Diagonal Electrophoresis Research Articles

Are Methionine Sulfoxide-Containing Proteins Related to Seed Longevity? A Case Study of Arabidopsisthaliana Dry Mature Seeds Using Cyanogen Bromide Attack and Two-Dimensional-Diagonal Electrophoresis.

In recent years, several reports pointed out the role of protein oxidation in seed longevity, notably regarding the oxidation of methionine (Met) residues to methionine sulfoxide (MetO) in proteins. To further consider this question, we present a handy proteomic method based on the use of two-dimensional diagonal electrophoresis (2Dd) and cyanogen bromide (CNBr) cleavage, which we refer to as 2Dd-CNBr. CNBr treatment of proteins causes the non-enzymatic hydrolysis of peptide bonds on the carboxyl side of reduced Met residues. However, Met oxidation causes a lack of cleavage, thus modifying the electrophoretic mobility of CNBr-induced peptides. This approach was first validated using bovine serum albumin as a model protein, which confirmed the possibility of distinguishing between oxidized and non-oxidized forms of Met-containing peptides in gels. Then, the 2Dd-CNBr method was applied to the Arabidopsis thaliana seed protein extract in a control (non-oxidized) condition and in an oxidized one (as obtained following hypochlorous acid treatment). Twenty-four oxidized Met residues in 19 proteins identified by mass spectrometry were found to be surface exposed in these proteins. In the three-dimensional environment of the oxidized Met, we detected amino acid residues that could be converted by oxidation (carbonylation) or by phosphorylation, suggesting a possible interplay between Met oxidation and the other protein modifications. The identification of the proteins oxidatively modified in Met residues revealed the finding that MetO-containing proteins are related to seed longevity. Based on these results, we suggest that the method presently described also has the potential for wider applications.

Phos-tag diagonal electrophoresis to analyze phosphoproteins

Phos-tag diagonal electrophoresis to analyze phosphoproteins

Phos-tag diagonal electrophoresis precisely detects the mobility change of phosphoproteins in Phos-tag SDS-PAGE

Phos-tag diagonal electrophoresis was developed to identify precisely a change in electrophoretic mobility of phosphoproteins in Phos-tag SDS-PAGE. Previously, if a single protein band was detected, it was impossible to determine whether mobility of the protein altered by Mn2+ Phos-tag in Phos-tag SDS-PAGE gels because SDS-PAGE and Phos-tag SDS-PAGE were performed on different gels. Moreover, when multiple protein bands were detected, it was difficult to determine whether the band with the highest mobility was altered mobility by Mn2+ Phos-tag. However, these problems were resolved by Phos-tag diagonal electrophoresis in which SDS-PAGE and Phos-tag SDS-PAGE patterns were provided on a single gel. Using this technique we identified phosphorylation states of various proteins such as α-lactalbumin, α- and β-casein, ovalbumin, basic 7S globulin, and 26S proteasome subunits. In the analyses of 26S proteasome subunits from humans and yeast, we could confirm that all subunits are phosphorylated, and find that the number of major proteins with different phosphorylation states is a few in each of the subunits despite having many phosphorylation sites. SignificancePreviously, Phos-tag SDS-PAGE has been developed to identify a change in electrophoretic mobility of phosphoproteins. However, we had a problem in this technique; it was often difficult to recognize the mobility shift by Mn2+ Phos-tag when we used separately SDS-PAGE and Phos-tag SDS-PAGE. Such a problem was resolved by Phos-tag diagonal electrophoresis in which SDS-PAGE and Phos-tag SDS-PAGE patterns are provided on a single gel. This technique was useful to identify phosphorylation states of various proteins.: Phos-tag diagonal electrophoresis, mass spectrometry, phosphoproteins, basic 7S globulin, proteasome

