An improved formulation of SYPRO Ruby protein gel stain: comparison with the original formulation and with a ruthenium II tris (bathophenanthroline disulfonate) formulation

SYPRO Ruby protein gel stain is compatible with a variety of imaging platforms since it absorbs maximally in the ultraviolet (280 nm) and visible (470 nm) regions of the spectrum. Dye localization is achieved by noncovalent, electrostatic and hydrophobic binding to proteins, with signal being detected at 610 nm. Since proteins are not covalently modified by the dye, compatibility with downstream proteomics techniques such as matrix-assisted laser desorption/ionisation-time of flight mass spectrometry is assured. The principal limitation of the original formulation of SYPRO Ruby protein gel stain, is that it was only compatible with a limited number of gel fixation procedures. Too aggressive a fixation protocol led to diminished signal intensity and poor detection sensitivity. This is particularly apparent when post-staining gels subjected to labeling with other fluorophores such as Schiff's base staining of glycoproteins with fluorescent hydrazides. Consequently, we have developed an improved formulation of SYPRO Ruby protein gel stain that is fully compatible with commonly implemented protein fixation procedures and is suitable for post-staining gels after detection of glycoproteins using the green fluorescent Pro-Q Emerald 300 glycoprotein stain or detection of beta-glucuronidase using the green fluorescent ELF 97 beta-D-glucuronide. The new stain formulation is brighter, making it easier to manually excise spots for peptide mass profiling. An additional benefit of the improved formulation is that it permits staining of proteins in isoelectric focusing gels, without the requirement for caustic acids.     

Quantitation of glycoproteins on electroblots using the biotin-streptavidin complex

A method for the detection and quantitation of glycoproteins on nitrocellulose electroblots is described. Protein mixtures may be solubilized in sodium dodecyl sulfate prior to labeling, which is especially useful when dealing with membrane proteins. Mild periodate oxidation produces aldehydes on the oligosaccharide moieties which are then specifically condensed with biotin aminocaproyl hydrazide. After polyacrylamide gel electrophoresis and transfer of proteins onto nitrocellulose membranes, biotinylated glycoproteins are detected with enzyme-linked streptavidin and quantitated by densitometric scanning. As little as 1 ng of alpha 1-acid glycoprotein can be detected by this method. The use of mild oxidation conditions renders the method highly selective for the detection of sialic acid-containing glycoproteins.     

An improved mechanically durable electrophoresis gel matrix that is fully compatible with fluorescence-based protein detection technologies

Unfortunately, conventional large-format polyacrylamide gels are mechanically fragile, often tearing during the subsequent manipulations required for visualization of the proteins. This problem is compounded when large-format two-dimensional gels are subjected to multiple staining procedures in order to detect different classes of proteins, such as total protein, phosphoproteins, and glycoproteins. A mechanically durable liquid polyacrylamide-based matrix has been developed that, upon polymerization, facilitates the handling of one-dimensional and two-dimensional gels. The matrix, referred to as Rhinohide liquid acrylamide, is stable as a refrigerated solution for up to one year, and forms a polymer-reinforced polyacrylamide gel suitable for electrophoresis, upon addition of catalysts. The matrix is superior to previously reported durable gel matrices in that it does not cause distortion of high-molecular-weight bands and does not suffer from other spot morphology artifacts, such as doubling of protein spots in the molecular weight dimension. The matrix is particularly valuable for the analysis of proteins applying multiple applications of fluorescent dyes, as required with serial staining of proteins for phosphorylation, glycosylation, and total protein expression, using Pro-Q Diamond phosphoprotein stain, Pro-Q Emerald glycoprotein stain and SYPRO Ruby protein gel stain, respectively.     

Global quantitative phosphoprotein analysis using Multiplexed Proteomics technology

