A high-throughput microscale method to release N-linked oligosaccharides from glycoproteins for matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis

This report describes a convenient method for the rapid and efficient release of N-linked oligosaccharides from low microgram amounts of glycoproteins. A 96-well MultiScreen assay system containing a polyvinylidene difluoride (PVDF) membrane is employed to immobilize glycoproteins for subsequent enzymatic deglycosylation. Recombinant tissue-type plasminogen activator (rt-PA) is used to demonstrate the deglycosylation of 0.1-50 micrograms of a glycoprotein. This method enabled the recovery of a sufficient amount of N-linked oligosaccharides released enzymatically with peptide N-glycosidase F (PNGaseF) from as little as 0.5 microgram rt-PA for subsequent analysis by matrix-assisted laser desorption/ionization time-of-flight (MALDI- TOF) mass spectrometry. The immobilization of rt-PA to the PVDF membrane did not sterically inhibit the PNGaseF-mediated release of oligosaccharides from rt-PA as determined by tryptic mapping experiments. Comparison of the oligosaccharides released from 50 micrograms of rt-PA by either the 96-well plate method or by a standard solution digestion procedure showed no significant differences in the profiles obtained by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Both neutral and sialylated oligosaccharide standards spiked into wells were recovered equally as determined by HPAEC-PAD. One advantage of this approach is that reduction and alkylation can be performed on submicrogram amounts of glycoproteins with easy removal of reagents prior to PNGaseF digestion. In addition, this method allows 60 glycoprotein samples to be deglycosylated in 1 day with MALDI-TOF or HPAEC-PAD analysis being performed on the following day.      

A single-sample method for determination of carbohydrate and protein contents glycoprotein bands separated by sodium dodecyl sulfate- polyacrylamide gel electrophoresis.

A method is described for determination of carbohydrate and protein contents of glycoproteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then electroblotted onto polyvinylidene difluoride (PVDF) membranes. Blots were stained, and appropriate pieces of PVDF membranes were excised, destained, and subjected to sequential hydrolysis with 0.2 M trifluoroacetic acid (TFA) for 1 h at 80 degrees C, then with 2 M TFA for 4 h at 100 degrees C, and finally with 6 M HCl at 100 degrees C for 24 h to release sialic acids, neutral sugars with hexosamines, and amino acids, respectively. In some instances preliminary methanolysis was used. Carbohydrates including sialic acids were quantitated by high pH anion exchange chromatography with pulsed amperometric detection. Protein content of the bands was determined as amino acids by the fluorescamine or ninhydrin method. In the calculation of results proper adjustments were made for small amounts of fucose released by hydrolysis with 0.2 M TFA at 80 degrees C, and for partial degradation of protein during hydrolysis with 2 M TFA at 100 degrees C. Recoveries of amino acids from hydrolysates of glycoproteins that had been electroblotted onto PVDF membranes equaled those of carbohydrates. This was possible because of preliminary hydrolysis of glycoproteins with TFA, as well as washing of wet, instead of dried, PVDF membranes after hydrolysis with 6 M HCl. The two modifications increased yields of amino acids by about 30%. The method was successfully applied to the determination of molar and weight percentage composition of human transferrin, band 3 protein, glycophorin A, and alpha(1)-acid glycoprotein. In each case the results obtained for directly hydrolyzed and electrophoresed/electroblotted glycoproteins were practically identical. We also determined the glucosamine content of band 4.1 protein of erythrocytes.     

Comparative study of different fluorescent dyes for the detection of proteins on membranes using the peroxyoxalate chemiluminescent reaction

We have previously shown that the bis(2,4,6-trichlorophenyl)oxalate (TCPO)-H(2)O(2) chemiluminescent reaction in acetone can be used for the detection of proteins labeled with the fluorescent reagent 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) on polyvinylidene difluoride (PVDF) membranes. To improve this method, in this work we have designed and constructed a cell that allows us to perform this chemiluminescent reaction on PVDF membranes with a homogeneous distribution of the reagents. Using this cell we have examined the analytical properties of several recently developed fluorescent protein dyes chemically different from MDPF. We have found that the metal chelate dye SYPRO Ruby can also be excited by the high-energy intermediate produced in the TCPO-H(2)O(2) reaction.     

