RESUMEN
Avidin functional affinity electrophoresis (AFAEP) is a variational method of affinity electrophoresis. In this technique, avidin is immobilized within a small area of the gel matrix by interaction with acrylamide and/or polyacrylamide either directly or through bifunctional linker glutaraldehyde during polymerization. Analytes can be heated with Tris-glycine sodium dodecyl sulfate (SDS) sample buffer so that biotinylated peptides/proteins are negatively charged and migrate electrophoretically towards the cathode through the avidin zone regardless of their isoelectric point (pI) values. Alternatively, if the behavior of the biotinylated analytes is known, the SDS treatment is not required. The polarity of the electrodes is set such that biotinylated analytes migrate electrophoretically through the avidin zone. This technique can work with or without SDS in gel running buffer. The AFAEP method allows the capture and concentration of biotinylated peptides/proteins. The values of this technique stem from a combination of merits of polyacrylamide gel electrophoresis and affinity technology.
Asunto(s)
Biotinilación/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Péptidos/aislamiento & purificación , Proteínas/aislamiento & purificación , Glicoproteínas/aislamiento & purificación , Humanos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
We present an improved protocol for coupling synthetic peptides to carrier proteins. In this protocol, dimethyl-formamide is used as the solvent to solubilize peptides instead of phosphate-buffered saline (PBS) or 6 M guanidine-HCl/0.01 M phosphate buffer (pH 7). Additionally, the last desalting or dialyzing step to remove uncoupled peptides as in the traditional method is eliminated. Finally, 3 ml of 0.1 M ammonium bicarbonate is added to the carrier protein conjugated peptide solution to help the lyophilization process. Coupling of Cys-containing synthetic peptides to keyhole limpet hemocyanin or bovine serum albumin using m-maleimidobenzoyl-N-hydroxysuccinimide ester are used as the test cases. This method produces high-quality antipeptide antibodies. Also, compared to the traditional method, this procedure is simpler and useful for peptides with solubility problems in PBS or 6 M guanidine-HCl.
Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/inmunología , Dimetilformamida , Inmunoglobulinas/biosíntesis , Péptidos/síntesis química , Secuencia de Aminoácidos , Proteínas Portadoras/metabolismo , Datos de Secuencia Molecular , Péptidos/inmunología , Péptidos/metabolismo , SolubilidadRESUMEN
A method, which utilizes microwave-assisted partial acid hydrolysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), to elucidate oligosaccharide composition of intact glycoproteins is presented here. Glycoproteins, such as ribonuclease B, avidin, alpha1-acid glycoprotein, and fetuin, are used as model systems to demonstrate this technique. Partial cleavage of oligosaccharides from whole intact glycoproteins with trifluoroacetic acid was observed after a short exposure to microwaves. Due to the high-resolution mass spectra obtained by MALDI-TOFMS from glycoproteins with molecular weights less than 20 kDa, the compositions of oligosaccharides are readily derived for ribonuclease B and avidin. The data agree with the proposed oligosaccharide structures of ribonuclease B (five glycoforms) and avidin (eight glycoforms). Larger glycoproteins such as alpha1-acid glycoprotein (many glycoforms) and fetuin (many glycoforms) exhibited only broad peaks with no glycoform resolution. Nevertheless, this method can be used successfully for analysis of glycoproteins with molecular weights greater than 20 kDa to determine the presence or absence of glycosylation.
Asunto(s)
Glicoproteínas/química , Microondas , Oligosacáridos/análisis , Oligosacáridos/química , Ácidos/química , Secuencia de Aminoácidos , Animales , Avidina/química , Clara de Huevo , Concentración de Iones de Hidrógeno , Hidrólisis , Datos de Secuencia Molecular , Ribonucleasas/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Microwave-assisted partial acid hydrolysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were used to study oligosaccharide structures of glycopeptides. Tryptic N-glycosylated peptides of horseradish peroxidase, with MH+ ions at m/z 2533, 2612, 3355, 3673, and 5647, were used as test cases. Within a microwave exposure with trifluoroacetic acid of 2 min, partial cleavages of the oligosaccharides of these tryptic N-glycosylated peptides were observed. The data showed that the most labile group within the oligosaccharides is the fucose (Fuc) residue, and that a majority of the end cleavage products are peptides with one N-acetylglucosamine (GlcNAc) residue linked to asparagine (Asn). In addition, the glycopeptides with m/z 3355 and 3673 carry an oligosaccharide (Xyl)Man3(Fuc)GlcNAc2, the glycopeptide at m/z 5647 carries two oligosaccharides (Xyl)Man3(Fuc)GlcNAc2, and the glycopeptides at m/z 2612 and 2533 carry (Xyl)Man3GlcNAc2 and (Fuc)GlcNAc, respectively. However, the glycosylation site of the m/z 2612 peptide at Asn286 is partially occupied. This simple and rapid method is particularly useful in identifying glycopeptides and obtaining monosaccharide compositions of glycopeptides.
