Enhanced protein electrophoresis technique for separating human skeletal muscle myosin heavy chain isoforms

Talmadge and Roy (J. Appl. Physiol. 1993, 75, 2337-2340) previously established a sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) protocol for separating all four rat skeletal muscle myosin heavy chain (MHC) isoforms (MHC I, IIa, IIx, IIb); however, when applied to human muscle, the type II MHC isoforms (Ila, IIx) are not clearly distinguished. In this brief paper we describe a modification of the SDS-PAGE protocol which yields distinct and consistent separation of all three adult human MHC isoforms (MHC I, IIa, IIx) in a minigel system. MHC specificity of each band was confirmed by Western blot using three monoclonal IgG antibodies (mAbs) immunoreactive against MHCI (mAb MHCs, Novacastra Laboratories), MHCI+IIa (mAb BF-35), and MHCIIa+IIx (mAb SC-71). Results provide a valuable SDS-PAGE minigel technique for separating MHC isoforms in human muscle without the difficult task of casting gradient gels.     

Electrophoretic and immunochemical evidence showing that marsupial limb muscles express the same fast and slow myosin heavy chains as eutherians

Limb muscles of eutherian (placental) mammals express a slow and three fast isoforms of myosin heavy chain (MyHC), but little is known about marsupial MyHCs. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of limb MyHCs from seven marsupial species, spanning two orders, revealed four components, each of which specifically cross-reacted in Western blots with a monoclonal antibody (mAb) against a corresponding eutherian MyHC. For all seven species, the relative mobility of the band identified by each mAb matched that in the rat, suggesting that the four are homologous to eutherian slow, 2B, 2X and 2A MyHCs, respectively, in the order of decreasing mobility. Immunohistochemical analysis of fast marsupial limb muscles identitied four different fiber populations whose relative fiber size spectra (IIA相似文献   

Method for Enhanced Separation of Cardiac Myosin Heavy Chain Isoforms by Nongradient SDS-PAGE

Abstract

Current methods of SDS-PAGE for cardiac myosin heavy-chain (MHC) isoforms are complex and require more than 24 h. The aim of the current study was to improve the methodology of gel electrophoresis for faster and efficient separation of MHC isoforms. Rat ventricle and soleus tissues were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) using tris-glycine buffer. Matrix-assisted laser desorption ionization (MALDI)–time of flight (TOF)–mass spectra (MS) of protein bands was done to identify α,β-MHC. Clear separation of α,β-MHC isoforms on SDS-PAGE was achieved and identity was confirmed by MALDI-TOF-MS. The smaller gel system drastically reduced the total run time to 2 h. The present method provides a simple, quick, and efficient protocol for cardiac α,β-MHC separation.     

Revisiting electroblotting of immobilized pH gradient gels: a new protocol for studying post-translational modification of proteins

Currently, one of the most important techniques in proteome analysis is two-dimensional electrophoresis that is widely used for separation of thousands of different protein spots. Nevertheless, characterization of special aspects in protein patterns, e.g., separation of protein isoforms generated by post-translational modifications, requires individual detection methods, e.g., immunoblotting. Blotting of proteins after fractionation in immobilized pH gradients has always caused some problems. In this paper we present an optimized protocol for immunoblotting after isoelectric focusing using immobilized pH gradient (IPG) strips cast on Net-Fix as an internal support that is permeable to electric current. The focusing procedure can be carried out in commonly used IPG systems, e.g., the IPGphor by Amersham Biosciences, where electrically assisted rehydration can be performed. This may be of interest for many laboratories, because the same system as used for the first dimension of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is involved. As an example, we describe separation and detection of up to seven isoforms of recombinant erythropoietin beta using semidry blotting of IPG strips and visualization by chemiluminescence detection.     

Effects of electrolyte modification and capillary coating on separation of glycoprotein isoforms by capillary electrophoresis

The capillary electrophoresis (CE) running electrolyte composition was optimized for the separation of selected glycoproteins. A good separation of the ovalbumin (OV) and alpha-acid glycoprotein (AAG) isoforms was achieved in 20 mmol x L(-1) N-(2-hydroxyethyl)piperazine-2'-(2-ethanesulfonic acid) (HEPES) at pH 7.0, in 20 mmol x L(-1) phosphate, pH 7.0, or in 25 mmol x L(-1) borate, pH 7.9. Various ways of suppression of the glycoprotein adsorption onto the capillary wall were compared. Alpha, omega-diamine alkanes and bis(aminoalkyl) amines were added to the CE buffers, the optimized concentration being 1 mmol x L(-1) in 20 mmol x L(-1) phosphate buffer. The OV and AAG isoforms could be separated using all the alpha,omega-diamine alkanes or bis(2-aminoethyl)amine. The length of the alkyl chain in the diaminoalkane did not influence the separation. The separation of the isoforms of pollen allergens was also tested. The effects of modification of the capillary wall by succinyl-poly-L-lysine and hydrophilic CElect-P1 capillary were compared. A decrease in the glycoprotein and protein adsorption resulted in an improved separation of the isoforms.     