Live-cell visualization of transmembrane protein oligomerization and membrane fusion using two-fragment haptoEGFP methodology

Protein interactions play key roles throughout all subcellular compartments. In the present paper, we report the visualization of protein interactions throughout living mammalian cells using two oligomerizing MV (measles virus) transmembrane glycoproteins, the H (haemagglutinin) and the F (fusion) glycoproteins, which mediate MV entry into permissive cells. BiFC (bimolecular fluorescence complementation) has been used to examine the dimerization of these viral glycoproteins. The H glycoprotein is a type II membrane-receptor-binding homodimeric glycoprotein and the F glycoprotein is a type I disulfide-linked membrane glycoprotein which homotrimerizes. Together they co-operate to allow the enveloped virus to enter a cell by fusing the viral and cellular membranes. We generated a pair of chimaeric H glycoproteins linked to complementary fragments of EGFP (enhanced green fluorescent protein)--haptoEGFPs--which, on association, generate fluorescence. Homodimerization of H glycoproteins specifically drives this association, leading to the generation of a fluorescent signal in the ER (endoplasmic reticulum), the Golgi and at the plasma membrane. Similarly, the generation of a pair of corresponding F glycoprotein-haptoEGFP chimaeras also produced a comparable fluorescent signal. Co-expression of H and F glycoprotein chimaeras linked to complementary haptoEGFPs led to the formation of fluorescent fusion complexes at the cell surface which retained their biological activity as evidenced by cell-to-cell fusion.     

Sensitive fluorescent detection of protein on nylon membranes

Detection of antigen immobilized on membranes, as in Western transfers and dot enzyme linked immunosorbent assays (ELISAs), often employ antibody-enzyme conjugates and chemiluminescent or precipitated colored reaction products. Although chemiluminescent markers are sensitive, they are time-consuming because of their required exposure to X-ray film and the presence of background artifacts sometimes limits their use. This report demonstrates that direct fluorescent detection technique using nylon membranes that has higher sensitivity than chemiluminescent methods is easier to perform and has a uniform, low background. An alkaline phosphatase conjugated antibody was compared with antibody conjugated to a fluorescent phycobiliprotein (allophycocyanin) for sensitivity in both Western transfers and dot ELISA assays using mouse IgG as the membrane-bound antigen. Direct fluorescent detection of antigen-antibody complexes on positively charged nylon membrane provided better sensitivity and lower background than similar conditions using enzyme amplification and chemiluminescent detection on either nylon or PVDF membranes. Processing time was reduced by the elimination of steps associated with substrate incubation, washing and X-ray film exposures required for chemiluminescence detection. These data support the view that direct fluorescent detection can represent a significant improvement in assay sensitivity and reduction in time compared with more traditional chemiluminescent detection techniques employed in the conduct of Western transfers and dot ELISA studies.     

Detection of lipopolysaccharides blotted to polyvinylidene difluoride membranes

Bacterial lipopolysaccharide (LPS) blotted to polyvinylidene difluoride (PVDF) membranes was detected by a technique adapted from current methodologies used to detect glycoproteins. PVDF-bound LPS was coupled to a hapten and localized on the membrane by Western blotting with an antibody-alkaline phosphatase conjugate specific for the hapten. Immobilon blots could be made reversibly transparent for photography and densitometry.     