Effect of high-speed shearing treatment on dehulled walnut proteins

To understand more clearly about true characteristics of walnut protein, dehulled walnut kernels were used to prepare proteins, thus influences of polyphenols on proteins were minimized. Based on solubility, glutelin was found to be the predominant walnut protein (67.64% of total walnut proteins), meanwhile albumin and globulin accounted for 32.29%. SDS-PAGE and two-dimensional electrophoresis results showed that polypeptide profiles for albumin, globulin and glutelin exhibited significant differences. Walnut protein contained major polypeptides of MWs 30–35 kDa (pI 5.5–7.5) and 17–22 kDa (pI 9.0–10.0), along with minor polypeptides of MWs 50–73 kDa (pI 6.0–7.5) and 11–13 kDa (pI about 6.3). Disulfide bonds-linked polypeptides in walnut protein were determined with diagonal electrophoresis, and intermolecular disulfide bonds among polypeptides with MWs 30–34 kDa and 17–22 kDa were dominant. Further analysis by MALDI-TOF/TOF-MS showed that main components of walnut proteins were vicilin and legumin-like proteins. Through high-speed shearing (15,000 rpm) treatment at 60 °C for 5 min, walnut protein solubility were greatly increased from 24.33% to 62.81% at pH 8.0 and from 50.93% to 76.65% at pH 9.0. High-speed shearing treatment, especially at basic pH, could be a better way to improve protein solubility, with decreased particle sizes and more globulin and glutelin solubilized.

Ultrasound treatment improved the physicochemical characteristics of cod protein and enhanced the stability of oil-in-water emulsion.

Effects of ultrasonic pretreatment on the physicochemical changes of cod protein and the characteristics of subsequent oil-in-water emulsions were investigated. Ultrasonic treatment led to a reduction of particle size of cod protein. With increasing ultrasonic power, a significant increase in surface hydrophobicity of cod protein was found, due to the decrease of aggregates size as well as protein conformational changes of protein, but a decrease in total sulfhydryl group content was observed, probably because of the formation of disulfide bonds. Ultrasonic pretreatment promoted the adsorption of cod protein on oil droplets, resulting in higher ratios of adsorbed proteins. Diagonal electrophoresis illustrated that larger aggregates in adsorbed proteins were composed of cod actin. Reducing SDS-PAGE showed that adsorbed proteins contained a large amount of cod actin and two faint bands of light chains of myosin, and non-adsorbed proteins were composed of actin, tropomyosin, and the three light chains of myosin. More homogenous microstructures with smaller size of oil droplets were observed for the emulsions stabilized by higher intensity ultrasound treated cod protein, and the coalescence effect was improved obviously. The enhancement of the stability of cod protein coated-oil emulsions might be due to the smaller size of oil droplets and reduced oil droplets attraction by higher ratios of adsorbed proteins.

Diagonal Electrophoresis for the Detection of Proteins Involved in Disulfide Bonds.

The formation and reduction of disulfide bonds through thiol-disulfide exchange is an important cellular mechanism for the regulation of protein activity and plays a crucial role in redox signaling. Thiol-disulfide exchange on key regulatory cysteine residues offers an efficient and reversible mechanism for regulatory control in response to alterations in the intracellular redox environment. Cysteines are one of the most rarely used amino acids in proteins, but when conserved in proteins, they usually play critical roles in protein structure, function, or regulation. Diagonal electrophoresis is a relatively simple technique to analyze the formation of protein disulfides by sequential nonreducing/reducing electrophoresis. Proteins that do not form disulfides migrate identically in both dimensions and form a diagonal after the second dimension; proteins that contained intra-chain disulfides lie above the diagonal, while proteins involved in inter-disulfides fall below the diagonal. This technique allows for the detection and subsequent identification of proteins with inter-disulfides and has been applied in combination with mass spectrometry to identify proteins involved in redox signaling cascades.

Novel Observations of Peroxiredoxin-2 Profile and Protein Oxidation in Skeletal Muscle from Pigs that Differ In Residual Feed Intake and Health Status

