser-145 phosphorylation site; 2) the appearance of the 59- and 61-kDa bands is dependent upon the presence of Yak1 kinase; and 3) Bcy1 protein is modified even in the absence of cAMP-dependent protein kinase catalytic subunits. Cells carrying the bcy1(ala145) allele exhibit non-wild type growth, indicating that these modifications may be functionally significant.

Influence of triiodothyronine on the polypeptide composition of the intestinal brush border membrane during amphibian metamorphosis.

Brush border fragments (BBF) were isolated from homogenates of intestinal epithelium prepared from four groups of tadpoles: premetamorphic larvae, thyrostatic larvae, spontaneously metamorphosed larvae, and triiodothyronine (T3)-induced froglets. Isolation was accomplished by a combination of both Ca2+ precipitation and differential centrifugation methods. These preparations were routinely enriched seven- to-eleven-fold for the two amphibian brush border marker enzymes, gamma-glutamyltransferase and maltase. Comparison by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining revealed the presence of a polypeptide of Mr 27,000 only after spontaneous and T3-induced metamorphosis. One-dimensional SDS-PAGE together with lectin staining showed six strongly concanavalin A reactive polypeptides (Mr 52,000, 57,000, 65,000, 80,000, 130,000 and 150,000) in both preparations examined. Immunoblot analyses allowed us to detect in both preparations the presence of villin (Mr 105,000), a cytoskeletal component of microvilli. Two-dimensional isoelectric focusing IEF/SDS-PAGE together with silver staining showed the polypeptides of Mr 41,500, 43,000, 60,500 and 101,000 to be specific components of the primary intestinal epithelium brush border. In contrast six polypeptides of Mr 27,000, 52,000, 58,000, 59,000 and 95,000 were only detected in intestinal BBF after spontaneous and T3-induced metamorphosis. Their presence is under the control of the thyroid hormone. The results provide new insight regarding the subcellular localization of polypeptides whose synthesis changes during spontaneous (Figiel et al., 1987) and T3-induced metamorphosis (Figiel et al., 1989).

Structural organization of the human glucocorticoid receptor determined by one- and two-dimensional gel electrophoresis of proteolytic receptor fragments.

The structural organization of the steroid-binding protein of the IM-9 cell glucocorticoid receptor was investigated by using one- and two-dimensional gel electrophoresis of proteolytic receptor fragments. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of receptor fragments isolated after trypsin digestion of immunopurified [3H]dexamethasone 21-mesylate ([3H]DM-) labeled receptor revealed the presence of a stable 26.5-kilodalton (kDa) steroid-containing, non-DNA-binding fragment, derived from a larger, less stable, 29-kDa fragment. The 26.5-kDa tryptic fragment appeared to be completely contained within a 41-kDa, steroid-containing, DNA-binding species isolated after chymotrypsin digestion of the intact protein. Two-dimensional electrophoretic analysis of the [3H]DM-labeled tryptic fragments resolved two (pI congruent to 5.7 and 7.0) 26.5-kDa and two (pI congruent equal to 5.7 and 6.8) 29-kDa components. This was the same number of isoforms seen in the intact protein, indicating that the charge heterogeneity of the steroid-binding protein is the result of modification within the steroid-containing, non-DNA-binding, 26.5-kDa tryptic fragment. Two-dimensional analysis of the 41-kDa [3H]DM-labeled chymotryptic species revealed a pattern of isoforms more complex than that seen either in the intact protein or in the steroid-containing tryptic fragments. These results suggest that the 41-kDa [3H]DM-labeled species resolved by one-dimensional SDS-PAGE after chymotrypsin digestion may be composed of several distinct proteolytic fragments.

Electrophoretic Analysis of Protoplast, Vacuole, and Tonoplast Vesicle Proteins in Crassulacean Acid Metabolism Plants

Protoplasts and vacuoles were isolated and purified in large numbers from the CAM plants Ananas comosus (pineapple) and Sedum telephium for protein characterization. Vacuoles were further fractionated to yield a tonoplast vesicle preparation. Polypeptides of protoplasts, vacuoles, and tonoplast vesicles were compared to whole leaf polypeptides from both plants by one-dimensional sodium dodecylsulfate-polyacrylamide gel electrophoresis. Approximately 100 vacuole polypeptides could be resolved of which 25 to 30% were enriched in the tonoplast vesicles. The proteins of protoplasts, vacuoles, and tonoplast vesicles from A. comosus were analyzed further by two-dimensional gel electrophoresis. When one-dimensional electrophoretograms of A. comosus polypeptides were stained with a glycoprotein-specific periodic acid Schiff stain, very few polypeptides appeared to be glycosylated, whereas a large number of glycosylated polypeptides were detected with a silver-based glycoprotein stain particularly in tonoplast vesicles. Analysis of the enzymic content of vacuoles from both plants indicated the presence of a variety of hydrolases, including bromelain as a major constituent of A. comosus. No substrate-specific ATPase, however, could be detected in vacuoles or tonoplast vesicles from either plant.