A high-affinity reversible protein stain for Western blots

We describe a reversible staining technique, using MemCode, a reversible protein stain by which proteins can be visualized on nitrocellulose and polyvinylidine fluoride (PVDF) membranes without being permanently fixed to the membrane itself. This allows subsequent immunoblot analysis of the proteins to be performed. The procedure is applicable only to protein blots on nitrocellulose and PVDF membranes. MemCode is a reversible protein stain composed of copper as a part of an organic complex that interacts noncovalently with proteins. MemCode shows rapid protein staining, taking 30s to 1 min for completion. The method is simple and utilizes convenient application conditions that are compatible with the matrix materials and the protein. The stain is more sensitive than any previously described dye-based universal protein staining system. The turquoise-blue-stained protein bands do not fade with time and are easy to photograph compared to those stained with Ponceau S. Absorbance in the blue region of the spectrum offers good properties for photo documentation and avoids interference from common biological chromophores. The stain on the protein is easily reversible in 2 min for nitrocellulose membrane and in 10 min for PVDF membrane with MemCode stain eraser. The stain is compatible with general Western blot detection systems, and membrane treatment with MemCode stain does not interfere with conventional chemiluminescent or chromogenic detection using horseradish peroxide and alkaline phosphatase substrates. The stain is also compatible with N-terminal sequence analysis of proteins.     

Application of electrophoretic methods for detection of protein-porphyrin complexes

Simple methods for detection and isolation of protein-porphyrin complexes were elaborated in our laboratory. They are based on the separation of protein-porphyrin complexes in native polyacrylamide gel and measurement of their fluorescence, with the use of two detection systems: the commercially available Gel Doc(TM) 2000 system, and a system specially designed for the purpose of these investigations, concerning protein-porphyrin interactions. The fluorescent complexes can be electro-transferred from the gel onto PVDF membrane, eluted and analyzed in order to identify the protein interacting with porphyrins.     

Detection of cellular sialic acid content using nitrobenzoxadiazole carbonyl-reactive chromophores

The selective ligation of hydrazine and amino-oxy compounds with carbonyls has gained popularity as a detection strategy with the recognition of aniline catalysis as a way to accelerate the labeling reaction in water. Aldehydes are a convenient functional group choice since there are few native aldehydes found at the cell surface. Aldehydes can be selectively introduced into sialic acid containing glycoproteins by treatment with dilute sodium periodate. Thus, the combination of periodate oxidation with aniline-catalyzed ligation (PAL) has become a viable method for detection of glycoconjugates on live cells. Herein we examine two fluorescent nitrobenzoxadiazole dyes for labeling of glycoproteins and cell surface glycoconjugates. We introduce a novel 4-aminooxy-7-nitro-benz-[2,1,3-d]-oxadiazole (NBDAO) (5) fluorophore, and offer a comparison to commercial dyes including the known 4-hydrazino-7-nitro-benz-[2,1,3-d]-oxadiazole (NBDH) (2) and Bodipy FL hydrazide. We confirm specificity for sialic acid moieties and that both dyes are suitable for in vitro and in vivo labeling studies using PAL and fluorescence spectroscopy. The dyes examined here are attractive labeling agents for microscopy, as they can be excited by a 488 nm laser line and can be made in a few synthetic steps. These carbonyl-reactive chromophores provide a one step alternative to avidin-biotin labeling strategies and simplify the detection of sialic acid in cells and glycoproteins.     

Double-layer fluorescent zymography for processing protease detection

We have developed a novel double-layer zymographic method for the detection of specific processing proteases of a target proprotease using a specific fluorescent substrate. The target processing proteases were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the gel was subsequently incubated with the target proenzyme used as the substrate. A cellulose acetate membrane was immersed in 10% glycerol and then soaked in the fluorescent substrate solution. The slab gel of the processing protease was covered with the fluorescent substrate membrane, making a double layer. The double layer was incubated at 37 degrees C, and the released fluorescent band, in which the processing protease was located, was detected using UV light. The advantages of the double-layer fluorescent zymographic method are as follows: (i) the specific detection of target proprotease using a specific substrate, (ii) a relatively rapid and sensitive method, (iii) effective detection using small amounts of crude material, and (iv) wide applications that include the detection of processing proteases and activators for target proteases. Typical examples used for the detection of the processing proteases, such as plasminogen activator, chymotrypsinogen activator, procaspase-3 processing protease and caspase-3 activators, using this new method are described in this article.     