A universal and rapid protocol for protein extraction from recalcitrant plant tissues for proteomic analysis

A simple and universally applicable protocol for extracting high-quality proteins from recalcitrant plant tissues is described. We have used the protocol with no modification, for a wide range of leaves and fruits. In all cases, this protocol allows to obtain good electrophoretic separation of proteins. As the protocol is rapid, universal, and compatible with silver staining, it could be used for routine protein extraction from recalcitrant plant tissues for proteomic analysis.     

Detection of transferrin isoforms in human serum: comparison of UV and ICP–MS detection after CZE and HPLC separations

Two methods for separation of transferrin (Tf) sialoforms, capillary electrophoresis (CE) and high performance liquid chromatography (HPLC) with conventional UV absorbance detection, have been investigated and compared. First, conditions affecting the separation of the Tf isoforms by capillary zone electrophoresis and HPLC were carefully optimized. The use of 15 mmol L⁻¹ borate buffer (pH 8.4) containing 3 mmol L⁻¹ diaminobutane (DAB) as additive enabled good separation of the Tf isoforms by CE (75 cm×50 μm i.d. fused silica capillary) at 25 kV. In HPLC, a gradient of ammonium acetate (from 0 to 250 mmol L⁻¹ in 45 min) buffered at pH 6 (Tris-HCl) proved suitable for separation of Tf isoforms on a Mono-Q HR 5/5 anion-exchange column. On-line specific detection of the iron associated with the different Tf isoforms, after Fe saturation, by inductively coupled plasma mass spectrometry (ICP–MS) was studied in detail to compare its analytical performance with UV detection. For both CE and HPLC an octapole reaction system (ORS) ICP–MS instrument was used to minimize polyatomic interferences on the ⁵⁶Fe major isotope. Limits of detection of the different isoforms were in the range of 0.02–0.04 μmol L⁻¹ Tf for HPLC–ICP (ORS)–MS. This hybrid technique proved more selective and reliable detection of transferrin isoforms with 2, 3, 4, 5, and 6 sialic acid residues (S₂, S₃, S₄, S₅, and S₆) in real serum samples. Interesting results from iron speciation of Tf in serum from healthy individuals and from pregnant women are given.     

Simultaneous detection of molecular weight and activity of adenylate kinases after electrophoretic separation

Adenylate kinases (AKs) are ubiquitous monomeric phosphotransferases catalyzing the reversible reaction, AMP + MgATP = ADP + MgADP, which plays a pivotal role in the energetic metabolism. In vertebrates, six AK isoforms are known. In this work, we report the detection of many AK isoforms directly on gel or NC after separation by denaturing electrophoresis and electroblotting, by an optimized protocol for the enzyme detection. The method allows to clarify the apparent MW of most of those AK isozymes that follow the cited reaction, especially onto NC where bands are sharper due to the absence of protein diffusion. In contrast, GTP:AMP phosphotransferases are not detectable. AK activity from many sources can be detected in both its reaction courses; ATP production appears as dark-blue bands, while ADP formation appears as nonfluorescent bands over a fluorescent background, under long-wavelength UV light. We show that nondenaturing gel electrophoresis is not the first choice for AK activity detection. Our method is different from the preceding reports on AK activity detection in bacteria after native polyacrylamide gel separations, in the absence of SDS or methanol. The procedure is also quantitative, allowing to determine the amount of enzyme present in samples.     