Systematic parallel analysis of the phosphorylation status of networks of interacting proteins involved in the regulatory circuitry of cells and tissues is certain to drive research in the post-genomics era for many years to come. Reversible protein phosphorylation plays a critical regulatory role in a multitude of cellular processes, including alterations in signal transduction pathways related to oncogene and tumor suppressor gene products in cancer. While fluorescence detection methods are likely to offer the best solution to global protein quantitation in proteomics, to date, there has been no satisfactory method for the specific and reversible fluorescent detection of gel-separated phosphoproteins from complex samples. The newly developed Pro-Q Diamond phosphoprotein dye technology is suitable for the fluorescent detection of phosphoserine-, phosphothreonine-, and phosphotyrosine-containing proteins directly in sodium dodecyl sulfate (SDS)-polyacrylamide gels and two-dimensional (2-D) gels. Additionally, the technology is appropriate for the determination of protein kinase and phosphatase substrate preference. Other macromolecules, such as DNA, RNA, and sulfated glycans, fail to be detected with Pro-Q Diamond dye. The staining procedure is rapid, simple to perform, readily reversible and fully compatible with modern microchemical analysis procedures, such as matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. Pro-Q Diamond dye technology can detect as little as 1-2 ng of beta-casein, a pentaphosphorylated protein, and 8 ng of pepsin, a monophosphorylated protein. Fluorescence signal intensity correlates with the number of phosphorylated residues on the protein. Through combination of Pro-Q Diamond phosphoprotein stain with SYPRO(R) Ruby protein gel stain, Multiplexed Proteomics technology permits quantitative, dichromatic fluorescence detection of proteins in 2-D gels. This evolving discovery platform allows the parallel determination of protein expression level changes and altered post-translational modification patterns within a single 2-D gel experiment. The linear responses of the fluorescence dyes utilized, allow rigorous quantitation of changes over an unprecedented 500-1000-fold concentration range.     

Proteome of the bacterium <Emphasis Type="Italic">Mycoplasma gallisepticum</Emphasis>

Using modern proteomic assays, we have identified the products of gene expression and posttranslational modifications of proteins of the bacterium Mycoplasma gallisepticum S6. Combinations of different technologies of protein separation by electrophoresis and mass-spectrometric analysis gave us a total of 446 proteins, i.e. 61% of the annotated proteins of this microorganism. The Pro-Q Diamond and Pro-Q Emerald dye technology was used for fluorescent detection of ten phosphoproteins and two glycoproteins. The acylation of proteins was studied by electrophoresis after in vivo labeling with different ¹⁴C-labeled fatty acids, followed by autoradiography. Sixteen acylated proteins were identified, with a quarter of them involved in plasma membrane construction and another quarter involved in cell energy metabolism.     

Pre‐staining of glycoprotein in SDS‐PAGE by the synthesis of a new hydrazide derivative

In this study, a new hydrazide derivative (UGF202) was synthesized and introduced as a highly sensitive and selective fluorescent probe to pre‐stain glycoproteins in 1D and 2D SDS‐PAGE. As low as 0.5–1 ng glycoproteins (transferrin, α1‐acid glycoprotein, avidin) could be selectively detected, which is comparable to that of Pro‐Q Emerald 300 stain, one of the most sensitive and commonly used glycoprotein staining kit. In addition, the specificity of the newly developed method was confirmed by the study of de‐glycosylation, glycoproteins affinity enrichment and LC‐MS/MS, respectively. According to the results, it is concluded that UGF202 pre‐stain can provide an alternative for the visualization of gel‐separated glycoproteins.     

Calcium oxalate dihydrate crystal induced changes in glycoproteome of distal renal tubular epithelial cells

Calcium oxalate dihydrate (COD) crystals can adhere onto the apical surface of renal tubular epithelial cells. This process is associated with crystal growth and aggregation, resulting in kidney stone formation. Glycoproteins have been thought to play roles in response to crystal adhesion. However, components of the glycoproteome that are involved in this cellular response remain largely unknown. Our present study therefore aimed to identify altered glycoproteins upon COD crystal adhesion onto tubular epithelial cells representing distal nephron, the initiating site of kidney stone formation. Madin-Darby Canine Kidney (MDCK) cells were maintained in culture medium with or without COD crystals for 48 h (n = 5 flasks per group). Cellular proteins were extracted, resolved by 2-DE and visualized by SYPRO Ruby total protein stain, whereas glycoproteins were detected by Pro-Q Emerald glycoprotein dye. Spot matching and quantitative intensity analysis revealed 16 differentially expressed glycoprotein spots, whose corresponding total protein levels were not changed by COD crystal adhesion. These altered glycoproteins were successfully identified by Q-TOF MS and/or MS/MS analyses, and potential glycosylation sites were identified by the GlycoMod tool. For example, glycoforms of three proteasome subunits (which have a major role in regulating cell-cell dissociation) were up-regulated, whereas a glycoform of actin-related protein 3 (ARP3) (which plays an important role in cellular integrity) was down-regulated. These coordinated changes implicate that COD crystal adhesion induced cell dissociation and declined cellular integrity in the distal nephron. Our findings provide some novel insights into the pathogenic mechanisms of kidney stone disease at the molecular level, particularly cell-crystal interactions.     