ObjectivesEfficiency of pork production is critical as demand for animal protein rises with increases in global populations and income. Feed efficiency and response to disease challenge are affected by stressors that increase reactive oxygen species in muscle. Reactive oxygen species are detrimental to growth, as they require the expenditure of energy for tissue repair that could be used for weight gain. Peroxiredoxin-2 (Prdx-2) is an antioxidant enzyme that converts hydrogen peroxide to water using reactive cysteines, thus mitigating oxidation. Prdx-2 can exist in multiple oxidation states (reduced, oxidized, and hyperoxidized) and quantenary structures (dimer and decamer). Prdx-2 concentration is greater in more color stable muscles and may be an indicator of tenderness. The objective of this study was to determine differences in Prdx-2 profile and protein oxidation between pigs that differed in feed efficiency and health status. Materials and MethodsPigs selected for differing feed efficiency based on residual feed intake (RFI) were used in a 2 × 2 factorial design for this study. At 50 ± 7 kg in body weight, high RFI (less efficient) and low RFI (more efficient) barrows were distributed between 2 rooms in the same building. One room was inoculated with a dual respiratory/enteric bacterial health challenge. At 21 d post-infection (projected peak of illness), pigs were necropsied (total = 24, n = 6 per group) and longissimus muscle samples were collected. Reduced and nonreduced sarcoplasmic protein samples (β-mercaptoethanol) were used for western blot analyses. Reduced samples were used to quantify total Prdx-2, while nonreduced samples were used to evaluate hyperoxidized peroxiredoxin, Prdx-2 decamer, and Prdx-2 profile in nonreducing gels (2 distinct bands analyzed). Carbonyl content and diagonal gel electrophoresis were performed on sarcoplasmic protein samples to determine protein oxidation. Statistical analysis was performed using SAS v. 9.4 (SAS Inst. Inc., Cary, NC) with fixed effects of RFI line, infection status, and RFI line × infection status interaction. Gel repetition and necropsy day were included as random effects. ResultsInfection status was significant for the Prdx-2 band comparison in nonreducing gels (P = 0.014) indicating potential differences in posttranslational modifications. Compared to low RFI pigs, high RFI pigs had greater total Prdx-2 (P = 0.035), greater Prdx-2 decamer (P = 0.0007), greater Prdx-2 in the second band of nonreducing gels (P = 0.0006), and less hyperoxidized peroxiredoxin relative to the total immunoreactive protein (P = 0.028). An interaction existed between RFI line and infection status for the Prdx-2 band comparison (P = 0.02), with high RFI challenged pigs having greater ratios, and high RFI control pigs having lesser ratios relative to low RFI pigs. Visual analysis of diagonal gels showed more proteins with intermolecular disulfide formation in low RFI muscle samples. No significant difference was seen for carbonyl content. ConclusionDifferences exist in Prdx-2 profile based on RFI line and infection status. High and low RFI pigs may respond differently to health challenges at the molecular level based on differences in antioxidant protein response. By defining Prdx-2 in livestock muscle, we may be better able to manage animals to meet demand for meat without increasing inputs.

YO-03 - Acute pancreatitis and cystinosis as experimental models of disulfide stress characterized by protein cysteinylation

Disulfide stress is a specific type of oxidative stress that is associated with protein cysteinylation. The aim of this research was to characterize experimental models of disulphide stress. Thus, the redox status of free thiols and protein cysteinylation was studied in acute pancreatitis as an in vivo model of inflammation and in cystinosis an in vitro model of cystine accumulation due to its dysfunctional lysosomal transport. Cystine and homocystine levels, and protein cysteinylation rose after taurocholate-induced acute pancreatitis. Oxidation of cysteines in mitochondrial sulfide quinone oxidoreductase and 60S ribosomal protein L7a was observed. Cysteinylated albumin was also detected. Serine/threonine protein phosphatase PP2Ac, and Keap-1 were identified as targets of disulfide stress in acute pancreatitis. PP2Ac exhibited an intramolecular disulfide that was detected by diagonal electrophoresis. Silencing of PP2Ac in 266 pancreatic cells triggered up-regulation of IL-6 under basal conditions and enhanced TNF-a-induced expression of Cxcl1 and Tnf-a. Silencing of lysosomal transporter, cystinosin, triggered an increase in cystine levels and cysteinylation in PANC-1 cells. In conclusion, disulfide stress triggers protein cysteinylation associated with oxidation of the cysteine/cysteine pair in acute pancreatitis and cystinosis. PP2A is a major target in acute inflammation.