Coupling of native IEF and extended X-ray absorption fine structure to characterize zinc-binding sites from pI isoforms of SOD1 and A4V pathogenic mutant

Extended X-ray absorption fine structure (EXAFS) has already provided high-resolution structures of metal-binding sites in a wide variety of metalloproteins. Usually, EXAFS is performed on purified metalloproteins either in solution or crystallized form but purification steps are prone to modify the metallation state of the protein. We developed a protocol to couple EXAFS analysis to metalloprotein separation using native gel electrophoresis. This coupling opens a large field of applications as metalloproteins can be characterized in their native state avoiding purification steps. Using native isoelectric focusing, the method enables the EXAFS analysis of metalloprotein pI isoforms. We applied this methodology to SOD1, wild-type, and Ala4Val mutant (A4V), a mutation found in amyotrophic lateral sclerosis (ALS) because decreased Zn affinity to SOD1 mutants is suggested to be involved in the pathogenesis of this neurodegenerative disease. We observed similar coordination structures for Zn in wild-type and mutant proteins, in all measured pI isoforms, demonstrating the feasibility of EXAFS on electrophoresis gels and suggesting that the Zn-binding site is not structurally modified in A4V SOD1 mutant.     

CE methods for analysis of isoforms of prostate-specific antigen compatible with online derivatization for LIF detection

Prostate-specific antigen (PSA) is the usual biomarker for prostate cancer (PCa). However, its lack of selectivity has lead to the search for new biomarkers. PSA glycosylation seems to depend on the pathophysiological conditions of the individual. Thus, methods to separate PSA isoforms (peaks) to study their role as PCa markers are needed. In this work, CE methods for PSA isoforms separation, based on the use of different dynamic coatings, are developed using UV detection. Three complementary CE methods allowing the separation of 8 or 9 PSA isoforms are selected. The longest method takes only 17?min, while the shortest one separates 9 isoforms in < 8?min. Depending on the isoforms of interest for their use as PCa biomarker, the CE method to be used can be chosen or various of them can be combined. A remarkable aspect of these methods is that the BGEs employed are devoid of compounds with primary amino groups, making the CE methods compatible with fluorescent on-column derivatization through amino residues. As a proof-of-concept, a preliminary result shows that LIF detection of labeled PSA analyzed by one of the three developed methods permits detection of glycoprotein isoforms.     

双消除法一锅简便合成1－丙炔基芳香化合物

报道了一种便捷合成1-丙炔基芳香化合物的新方法. 在充分探讨反应条件的基础上, 利用硫试剂乙基苯基砜与芳香醛组合, 采用双消除反应成功地一锅合成了一系列1-丙炔基芳香化合物1a～1s. 运用此法不仅合成出一些含有醚键、萘环、杂环以及含有两个1-丙炔基的化合物, 重要的是芳环上含有卤原子(Br和I)的化合物也能顺利地合成出来. 该方法原料易得, 操作简单, 产物容易分离纯化并且产率理想.     

Analysis of alpha-1-acid glycoprotein isoforms using CE-LIF with fluorescent thiol derivatization

The analysis of glycoprotein isoforms is of high interest in the biomedical field and clinical chemistry. Many studies have demonstrated that some glycoprotein isoforms could serve as biomarkers for several major diseases, such as cancers and vascular diseases, among others. Capillary zone electrophoresis (CZE) is a well-established technique to separate glycoprotein isoforms, however, it suffers from limited sensitivity when UV-Vis detection is used. On the other hand, with laser-induced fluorescence (LIF) detection, derivatization reaction to render the proteins fluorescent can destroy the resolution of the isoforms. In this work, a derivatization procedure through the thiol groups of glycoproteins using either 5-(iodoacetamide) fluorescein (5-IAF) or BODIPY iodoacetamide is presented with the model protein of alpha-1-acid glycoprotein (AGP). The derivatization process presented enabled high-resolution analysis of AGP isoforms by CZE-LIF. The derivatization procedure was successfully applied to label AGP from samples of serum and secretome of artery tissue, enabling the separation of the AGP isoforms by CE-LIF in natural samples at different concentration levels.     

High-resolution separations of protein isoforms with liquid chromatography time-of-flight mass spectrometry using polymer monolithic capillary columns

The separation of intact proteins, including protein isoforms arising from various amino-acid modifications, employing a poly(styrene-co-divinylbenzene) monolithic capillary column in high-performance liquid chromatography coupled on-line to a time-of-flight mass spectrometer (MS) is described. Using a 250 mm × 0.2 mm monolithic capillary column high-sensitivity separations yielding peak capacities of >600 were achieved with a 2h linear gradient and formic acid added in the mobile phase as ion-pairing agent. The combination of high-resolution chromatography with high-accuracy MS allowed to distinguish protein isoforms that differ only in their oxidation and biotinylation state allowing the separation between structural isoforms. Finally, the potential to separate proteins isoforms due to glycosylation is discussed.     