Site-directed immobilization of glycoproteins on hydrazide-containing solid supports

Methods are described for the preparation and use of solid supports containing hydrazide functions for the immobilization of glycoproteins specifically through the oligosaccharide moieties. The solid supports are prepared from commercial "active ester" agarose by reaction with hydrazine hydrate. Glycoproteins are oxidized with sodium periodate, resulting in the production of aldehydes on the oligosaccharide moieties. Oxidized glycoprotein is then reacted with the hydrazide-derivatized solid support to produce stable hydrazone linkages. Data are presented for the optimization of binding of oxidized glycoprotein to hydrazide-derivatized agarose. Agarose hydrazide/glycoprotein gels were shown to be stable from pH 3 to 10 and activity studies using immobilized avidin show that this method of immobilization results in an increased "specific activity" of bound protein when compared with standard methods of immobilization.     

Electrophoretic analysis of four high molecular weight sialoglycoproteins produced by metastatic human colon carcinoma cells

We have found that polyacrylamide gel electrophoresis in 3% gels in the presence of sodium dodecyl sulfate is suitable for the separation of cellular glycoproteins having molecular weights ranging from 200,000 to 1,000,000. The gels secured on a rigid support (Gelbond) allow blotting techniques with lectins and antibodies for the detection of glycoproteins. Using these methods we have separated lysates of HT-29 human colon carcinoma cells and detected at least four distinct high molecular weight sialoglycoproteins having molecular weights of 900,000, 740,000, 560,000, and 450,000. The expression of the 900,000 component, as revealed by wheat germ agglutinin binding, was much higher in a subline of HT-29 cells established from liver metastases in a nude mouse than it was in the parental cells. The relative intensity of wheat germ agglutinin binding to these four sialoglycoprotein components differs depending upon their growth phase in vitro. These glycoproteins were also detectable by the binding of peanut agglutinin, provided the glycoproteins were previously treated in the gels with mild acid to remove the sialic acid from their carbohydrate chains, suggesting that mucin-type carbohydrate chains are present on these glycoproteins. The same set of glycoproteins can be detected by metabolic labeling of the cells with [3H]glucosamine in tissue culture. Very similar glycoprotein profiles are revealed by metabolic labeling of fresh colon carcinoma tissues with [3H]glucosamine in vitro.     

Polyacrylic polyhydrazides as reagents for detection of glycoproteins

Glycoproteins immobilized on membranes can be detected with high selectivity and sensitivity by the four-step procedure described in this work. The glycoproteins are first oxidized by sodium periodate and then polyacrylic polyhydrazides are coupled to the aldehyde groups generated in the sugar part of the glycoproteins. In the third step, a glycoenzyme, such as horseradish peroxidase, is coupled to the remaining hydrazide groups on the polymer through the aldehydes formed in its glycan chains. In the last step, the visualization of glycoproteins is achieved through the reaction product of the bound glycoenzyme. The sensitivity of the glycoprotein detection is most critically dependent on the hydrazide reagent. Thus, dihydrazides were not satisfactory, a trihydrazide was better, and polyhydrazides were the best. Two different polyhydrazides were used. One was based on acrylamide and the other on N-acryloyl-tris(hydroxymethyl)aminomethane. The second one proved to be superior because it gave higher sensitivity with no detectable background staining. We have also investigated the influence of various reaction conditions on staining of glycoproteins having oligomannose and N-acetyllactosamine type glycan chains. Some of them, invertase and fetuin, could be detected with sensitivity similar to that of silver staining in gels and colloidal gold staining on the membranes. The detection of small quantities of Endo H-deglycosylated glycoproteins was possible under standard conditions only if several N-acetylglucosamine residues remained bound to the protein.     

Description and application of an immunological detection system for analyzing glycoproteins on blots

By introducing the steroid hapten digoxigenin specifically into sugars, a sensitive detection system for glycoproteins on blots has been developed. Sugars are oxidized to obtain aldehyde groups, which then react with digoxigenin-succinyl--amido caproic acid hydrazide. A high-affinity antibody, conjugated to alkaline phosphatase, is used for the detection of the incorporated digoxigenin.This system allows the detection of nanogram-amounts of glycoproteins on blots, and it's specificity allows a clear distinction of a glycoprotein from a non-glycoprotein. In combination with endo- and exoglycosidases it is very useful for determining the type of carbohydrate linkage in a glycoprotein, and by varying the oxidation conditions, specific labeling of sialic acids and terminal galactoses can be achieved.     