58 - DUOX1-Dependent Disulfide Bonding of Src Kinase in the Airway Epithelium

The non-receptor tyrosine kinase Src is ubiquitously expressed and is involved in regulation of cell proliferation, differentiation, and motility. Strongly linked with cancer progression, Src also contributes to non-malignant diseases such as allergic asthma. Regulation of Src activity is incompletely understood, but critically involves tyrosine (de)phosphorylation events that mediate Src “unclamping” and interactions with regulatory or substrate proteins as well as kinase activation. In addition, more recent studies indicate that Src-dependent signaling is also regulated by redox-dependent mechanisms involving reversible oxidation of one or more conserved cysteine residues. Indeed, our previous studies indicate that epithelial wound responses or innate response to airborne allergens are mediated by oxidant-mediated activation of Src by the NADPH oxidase DUOX1. However, the precise mechanisms by which cysteine oxidation regulates Src activity are still not clear, and diverse cysteine residues have been implicated. In addition to its potential role in regulating Src tyrosine kinase activity, evidence is emerging that cysteine oxidation within Src can promote intermolecular disulfide bonding, e.g. between Src monomers, with its negative regulator C-terminal Src Kinase (CSK), or its substrate cortactin. The aim of the present study was to determine whether DUOX1-dependent activation of Src oxidation in airway epithelial cells results in intermolecular disulfide cross-linking with other proteins, and to characterize Src interacting partners. Using diagonal electrophoresis to visualize intermolecular disulfides, we observed that stimulation of H292 epithelial cells with H2O2 or with ATP, which activates DUOX1, induces disulfide-based cross-linking of Src with a number of proteins. This was confirmed after immunoprecipitation of Src, and interacting proteins are being identified using mass spectrometry. We also used recombinant Src and various Cys-to-Ala variants to determine oxidant-mediated cross-linking between Src monomers, with indicated intermolecular crosslinking between Src monomers, which was diminished with Cys mutants. We seek to extend these findings in studies with epithelial cells expressing different Src Cys-to-Ala variants to gain further insight into the significance of specific cysteine oxidation events in Src-dependent activation of various signaling pathways. Supported by NIH R01 ES021476, NIH R01 HL085646, and NIH F32 HL129706.

Diagonal two-dimensional electrophoresis (D-2DE): a new approach to study the effect of osmotic stress induced by polyethylene glycol in durum wheat (Triticum durum Desf.).

Acclimatization to stress is associated with profound changes in proteome composition. The use of plant cell and tissue culture offers a means to investigate the physiological and biochemical processes involved in the adaptation to osmotic stress. We employed a new proteomic approach to further understand the response of calli to dehydration induced by polyethylene glycol (PEG6000). Calli of three durum wheat genotypes Djenah Khetifa, Oued Zenati and Waha were treated with two concentrations of polyethylene glycol to mimic osmotic stress. Changes in protein relative abundance were analyzed using a new electrophoretic approach named diagonal two-dimensional electrophoresis (D-2DE), combined with mass spectrometry. Total proteins were extracted from 30-day-old calli from three durum wheat genotypes that showed contrasting levels of drought stress tolerance in the field. The combination of one-dimensional electrophoresis and D-2DE gave a specific imprint of the protein extracts under osmotic stress, as well as characterizing and identifying individual target proteins. Of the variously expressed proteins, three were selected (globulin, GAPDH and peroxidase) and further analyzed using qRT-PCR at the transcriptome level in order to compare the results with the proteomic data. Western blot analysis was used to further validate the differences in relative abundance pattern. The proteins identified through this technique provide new insights as to how calli respond to osmotic stress. Our method of study provides an original and relevant approach of analyzing the osmotic-responsive mechanisms at the cellular level of durum wheat with agronomic perspectives.

Release behavior of non-network proteins and its relationship to the structure of heat-induced soy protein gels.

Heat-induced soy protein gels were prepared by heating protein solutions at 12%, 15% ,or 18% for 0.5, 1.0, or 2.0 h. The release of non-network proteins from gel slices was conducted in 10 mM pH 7.0 sodium phosphate buffer. SDS-PAGE and diagonal electrophoresis demonstrated that the released proteins consisted of undenatured AB subunits and denatured proteins including monomers of A polypeptides, disulfide bond linked dimers, trimers, and polymers of A polypeptides, and an unidentified 15 kDa protein. SEC-HPLC analysis of non-network proteins revealed three major protein peaks, with molecular weights of approximately 253.9, 44.8, and 9.7 kDa. The experimental data showed that the time-dependent release of the three fractions from soy protein gels fit Fick's second law. An increasing protein concentration or heating time resulted in a decrease in diffusion coefficients of non-network proteins. A power law expression was used to describe the relationship between non-network protein diffusion coefficient and molecular weight, for which the exponent (α) shifted to higher value with an increase in protein concentration or heating time, indicating that a more compact gel structure was formed.