Origin of periodic acid-Schiff-reactive glycoprotein in human eccrine sweat.

To evaluate the possible involvement of ductal blockade with periodic acid-Schiff (PAS)-positive materials in the mechanism of hidromeiosis in humans, skin slices were incubated with methacholine for 2 h and PAS-positive materials localized histologically in the ductal lumen. In 20% of the glands complete ductal blockade with PAS-positive materials was noted. The characteristics and origin of such PAS-positive glycoproteins in human sweat were then studied using various electrophoretic techniques. One-dimensional sodium dodecylsulfate-polyacrylamide gel electrophoresis (1-D SDS-PAGE) demonstrated considerable individual variation in the electrophoretic pattern; however, four major bands at 45, 28, 20, and 18K shared by different individuals, were PAS positive. Further studies using two-dimensional SDS-PAGE, immunodiffusion and immunoaffinity chromatography demonstrated that the PAS-positive glycoproteins are not derived directly from serum because they are electrophoretically and antigenically distinct from serum proteins, including alpha 1-glycoprotein, alpha 2-HS-glycoprotein, and alpha 1-antitrypsin. Since only dark cell granules are densely stained in the histochemical PAS staining, and because antiserum produced against the PAS-positive band selectively stained cells facing the secretory coil lumen (which are most likely dark cells), it is suggested that PAS-positive sweat glycoproteins are derived predominantly from the dark cells.

Two-dimensional isoelectric focussing/sodium dodecyl sulphate polyacrylamide gel electrophoretic mapping and some molecular characteristics of the proteins of the adult guinea-pig small intestinal microvillus membrane

(1) The adult guinea-pig small intestinal microvillus membrane was purified approximately 25-fold by both cation-precipitation and differential centrifugation methods. Comparison by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed no substantial differences in polypeptide composition between the two preparations. (2) One-dimensional SDS-PAGE and two-dimensional isoelectric focussing (IEF)/SDS-PAGE, together with Coomassie-blue, silver and lectin-staining, showed three major high molecular weight polypeptides, M r, 108 000, 116 000 and 127 000, as well as a 47 kDa protein (actin), as major constituents of the membrane. The proteins of M r 108 000 and 116 000 were strongly concanavalin A reactive. A detailed two-dimensional IEF/SDS-PAGE map of the membrane was constructed. (3) Sodium carbonate treatment showed the two concanavalin A-reactive glycoproteins, M r 108 000 and 116 000, comprising the sucrase-isomaltase complex, to be loosely-associated ‘extrinsic’ microvillus membrane proteins. Two proteins, M r 127 000 and 135 000, were tightly-associated ‘intrinsic’ microvillus proteins. (4) Despite regional differences in specific activity of some small intestinal microvillar enzymes, most noticeably enterokinase (EC 3.4.21.9) and dipeptidyl peptidase IV (EC 3.4.14.x), no substantial regional differences were seen in microvillus membrane polypeptide composition. In contrast, a substantial increase in the major high molecular weight proteins of M r 108 000 and 116 000 accompanied a 10-fold rise in sucrase-isomaltase activity, and loss of a major protein of M r 131 000 accompanied the complete loss of lactase activity from the membrane during postnatal development.

Genetic variation in the subunits of globulin-1 storage protein of French bean

Charge and molecular weight heterogeneity of globulin-1 (G1) polypeptides of the bean, Phaseolus vulgaris L., were revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Different bean cultivars were classified into three groups: 'Tendergreen', 'Sanilac', and 'Contender' on the basis of their protein subunit composition. Nine distinct major bands: α51,α49, α48.5,β48(T), β48(S), β47, γ45.5, γ45(S), and γ45(C), and two minor bands: γ46(T) and γ46(S) were found to account for the three profiles seen on one-dimensional SDS-PAGE. Two-dimensional analysis revealed these eleven protein bands to be composed of a minimum of fourteen distinct protein subunits. The 'Tendergreen' and 'Sanilac' types differ in their G1 polypeptide composition. The protein patterns of the 'Contender' types are intermediate, containing many protein subunits found in the patterns of the 'Tendergreen' and 'Sanilac' types suggesting a genetic and evolutionary relationship.