Prestaining of glycoproteins in sodium dodecyl sulfate polyacrylamide gels by dansylhydrazine

A new fluorescent prestaining method for gel‐separated glycoproteins in 1D and 2D SDS‐PAGE was developed by using dansylhydrazine in this study. The prestained gels could be easily imaged after electrophoresis without any time‐consuming steps needed for poststains. As low as 4–8 ng glycoproteins (transferrin, α1‐acid glycoprotein) could be selectively detected, which is comparable to that of Pro‐Q Emerald 488, one of the most commonly used glycoprotein stain. In addition, a subsequent study of deglycosylation, glycoprotein affinity isolation, and LC‐MS/MS analysis was performed to confirm the specificity of the newly developed method.     

Fluorescent labeling of the carbohydrate moieties of human chorionic gonadotropin and alpha 1-acid glycoprotein

A method for covalent attachment of a fluorescent molecule to the carbohydrate moieties of glycoproteins is described. The glycoproteins were oxidized with periodate under mild conditions selective for sialic acid (Van Lenten, L. and Ashwell, G. (1971) J. Biol. Chem. 246, 1889--1894). The resulting aldehydes were condensed with either dansylhydrazine, dansylethylenediamine, or fluoresceinamine followed by reduction with NaCNBH3 and NaBH4. Conjugates prepared with dansylhydrazine were found to be insufficiently stable for spectroscopic analysis, whereas the primary amines produced stable conjugates whose fluorescence polarization (P) was constant for several hours at 37 degrees C. The degree of labeling correlated roughly with the sialic acid contents of the vaious glycoproteins. Very little covalent incorporation was observed with albumin (which is devoid of carbohydrate) or with asialo alpha 1-acid glycoprotein. Exclusion chromatography in the presence of a dissociating agent was sometimes required to remove significant amounts of noncovalently adsorbed dye. Fluorescent-labeled alpha subunits of human chorionic gonadotropin were shown to recombine normally with native beta subunits. However, the labeling procedure appeared to compromise the ability of the beta subunits to recombine. Electrophoretic analysis produced evidence of covalent cross-linking between subunits following periodate oxidation of the intact gonadotropin. The possibility that primary amine groups of the protein compete with added fluorescent amines for reaction with periodate-generated aldehydes is discussed.     

Estimating glycoprotein carbohydrate chain structures by lectin reactivities in polyacrylamide gels

Complex mixtures of cellular glycoproteins contain a myriad of different carbohydrate chains that cannot be easily analyzed without rigorous purification of each individual glycoprotein. We have analyzed the carbohydrate chains in complex mixtures of cellular glycoproteins by separation using sodium dodecyl-sulfate polyacrylamide gel electrophoresis and interacting the gels with several 125I-labeled lectins. By use of in situ chemical modifications of the glycoproteins after their electrophoretic separation together with the known carbohydrate-binding specifities of several lectins, it has been possible to estimate glycoprotein carbohydrate chain structures. As an example we have examined the cellular glycoproteins of a ovary-colonizing metastatic variant of B16 melanoma and report the types of carbohydrate chains that are found on various melanoma glycoproteins.     

Optimization of erythrocyte membrane glycoprotein fluorescent labeling with dansylhydrazine after polyacrylamide gel electrophoresis

An improved procedure for the labeling of glycoproteins with dansylhydrazine subsequent to electrophoresis in polyacrylamide gels is reported. This procedure is derived from the work of Eckhardt et al. (1976, Anal. Biochem. 73, 192-197) and Weber and Hof (1975, Biochem. Biophys. Res. Commun. 65, 1298-1302) who showed that dansylhydrazine may be condensed with the aldehyde groups of oxidized glycoprotein carbohydrates and the resulting hydrazones reduced with dimethylamine borane and/or sodium borohydride. Using the known distribution of erythrocyte membrane glycoproteins as a benchmark the effect of variation of a number of process parameters was investigated and an optimal procedure identified. The procedure is shown to be relatively insensitive to moderate variations in reagent composition, pH, and time of incubation with dansylhydrazine solution or reducing agents. It is also shown that labeling patterns may be preserved in dried gels if dimethylsulfoxide is replaced or omitted from all of the process solutions and destaining is effected with 1 M sodium acetate, pH 5.6. While specifically developed for the labeling of erythrocyte membrane proteins, the procedure is demonstrated to be applicable to other glycoprotein containing preparations.     

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.     