対角線電気泳動法を用いた健常人尿中Tamm-Horsfall protein解析

対角線電気泳動法を用いた健常人尿中Tamm-Horsfall protein解析

Identification of phosphorylated form of 2′, 3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) as 46 kDa phosphoprotein in brain non-synaptic mitochondria overloaded by calcium

In our previous studies phosphorylation of several membrane-bound proteins in brain and liver mitochondria were found to be regulated by Ca(2+) as a second messenger. One of the proteins, the 46kDa phosphoprotein was found to be highly phosphorylated when Ca(2+)-induced permeability transition pore (mPTP) was opened in rat brain mitochondria (RBM). In the present study the 46kDa phosphoprotein was identified as 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) after purification by 2D diagonal electrophoresis following mass spectrometric analysis and Western blot probed with anti-CNP antibody. CNPase was discovered in immunoprecipitates of mitochondria, phosphorylated under both conditions (control and with opened mPTP). Status phosphorylation of CNPase was found to be higher in the inmmunoprecipiates of calcium-overloaded RBM. The phospohoserine and phosphotyrosine residues were detected in phosphorylated 46kDa band (CNPase) as well as in CNPase immunoprecipitates indicating possible participation of tyrosine and serine protein kinases in phosphorylation of CNPase in mitochondria. The levels of phospo-Ser and phospho-Tyr were increased in RBM with mPTP opened. It was found that CNPase substrate, 2',3'-cAMP (5μM) and, a non-competitive CNPase inhibitor, atractyloside (5μM), were able to increase the level of CNPase phosphorylation in calcium-overloaded mitochondria, while CsA (mPTP blocker) was able to strong suppress the phosphorylation of the enzyme. Collectively, our results provide evidence that Ca(2+)-stimulated and mPTP-associated CNPase phosphorylation might be an important stage of mPTP regulation in mitochondria, revealing a new function of CNPase outside of myelin structure.

Disulfide stress: a novel type of oxidative stress in acute pancreatitis

Glutathione oxidation and protein glutathionylation are considered hallmarks of oxidative stress in cells because they reflect thiol redox status in proteins. Our aims were to analyze the redox status of thiols and to identify mixed disulfides and targets of redox signaling in pancreas in experimental acute pancreatitis as a model of acute inflammation associated with glutathione depletion. Glutathione depletion in pancreas in acute pancreatitis is not associated with any increase in oxidized glutathione levels or protein glutathionylation. Cystine and homocystine levels as well as protein cysteinylation and γ-glutamyl cysteinylation markedly rose in pancreas after induction of pancreatitis. Protein cysteinylation was undetectable in pancreas under basal conditions. Targets of disulfide stress were identified by Western blotting, diagonal electrophoresis, and proteomic methods. Cysteinylated albumin was detected. Redox-sensitive PP2A and tyrosine protein phosphatase activities diminished in pancreatitis and this loss was abrogated by N-acetylcysteine. According to our findings, disulfide stress may be considered a specific type of oxidative stress in acute inflammation associated with protein cysteinylation and γ-glutamylcysteinylation and oxidation of the pair cysteine/cystine, but without glutathione oxidation or changes in protein glutathionylation. Two types of targets of disulfide stress were identified: redox buffers, such as ribonuclease inhibitor or albumin, and redox-signaling thiols, which include thioredoxin 1, APE1/Ref1, Keap1, tyrosine and serine/threonine phosphatases, and protein disulfide isomerase. These targets exhibit great relevance in DNA repair, cell proliferation, apoptosis, endoplasmic reticulum stress, and inflammatory response. Disulfide stress would be a specific mechanism of redox signaling independent of glutathione redox status involved in inflammation.