Characterization of carbohydrates using highly fluorescent 2-aminobenzoic acid tag following gel electrophoresis of glycoproteins.

Application of the most sensitive fluorescent label 2-aminobenzoic acid (anthranilic acid, AA) for characterization of carbohydrates from the glycoproteins ( approximately 15 pmol) separated by polyacrylamide gel electrophoresis is described. AA label is used for the determination of both monosaccharide composition and oligosaccharide map. For the monosaccharide determination, bands containing the glycoprotein of interest are excised from the polyvinylidene fluoride (PVDF) membrane blots, hydrolyzed in 20% trifluoroacetic acid, derivatized, and analyzed by C-18 reversed-phase high-performance liquid chromatography. For the oligosaccharide mapping, bands were digested with peptide N-glycosidase F (PNGase F) in order to release the N-linked oligosaccharides, derivatized, and analyzed by normal-phase anion-exchange chromatography. For convenience, the PNGase F digestion was performed in 1:100 diluted ammonium hydroxide overnight. The oligosaccharide yield from ammonium hydroxide-PNGase F digestion was better or equal to all the other reported procedures, and the presumed "oligosaccharide-amine" product formed in the reaction mixture did not interfere with labeling of the oligosaccharides under the conditions used for derivatization. Sequencing of oligosaccharides can be performed using the same mapping method following treatment with an array of glycosidases. In addition, the mapping method is useful for determining the relative and simultaneous distribution of sialic acid and fucose.     

Transfer Buffer Containing Methanol Can Be Reused Multiple Times in Protein Electrotransfer

We investigated the feasibility of repeated use of transfer buffer containing methanol in electrotransfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to polyvinylidene difluoride (PVDF) membrane using a prestained protein marker of broad molecular sizes. Transfer of the antitumor protein p53 in HEK293T cell extracts, using fresh and used transfer buffer, followed by detection with anti-p53 antibody was also performed to test detectability in immunoblot. Results from these experiments indicate that the transfer buffer can be reused at least five times and maintain a similar extent of protein transfer to PVDF membrane. Repeated use of the transfer buffer containing methanol will significantly reduce the volume of hazardous waste generated and its disposal cost as well as its adverse effect on environment.     

Membrane chromatographic immunoassay method for rapid quantitative analysis of specific serum antibodies

This paper discusses a membrane chromatographic immunoassay method for rapid detection and quantitative analysis of specific serum antibodies. A type of polyvinylidine fluoride (PVDF) microfiltration membrane was used in the method for its ability to reversibly and specifically bind IgG antibodies from antiserum samples by hydrophobic interaction. Using this form of selective antibody binding and enrichment an affinity membrane with antigen binding ability was obtained in-situ. This was done by passing a pulse of diluted antiserum sample through a stack of microporous PVDF membranes. The affinity membrane thus formed was challenged with a pulse of antigen solution and the amount of antigen bound was accurately determined using chromatographic methods. The antigen binding correlated well with the antibody loading on the membrane. This method is direct, rapid and accurate, does not involve any chemical reaction, and uses very few reagents. Moreover, the same membrane could be repeatedly used for sequential immunoassays on account of the reversible nature of the antibody binding. Proof of concept of this method is provided using human hemoglobin as model antigen and rabbit antiserum against human hemoglobin as the antibody source.     