Profiling of erythropoietin products by capillary electrophoresis with native fluorescence detection

The potential of CE with native fluorescence detection (Flu) for the profiling of the therapeutic protein erythropoietin (EPO) was studied. EPO is a highly heterogeneous glycoprotein comprising a large number of isoforms. CE was applied to induce separation among the various glycoforms. Native Flu of EPO provided high detection selectivity yielding good signal‐to‐noise ratios and stable baselines, particularly when compared to conventional UV absorbance detection. In order to enhance EPO isoform resolution, CE was performed using a capillary with a neutral coating in combination with a simple BGE of 2.0 M acetic acid (pH 2.1). CE‐Flu analysis of the EPO biological reference preparation of the European Pharmacopeia resulted in a highly detailed glycoform profile. Migration time RSDs for selected EPO isoforms were less than 0.22% and 0.80% for intraday and interday repeatability, respectively. RSDs for relative peak intensity of the major EPO isoforms were less than 3%. The achieved resolution, migration time stability, and sensitivity allowed discrimination of different EPO products (EPO‐α and EPO‐β) based on the recorded glycoform pattern. The developed CE‐Flu method is relatively straightforward, and shows potential for quality control in biopharmaceutical production.     

On-line immunoaffinity capillary electrophoresis based on magnetic beads for the determination of alpha-1 acid glycoprotein isoforms profile to facilitate its use as biomarker

An immunoaffinity purification method coupled on-line to capillary electrophoresis (IACE) which allows the determination of several isoforms of intact alpha-1 acid glycoprotein (AGP) in serum samples using UV detection is developed. The immunoaffinity step is based on anti-AGP antibodies (Abs) covalently bound to magnetic beads (MBs) which are captured at the inlet end of the capillary using permanent magnets placed inside the cartridge of the CE instrument. The on-line method includes injection of the MBs with the Ab bound (MBs–Ab) and their trapping by the magnets at the entrance of the separation column, injection of serum sample and capture of AGP by the Abs, release of captured AGP, focus of desorbed protein, separation of AGP isoforms, and removal of MBs–Ab. The optimization of the different factors involved in each step allowed purification, separation and detection of AGP isoforms in a single electrophoretic analysis in about 1 h. Automation, sample and reagents consumption as well as analysis time was improved compared to off-line alternatives which use purification of AGP in an immunochromatographic column and CE separation of AGP isoforms in two independent operations. The analytical methodology developed allows the separation of 10 AGP isoforms in serum samples from a healthy donor. For a serum sample, precision (expressed as relative standard deviation) in terms of corrected area percentage was better than 0.5% for each peak accounting for more than 10% of total AGP and it was better than 4.0% in terms of relative migration time of each AGP isoform considering the whole process.     

金属硫蛋白异构体及亚型异构体的液相色谱分离与质谱鉴别

建立了金属硫蛋白(MT)异构体及亚型异构体的色谱分离与质谱鉴别方法。将金属硫蛋白混合物通过弱阴离子DEAE Sephadex A-25离子交换柱,结合离线电感耦合等离子体质谱(ICP-MS)对锌诱导金属硫蛋白的两个异构体MT-1和MT-2进行分离和检测;利用Sephadex G-25凝胶排阻色谱柱对得到的两个金属硫蛋白异构体进行脱盐;探索脱盐后的金属硫蛋白异构体在不同色谱条件下的C18反相色谱柱上的保留行为,进而实现各个亚型异构体的分离;通过在线电喷雾质谱检测实现了对金属硫蛋白各个亚型异构体的鉴别。结果表明,通过优化色谱条件,由离子交换色谱及凝胶排阻色谱得到的金属硫蛋白各亚型异构体在酸性条件下均得到了良好的分离,质谱检测结果与前人的文献报道结果一致。该方法可使金属硫蛋白各异构体均达到最佳的分离效果。     

A fast and reliable route integrating calibration and analysis protocols for water-soluble vitamin determination on microchip-electrochemistry platforms

A novel analytical route to determine water-soluble vitamins (B group and C) using single channel microchip-electrochemistry platforms is presented. The electrochemical detection protocol was carefully optimized, and it was shown that it was crucial to use 1 M nitric acid in the detector compartment to detect folic acid. A phosphate buffer (pH 6, 10 mM) and a separation voltage of 2 kV gave the complete separation of vitamins in less than 130 s, with good reproducibility (RSDs less than 10%) and accuracy (error less than 9%). In addition, a methodological innovation integrating calibration and analysis of water-soluble vitamins on the chip is also proposed. The strategy consisted in sequentially using both reservoirs (named calibration and analysis reservoirs) as well as a calibration factor (defined as signal/concentration of analyte). The analytical route required 350 s in the overall protocol (employing 130 s in calibration plus 130 s in analysis), an improvement over the times used in both conventional and microchip protocols.     