Plasma membrane-associated malate dehydrogenase of maize (Zea mays L.) roots: Native versus recombinant protein

Malate dehydrogenase (MDH, EC 1.1.1.37) is involved in several cellular processes including plant development, nutrient uptake and oxidative stress. Evidence for a plasma membrane-associated MDH has been presented for maize (Zea mays L.) roots. In the present study isoenzymes of MDH were purified from highly enriched plasma membrane preparations of maize and compared with soluble isoenzymes (Km, pH optima, pI and molecular masses). Modified SDS-PAGE analyses revealed monomers of 41 kDa for membrane-associated MDH, whereas monomers (35 kDa) and dimers (70 kDa) were detected for soluble isoenzymes. Membrane-associated MDH of cauliflower (Brassica oleracea L.) inflorescences and spinach (Spinacia oleracea L.) leaves showed molecular masses similar to the membrane-associated MDH of maize. The specific maize MDH involved was identified by mass spectrometry (ESI-QTOF-MS/MS, MALDI-TOF-MS). The corresponding gene was cloned and the protein was characterised after heterologous expression in Escherichia coli. Enzyme kinetics and properties of the recombinant and native proteins were compared. The function of thiol groups and the presence of disulphide bonds were analysed by the effect of N-ethylmaleimide, diagonal electrophoresis and labelling. Semiquantitative reverse transcription polymerase chain reaction of maize root transcripts demonstrated a constitutive expression of the gene encoding plasma membrane-associated MDH.

OmpA‐like protein influences cell shape and adhesive activity of Tannerella forsythia

Tannerella forsythia, a gram-negative fusiform rod, is implicated in several types of oral anaerobic infections. Most gram-negative bacteria have OmpA-like proteins that are homologous to the OmpA protein in Escherichia coli. We identified an OmpA-like protein in T. forsythia encoded by the tf1331 gene as one of the major proteins by mass spectrometric analysis. Two-dimensional, diagonal electrophoresis showed that the OmpA-like protein formed a dimeric or trimeric structure via intermolecular disulfide bonds. A biotin labeling experiment revealed that a portion of the protein was exposed on the cell surface, even though T. forsythia possesses an S-layer at the outermost cell surface. Using a tf1331-deletion mutant, we showed that the OmpA-like protein affected cell morphology. The length of the mutant cell was reduced almost by half. Cell swelling was observed in more than 40% of the mutant cells. Moreover, the mutant exhibited decreased adhesion to fibronectin, retarded autoaggregation, and reduced cell surface hydrophobicity. These results suggest that the OmpA-like protein in T. forsythia plays an important role in cellular integrity and adhesive function.

Electrophoresis release of hemoglobin from living red blood cells

Nowadays, proteomics research is almost always made by lysing cells to release protein first and then to analyze the protein solution in many ways, the lytic product of red blood cells is also called hemolysate, the main components of which are hemoglobin A 1 , A 2 , A 3 and carbonic anhydrase (CA), etc. In our laboratory, intact (unlysed) living red blood cells are used for direct electrophoresis so as to observe the released hemoglobin (Hb). Thus an electophoresis release experiment has been established with starch-agarose mixed gel. Electrophoresis release can be classified as initial release and re-release. In the former, the electropheresis is completed by switching on once when the release of Hb can be seen. In the latter, the electrophoresis is switched off and then switched on, when Hb release can be seen again. In our laboratory, HbA 2 phenomenon was observed in the initial release experiment and HbA 2 and HbA 1 interaction was seen in Hb cross eletrophoresis. In a two-way diagonal electrophoresis of the initial release type, HbA 2 and HbA 1 were found to exist in binding in the red blood cells. SDS-PAGE and mass spectrometry were conducted for the released products in the HbA 2 phenomenon of the electrophoresis and it was found that Prx-2 (peroxiredoxin-2) may be involved in the formation of HbA 2 phenomenon. In the re-release experiment, it was found that Hb mostly had “multi-band re-release phenomenon”. Based on this research, we designed a series of relevant experiments such as isotonic re-release, hypotonic re-release, full re-release from isolonic to hypotonic, two-way diagonal electrophoresis of re-release type, as well as comparative study of re-release of two kinds of red blood cells (red blood cells in whole blood and those isolated from it) and the influence of plasma elements on the re-release of Hb in the red blood cells. These methods have provided certain bases for research into the existence states of in vivo intracellular proteins and opened up new approaches and new fields of research.