Rapid and simple single nanogram detection of glycoproteins in polyacrylamide gels and on electroblots

The fluorescent hydrazide, Pro-Q Emerald 300 dye, may be conjugated to glycoproteins by a periodic acid Schiff's (PAS) mechanism. The glycols present in glycoproteins are initially oxidized to aldehydes using periodic acid. The dye then reacts with the aldehydes to generate a highly fluorescent conjugate. Reduction with sodium metabisulfite or sodium borohydride is not required to stabilize the conjugate. Though glycoprotein detection may be performed on transfer membranes, direct detection in gels avoids electroblotting and glycoproteins may be visualized within 2-4 h of electrophoresis. This is substantially more rapid than PAS labeling with digoxigenin hydrazide followed by detection with an antidigoxigenin antibody conjugate of alkaline phosphatase, or PAS labeling with biotin hydrazide followed by detection with horseradish peroxidase or alkaline phosphatase conjugates of streptavidin, which require more than eight hours to complete. Pro-Q Emerald 300 dye-labeled gels and blots may be poststained with SYPRO Ruby dye, allowing sequential two-color detection of glycosylated and nonglycosylated proteins. Both fluorophores are excited with mid-range UV illumination. Pro-Q Emerald 300 dye maximally emits at 530 nm (green) while SYPRO Ruby dye maximally emits at 610 nm (red). As little as 300 pg of alpha 1-acid glycoprotein (40% carbohydrate) and 1 ng of glucose oxidase (12% carbohydrate) or avidin (7% carbohydrate) are detectable in gels after staining with Pro-Q Emerald 300 dye. Besides glycoproteins, as little as 2-4 ng of lipopolysaccharide is detectable in gels using Pro-Q Emerald 300 dye while 250-1000 ng is required for detection with conventional silver staining. Detection of glycoproteins may be achieved in sodium dodecyl sulfate-polyacrylamide gels, two-dimensional gels and on polyvinylidene difluoride membranes.     

Sample centrifugation onto membranes for sequencing

This paper presents a new method for adsorption of proteins in solution onto a polyvinylidene diflouride (PVDF) membrane using centrifugation. The technique uses a low molecular weight cut-off membrane (LMW) placed underneath a PVDF membrane. The paired membranes are placed in a receptacle which in turn fits into a microcentrifuge tube. During sample centrifugation, the LMW acts to increase the amount of protein that is concentrated and adsorbed onto the hydrophobic surface of the PVDF membrane. By alternating between two receptacle sizes, this method can accommodate large (greater than 10 micrograms) and small (less than 10 micrograms) amounts of sample. This paper demonstrates sample recovery for a variety of proteins as quantitated by radioactivity and amino acid analysis after centrifugation onto PVDF. Amino acid and sequence analysis results demonstrate the efficiency with which interfering buffers and sodium dodecyl sulfate are removed as a result of sample centrifugation and washing. Finally, we demonstrate the utility of this technique with samples in the low picomole range to obtain useful sequence information following electrophoretic isolation of cyanogen bromide fragments purified by high performance electrophoresis chromatography.     

Orientation of transmembrane polypeptides as revealed by antibody quenching of fluorescence

We describe here a new method, based on fluorescent techniques, for the determination of the orientation of membrane protein molecules present in vesicles. The method consists of: (a) attachment of a fluorescein derivative to sugar residues of glycoproteins and glycolipids in the cell membrane, and (b) the use of anti-fluorescein antibody, a highly efficient quencher of fluorescein fluorescence, for the quantitative evaluation of sidedness of transmembrane orientation of protein molecules in vesicles. Since antibody molecules do not permeate membranes, quenching is limited exclusively to sites exposed at the external surface of the vesicles. Addition of antibody to a fluorescently-labeled cell suspension results in a full and immediate quenching of the fluorescent signal. The method is highly sensitive (pM protein concentration), rapid and readily applicable to various vesicle preparations. With this method we assessed the orientation of vesicles derived from red blood cell membranes (ghosts) in isotonic medium and followed their inversion from right-side-out to inside-out orientation upon incubation in alkaline, low ionic strength medium.     