Evaluation of capillary isoelectric focusing in glycerol-water media with a view to hydrophobic protein applications

Capillary isoelectric focusing (CIEF) separations are usually performed with neutral coated fused-silica capillaries in aqueous anticonvective media. Glycerol, a very viscous solvent (eta = 945 mPa x s at 25 degrees C), known to help stabilize any kind of proteins and solubilize hydrophobic ones, was tested as an alternative to using commercial gels. Viscosity and electroosmotic mobility were measured as a function of gel or glycerol content in water, and a 30:70 v/v glycerol-water medium appeared as a good compromise for performing CIEF in a bare fused-silica capillary without imposing too high a viscosity. To demonstrate the feasibility of this new CIEF system, a standard mixture of nine model proteins was separated according to their pI with a good agreement between experimental and literature aqueous pIs. Moreover, better resolution was achieved with this system than with the conventional aqueous CIEF system, as two of the model proteins could not be separated in the latter system. Glycerol-water CIEF in bare silica capillary was next applied to the separation of horse radish peroxidase, a complex mixture of protein isoforms. The good concordance with the separation obtained by the conventional CIEF system indicated the adequacy of this new system. Finally, as anticipated from the results obtained for the separation of bacteriorhodopsin, a membrane protein, glycerol-water CIEF performed in bare silica capillary appears to be a promising alternative to conventional aqueous CIEF for hydrophobic protein characterization, under their native form.     

Detection of isoforms of recombinant human erythropoietin by various plant lectins after isoelectric focusing

A screening method to determine the binding behavior of lectins toward recombinant human erythropoietin (rHuEPO) was developed. Twenty-three different lectins were tested for this purpose. rHuEPO isoforms were separated by isoelectric focusing using the International Olympic Committee (IOC) and World Anti-Doping Agency (WADA) accredited method for the direct detection of the prohibited doping substance erythropoietin (EPO). For the visualization of the rHuEPO isoforms lectins were used instead of antibodies. Optimization of the screening protocol enabled the detection of a maximum number of rHuEPO isoforms. By means of this protocol information about the binding properties of a lectin toward each individual rHuEPO isoform was accessible. All evaluated lectins showed significant differences in their binding behavior. The most intense response was obtained with WGA, DSL, PHA-E, LEL, PSA, and LCA. While WGA, DSL, PHA-E, and LEL were able to bind all isoforms detected by the standard antibody, LCA and PSA demonstrated a clear preference for rHuEPO isoforms located in the more basic region of the electropherogram. Further lectins tested were ConA, succWGA, PHA-L, RCA, SNA, MAA, STL, ECL, GSL-II, SJA, SBA, UEA-I, Jacalin, PNA, DBA, GSL-I, and VVA. Compared to the lectins mentioned above, they showed reduced sensitivity. Endogenous and recombinant EPO only differ in the composition of their N- and O-glycan moieties. As lectins possess the unique ability to recognize subtle differences in glycan substructures, they represent an interesting approach for their structural characterization. Furthermore, they might be useful for affinity enrichment/purification of rHuEPO in doping control.     

Arene diazonium saccharin intermediates: a greener and cost-effective alternative method for the preparation of aryl iodide

In the current protocol, the arene diazonium saccharin derivatives were initially produced from various substituted aromatic amines; subsequently, these intermediates were treated with a greener organic iodide for the preparation of the aryl iodide. We tried to choose low-cost, commercially available, biodegradable, recoverable, ecofriendly, and safe reagents and solvents. The arene diazonium saccharin intermediates could be stored in the liquid phase into a refrigerator for a long time with no significant loss activity. The outstanding merits of the current protocol (a) included the partial recovering of saccharin and tetraethylammonium salt, (b) reduce the use of solvents and the reaction steps due to eliminating separation and purification of intermediates, (c) good yield of the sterically hindered substrates, and (d) avoid the generation of heavy metal or corrosive waste.