Interactions of hemoglobin in live red blood cells measured by the electrophoresis release test

To elucidate the protein-protein interactions of hemoglobin (Hb) variants A and A(2), HbA was first shown to bind with HbA(2) in live red blood cells (RBCs) by diagonal electrophoresis and then the interaction between HbA and HbA(2) outside the RBC was shown by cross electrophoresis. The starch-agarose gel electrophoresis of hemolysate, RBCs, freeze-thawed RBCs and the supernatant of freeze-thawed RBCs showed that the interaction between HbA and HbA(2) was affected by membrane integrity. To identify the proteins involved in the interaction, protein components located between HbA and HbA(2) in RBCs (RBC HbA-HbA(2)) and hemolysate (hemolysate HbA-HbA(2)) were isolated from the starch-agarose gel and separated by 5-12% SDS-PAGE. The results showed that there was a ≈22 kDa protein band located in the RBC HbA-HbA(2) but not in hemolysate HbA-HbA(2). Sequencing by LC/MS/MS showed that this band was a protein complex that included mainly thioredoxin peroxidase B, α-globin, δ-globin and β-globin. Thus, using our unique in vivo whole blood cell electrophoresis release test, Hbs were proven for the first time to interact with other proteins in the live RBC.

Analysis of outer membrane porin complexes of Neisseria meningitidis in wild‐type and specific knock‐out mutant strains

The structure of the porin complexes of Neisseria meningitidis was assessed in the vaccine strain H44/76 and its homologous mutants lacking the main porins (PorA and PorB) and other outer membrane (OM) components (RmpM and FetA). The analysis using 1-D blue native (BN) electrophoresis, 2-D BN/SDS-PAGE and 2-D diagonal electrophoresis, followed by LC/MS-MS (for 1-D gels) or MALDI-TOF (for 2-D gels) revealed at least six porin complexes in the wild-type strain with molecular masses (MW) ranging from 145 to 195 kDa and variable composition: The two higher MW complexes are formed by PorA, PorB and RmpM, the following three are formed by PorA and PorB, and the lower MW one is formed by only PorB. Complexes in the mutants lacking either PorA, PorB or RmpM, but not those in the mutant lacking FetA, were alterered respect to those in the wild-type strain. The most evident alteration was seen in the mutant lacking PorB, in which PorA formed only a high MW complex (approximately 800 kDa). Our results suggest that PorA and PorB could form a 'basic' template for the transportation systems in the OM of the meningococci. Other proteins (such as RmpM) could be transiently associated to the porin complexes, depending on the specific tranport needs at different stages of the meningococcal life cycle, resulting in a dynamic net of pores of variable composition.

Studying the protein–protein interactions in the postsynaptic density by means of immunoabsorption and chemical crosslinking

Postsynaptic densities (PSDs), isolated from porcine cerebral cortices, are large disk-shaped aggregates consisting of hundreds of different proteins. To study the protein-protein interactions in such complex supramolecules, we developed a procedure to break up the PSD's overall structure, while preserving some interactions between individual proteins. Using the resulting PSD sample and an indirect immunoabsorption procedure, PSD-95 was isolated along with the α- and β-subunits of calcium calmodulin-dependent protein kinase II (CaMKIIα and CaMKIIβ), α-tubulin, β-tubulin, and Chapsyn110. Similarly, CaMKIIα was isolated along with CaMKIIβ, α-tubulin, β-tubulin, and small amounts of PSD-95. The proteins isolated from PSDs treated with a cleavable bifunctional crosslinking reagent were further subjected to diagonal gel electrophoresis analysis, and the results indicated that CaMKIIα resides next to α-tubulin in the PSD. Overall, the results obtained here suggest that within the PSD, large aggregates of CaMKIIα, CaMKIIβ, α-tubulin, and β-tubulin may occur that indirectly associate with PSD-95 and Chapsyn110. Such a protein organization would allow interactions with F-actin in the cytoplasm and with proteins, such as N-methyl-D-aspartate receptors, which reside on the postsynaptic membrane. Furthermore, it would facilitate binding to proteins such as the various microtubule-associated proteins that reside in the core region of the PSD.