Simple fluorimetric method for quantification of sialic acids in glycoproteins

A simple and rapid fluorimetric method was developed for detection and quantitative analysis of sialic acids in glycoproteins. Sialic acid residues in glycoproteins were specifically oxidized with periodate at 0 degrees C for 45 min. Formaldehyde generated from carbon 9 (C-9) of sialic acid was converted specifically to fluorescent dihydropyridine derivative with acetoacetanilide and ammonia at room temperature for 10 min. The reaction products indicate intense fluorescence with excitation and emission maxima at 388 and 471 nm, respectively. When the reaction was conducted in approximately a 1-ml volume, the linearity of the calibration exhibited between 2 and 180 microg of bovine fetuin, or between 0.3 and 27 nmol of N-acetylneuraminic acid, as a model glycoprotein. The limit of detection, based on three times the standard deviation of the reagent blank, was 0.5 microg of fetuin. The proposed method was applied to determination of sialic acids in various glycoprotein samples. This proposed method is simple and obviates the heating and extraction steps. It is highly specific to sialic acids in glycoproteins and indicates no fluorescence of neutral glycoproteins.     

Chemoenzymatic Site-Specific Labeling of Influenza Glycoproteins as a Tool to Observe Virus Budding in Real Time

The influenza virus uses the hemagglutinin (HA) and neuraminidase (NA) glycoproteins to interact with and infect host cells. While biochemical and microscopic methods allow examination of the early steps in flu infection, the genesis of progeny virions has been more difficult to follow, mainly because of difficulties inherent in fluorescent labeling of flu proteins in a manner compatible with live cell imaging. We here apply sortagging as a chemoenzymatic approach to label genetically modified but infectious flu and track the flu glycoproteins during the course of infection. This method cleanly distinguishes influenza glycoproteins from host glycoproteins and so can be used to assess the behavior of HA or NA biochemically and to observe the flu glycoproteins directly by live cell imaging.     

Orientation of transmembrane polypeptides as revealed by antibody quenching of fluorescence

We describe here a new method, based on fluorescent techniques, for the determination of the orientation of membrane protein molecules present in vesicles. The method consists of: (a) attachment of a fluorescein derivative to sugar residues of glycoproteins and glycolipids in the cell membrane, and (b) the use of anti-fluorescein antibody, a highly efficient quencher of fluorescein fluorescence, for the quantitative evaluation of sidedness of transmembrane orientation of protein molecules in vesicles. Since antibody molecules do not permeate membranes, quenching is limited exclusively to sites exposed at the external surface of the vesicles. Addition of antibody to a fluorescently-labeled cell suspension results in a full and immediate quenching of the fluorescent signal. The method is highly sensitive (pM protein concentration), rapid and readily applicable to various vesicle preparations. With this method we assessed the orientation of vesicles derived from red blood cell membranes (ghosts) in isotonic medium and followed their inversion from right-side-out to inside-out orientation upon incubation in alkaline, low ionic strength medium.     

Sequential analysis of N- and O-linked glycosylation of 2D-PAGE separated glycoproteins

A robust method has been developed that allows analysis of both N- and O-linked oligosaccharides released from glycoproteins separated using 2D-PAGE and then electroblotted to PVDF membrane. This analysis provides efficient oligosaccharide profiling applicable to glycoproteomic analysis. The method involves the enzymatic release of N-linked oligosaccharides using PNGase F followed by the chemical release of O-linked oligosaccharides using reductive beta-elimination and analysis using LC-ESI-MS. Oligosaccharides from the major plasma glycoproteins with a pI between 4 and 7 were characterized from the glycoforms of haptoglobin, alpha2-HS-glycoprotein, serotransferrin, alpha1-antitrypsin, and alpha1-antichymotrypsin. It was shown that the separation of protein glycoforms evident in 2D-PAGE is partially due to the combined sialylation of the O-linked and N-linked oligosaccharides. Bi-, tri- and tetra-antennary N-linked structures, which had differing levels of sialylation and fucosylation, were found to be present on the glycoproteins analyzed, together with O-linked oligosaccharides such as mono-, and disialylated T-antigen and a disialylated core type 2 hexasaccharide. In addition, N-linked site-specific information was obtained by MALDI-MS analysis using tryptic digestion after PNGase F release of the oligosaccharides.