Selective labeling of functional groups on membrane proteins or glycoproteins using reactive biotin derivatives and 125I-streptavidin

Amino groups, sulfhydryl groups or oxidation-induced aldehydes on erythrocyte membrane proteins and/or glycoproteins, were reacted with biotinyl N-hydroxysuccinimide ester (BNHS), 3-(N-maleimido-propionyl) biocytin (MPB) or biocytin hydrazide (BCHZ), respectively. The detergent-lysed biotinylated samples were subjected to SDS-polyacrylamide gel electrophoresis, and the proteins were transferred onto nitrocellulose membranes. The blot was then incubated with a solution containing 125I-streptavidin, and processed for autoradiography. The advantages of this approach over previously reported procedures for labeling the three functional groups include the following: extremely high sensitivity; short exposure times of autoradiograms and relatively low levels of radioactivity; single-step radiolabeling procedures subsequent to processing and handling of gels and no background labeling in control samples.     

Enzyme-based detection of glycoproteins on blot transfers using avidin-biotin technology

Avidin-biotin technology has been employed for the improved nonradioactive detection of glycoproteins on blots. Periodate oxidation of samples on blots converts the glycoprotein-based carbohydrate residues to the corresponding aldehydes. The latter undergo interaction with preformed complexes consisting of either avidin hydrazide or streptavidin hydrazide combined with biotinylated alkaline phosphatase. The sensitivity of the new assay exceeds the previously described enzyme hydrazide method by a factor of at least 10. The approach can be rendered selective for sialoglycoproteins, and approximately 12 sugar-containing bands could be observed in erythrocyte membrane preparations. Problems of nonspecific binding and high levels of background label were alleviated using a nonglycosylated basic protein (lysozyme) for quenching.     

Glycoprofiling of the Human Salivary Proteome

Introduction  Glycosylation is an important component for a number of biological processes and is perhaps the most abundant and complicated of the known post-translational modifications found on proteins. Methods  This work combines two-dimensional (2-D) polyacrylamide gel electrophoresis and lectin blotting to map the salivary glycome and mass spectrometry to identity the proteins that are associated with the glycome map. A panel of 15 lectins that recognize six sugar-specific categories was used to visualize the type and extent of glycosylation in saliva from two healthy male individuals. Lectin blots were compared to 2-D gels stained either with Sypro Ruby (protein stain) or Pro-Q Emerald 488 (glycoprotein stain). Results  Each lectin shows a distinct pattern, even those belonging to the same sugar-specific category. In addition, the glycosylation profiles generated from the lectin blots show that most salivary proteins are glycosylated and that the profiles are more widespread than is demonstrated by the glycoprotein-stained gel. Finally, the coreactivity between lectins was measured to determine what types of glycan structures are associated with one another and also the population variation of the lectin reactivity for 66 individuals were reported. Conclusions  This starting 2-D gel glycosylation reference map shows that the scientifically accepted, individual oligosaccharide variability is not limited to a few large glycoproteins such as MUC5B, but are found on most members of the salivary proteome. Finally, in order to see the full range of oligosaccharide distribution, multiple reagents or lectins are needed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A sensitive method for determining the phosphorylation status of natriuretic peptide receptors: cGK-Ialpha does not regulate NPR-A

Natriuretic peptide receptor A (NPR-A) and natriuretic peptide receptor B (NPR-B) are transmembrane guanylyl cyclases that catalyze the synthesis of cGMP in response to natriuretic peptides. Phosphorylation and dephosphorylation regulate these receptors and have been traditionally studied by (32)PO(4) labeling of transfected cells. However, this approach cannot be used to determine the phosphorylation state of receptors isolated from unlabeled sources. Here, we use Pro-Q Diamond and SYPRO Ruby dyes to quantify the phosphorylation status and protein levels, respectively, of natriuretic peptide receptors from tissues and cells. Strong Pro-Q Diamond signals for NPR-A and NPR-B were obtained when receptors were isolated from lung tissue, liver tissue and overexpressing cells. The level of NPR-A Pro-Q staining was also high in kidney but was much lower in heart tissue. In contrast, the SYPRO Ruby protein signal was weaker and more variable. In a direct comparison, Pro-Q Diamond staining was as sensitive as but more specific than the (32)PO(4) labeling method. The two approaches were highly correlated (R(2) = 0.98). We exploited these techniques to measure the effect of cGMP-dependent protein kinase Ialpha on the phosphate content and guanylyl cyclase activity of NPR-A. Neither value was significantly affected in cells overexpressing cGK-Ialpha or in tissues from mice lacking cGK-I. We conclude that cGK-I does not regulate the cyclase activity or phosphorylation state of NPR-A. Furthermore, we find that Pro-Q Diamond staining is a sensitive method for measuring the phosphate levels of natriuretic peptide receptors, but protein levels are best detected by Western blot analysis, not SYPRO Ruby staining.