Asunto(s)
Ácidos/química , Glicopéptidos/química , Microondas , Oligosacáridos/análisis , Oligosacáridos/química , Secuencia de Aminoácidos , Conformación de Carbohidratos , Peroxidasa de Rábano Silvestre/química , Hidrólisis , Datos de Secuencia Molecular , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Modifications to antibody affinity electrophoresis for improved detection of proteins have been developed. The bifunctional linker glutaraldehyde is added to the polyacrylamide gel solution for better incorporation of the bait antibody into a distinct region of a 10% w/v polyacrylamide gel. The addition of glutaraldehyde alleviates the need of an electrophoresis buffer with a specific pH. The protein sample to be analyzed is treated with 2% w/v sodium dodecyl sulfate (SDS) to ensure that they carry a negative charge. The negative charge will allow the proteins to migrate towards the cathode and hence pass through the area embedded with the bait antibody. It is observed that electrophoretic migration of bovine serum albumin (BSA) or protein G ceases upon encounter with anti-BSA whereas proteins ovalbumin, beta-lactoglobulin A, and myoglobin migrate freely. However, the addition of 0.1% w/v SDS in the native gel running buffer disrupts the antibody-antigen bond and neither BSA nor protein G can be captured by anti-BSA.
Asunto(s)
Reacciones Antígeno-Anticuerpo , Antígenos/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida/métodos , Proteínas/aislamiento & purificación , Anticuerpos/química , Antígenos/química , Glutaral/química , Inmunoglobulina G/química , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/aislamiento & purificación , Proteínas/química , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/aislamiento & purificación , Dodecil Sulfato de Sodio/químicaRESUMEN
Avidin functional affinity electrophoresis (AFAEP) is a new method of affinity electrophoresis. In this technique, bifunctional linker glutaraldehyde is added to the polyacrylamide gel solution to embed avidin within the gel matrix by interaction with the amino/amide groups. Samples are heated with triglycine sodium dodecyl sulfate (SDS) sample buffer to ensure that biotinylated proteins biotinylated peptides are negatively charged and migrate electrophoretically toward the cathode through the avidin zone regardless of their pI values. The AFAEP method allows the capture and concentration of biotinylated proteins or biotinylated peptides irrespective of the use of SDS in both the sample buffer and the gel running buffer.
Asunto(s)
Avidina/metabolismo , Tampones (Química) , Electroforesis en Gel de Poliacrilamida/métodos , Péptidos/química , Péptidos/metabolismo , Proteínas/química , Proteínas/metabolismo , Dodecil Sulfato de Sodio/farmacología , Animales , Biotinilación , Bovinos , Pollos , Humanos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The protective antigen (PA) of Bacillus anthracis plays a crucial role in the pathogenesis of the anthrax disease. The fourth domain of PA (PA-D4) is responsible for initial binding of the anthrax toxin to the cellular receptor, and thus, is an attractive target for structure-based drug therapies. A synthetic gene for PA-D4 has been prepared by recursive PCR. PA-D4 has been expressed as a fusion protein in Escherichia coli. PA-D4 has been purified to near homogeneity and its identity has been verified by mass spectrometry. The recombinant PA-D4 exhibits CD and NMR spectra that suggest that it is folded and amenable for biophysical studies. Moreover, recombinant PA-D4 binds to HeLa cells, which suggests that recombinant PA-D4 is functional to bind to its cellular receptor.