Hydrazide Beads Research Articles

The proteome of the blood–brain barrier in rat and mouse: highly specific identification of proteins on the luminal surface of brain microvessels by in vivo glycocapture

BackgroundThe active transport of molecules into the brain from blood is regulated by receptors, transporters, and other cell surface proteins that are present on the luminal surface of endothelial cells at the blood–brain barrier (BBB). However, proteomic profiling of proteins present on the luminal endothelial cell surface of the BBB has proven challenging due to difficulty in labelling these proteins in a way that allows efficient purification of these relatively low abundance cell surface proteins.MethodsHere we describe a novel perfusion-based labelling workflow: in vivo glycocapture. This workflow relies on the oxidation of glycans present on the luminal vessel surface via perfusion of a mild oxidizing agent, followed by subsequent isolation of glycoproteins by covalent linkage of their oxidized glycans to hydrazide beads. Mass spectrometry-based identification of the isolated proteins enables high-confidence identification of endothelial cell surface proteins in rats and mice.ResultsUsing the developed workflow, 347 proteins were identified from the BBB in rat and 224 proteins in mouse, for a total of 395 proteins in both species combined. These proteins included many proteins with transporter activity (73 proteins), cell adhesion proteins (47 proteins), and transmembrane signal receptors (31 proteins). To identify proteins that are enriched in vessels relative to the entire brain, we established a vessel-enrichment score and showed that proteins with a high vessel-enrichment score are involved in vascular development functions, binding to integrins, and cell adhesion. Using publicly-available single-cell RNAseq data, we show that the proteins identified by in vivo glycocapture were more likely to be detected by scRNAseq in endothelial cells than in any other cell type. Furthermore, nearly 50% of the genes encoding cell-surface proteins that were detected by scRNAseq in endothelial cells were also identified by in vivo glycocapture.ConclusionsThe proteins identified by in vivo glycocapture in this work represent the most complete and specific profiling of proteins on the luminal BBB surface to date. The identified proteins reflect possible targets for the development of antibodies to improve the crossing of therapeutic proteins into the brain and will contribute to our further understanding of BBB transport mechanisms.

Post‐translational modification and phenotype

Post‐translational modification and phenotype

Carbohydrate Background Removal in Metabolomics Samples.

NMR-basedmetabolomics is a powerful tool to comprehensively monitor chemical processes in biological systems. Key to its success is the accurate and complete metabolite identification and quantification. Due to the inherent complexity of most metabolic mixtures, NMR peak overlap can make data analysis of 1D or even 2D NMR spectra challenging, especially for the 1H spectral region from 3.2-4.5 ppm that is dominated by carbohydrates and their derivatives. To address this problem, we present an effective method for carbohydrate signal removal in complex metabolomics samples by oxidation via the addition of sodium periodate (NaIO4). In an optional step, reaction products can be removed with hydrazide beads. The treated samples show substantially simplified 1D and 2D NMR spectra with their carbohydrate peaks removed, whereas noncarbohydrate peaks remain mostly unaffected. This allows the unrestricted detection of those metabolites that are otherwise obscured by carbohydrate signals. The method was first tested for metabolite model mixtures and then applied to urine and serum samples. It revealed a significant number of noncarbohydrates that were made unambiguously observable and identifiable by this method. The proposed protocol is simple and it is suitable for high-throughput sample treatment for the comprehensive metabolite identification in a broad range of samples.

Chemoenzymatic Approach for the Proteomics Analysis of Mucin-Type Core-1 O-Glycosylation in Human Serum.

Human serum is a complex body fluid that contains various N-linked and O-linked glycoproteins. Compared with N-linked glycoproteins, the serum O-linked glycoproteins are not well-studied due to their high heterogeneity and their low abundance. Herein, we presented a novel chemoenzymatic method to analyze core-1 type of O-GalNAcylation in human serum. In this approach, the tryptic digest of serum was first subjected to PNGase F treatment to release the N-glycan and was then treated with strong acid to release sialic acid residues from mucin-type O-glycans. In this way, the internal Gal/GalNAc residues were exposed and were oxidized by the galactose oxidase to carry the aldehyde groups. The oxidized O-GalNAcylated peptides were then captured by hydrazide beads and eluted with methoxylamine for LC-MS/MS analysis. The de-N-deglycosylation decreased the abundance of N-glycopeptides, the desialylation simplified the O-glycans and the enzymatic oxidization conferred the enrichment specificity. We have demonstrated that this method was fitted to analyze O-GalNAcylated peptides with high confidence. This method was applied to analyze human serum, which resulted in the identification of 59 O-GalNAc modified peptide sequences corresponding to 38 glycoproteins from 50 μL of serum. This method is expected to have broad applications in the analysis of O-glycoproteome.

Differentiation of Sialyl Linkage Isomers by One-Pot Sialic Acid Derivatization for Mass Spectrometry-Based Glycan Profiling.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been used for high-throughput glycan profiling analysis. In spite of the biological importance of sialic acids on nonreducing ends of glycans, it is still difficult to analyze glycans containing sialic acid residues due to their instability and the presence of linkage isomers. In this Article, we describe a one-pot glycan purification/derivatization method employing a newly developed linkage-specific sialic acid derivatization for MS-based glycan profiling with differentiation of sialyl linkage isomer. The derivatization, termed sialic acid linkage specific alkylamidation (SALSA), consists of sequential two-step alkylamidations. As a result of the reactions, α2,6- and α2,3-linked sialic acids are selectively amidated with different length of alkyl chains, allowing distinction of α2,3-/α2,6-linkage isomers from given mass spectra. Our studies using N-glycan standards with known sialyl linkages proved high suitability of SALSA for reliable relative quantification of α2,3-/α2,6-linked sialic acids compared with existing sialic acid derivatization approaches. SALSA fully stabilizes both α2,3- and α2,6-linked sialic acids by alkylamidation; thereby, it became possible to combine SALSA with existing glycan analysis/preparation methods as follows. The combination of SALSA and chemoselective glycan purification using hydrazide beads allows easy one-pot purification of glycans from complex biological samples, together with linkage-specific sialic acid stabilization. Moreover, SALSA-derivatized glycans can be labeled via reductive amination without causing byproducts such as amide decomposition. This solid-phase SALSA followed by glycan labeling has been successfully applied to human plasma N-glycome profiling.

Comprehensive Profiling of Glycosphingolipid Glycans Using a Novel Broad Specificity Endoglycoceramidase in a High-Throughput Workflow.

The biological function of glycosphingolipids (GSLs) is largely determined by their glycan headgroup moiety. This has placed a renewed emphasis on detailed GSL headgroup structural analysis. Comprehensive profiling of GSL headgroups in biological samples requires the use of endoglycoceramidases with broad substrate specificity and a robust workflow that enables their high-throughput analysis. We present here the first high-throughput glyco-analytical platform for GSL headgroup profiling. The workflow features enzymatic release of GSL glycans with a novel broad-specificity endoglycoceramidase I (EGCase I) from Rhodococcus triatomea, selective glycan capture on hydrazide beads on a robotics platform, 2AB-fluorescent glycan labeling, and analysis by UPLC-HILIC-FLD. R. triatomea EGCase I displayed a wider specificity than known EGCases and was able to efficiently hydrolyze gangliosides, globosides, (n)Lc-type GSLs, and cerebrosides. Our workflow was validated on purified GSL standard lipids and was applied to the characterization of GSLs extracted from several mammalian cell lines and human serum. This study should facilitate the analytical workflow in functional glycomics studies and biomarker discovery.

Highly Efficient Release of Glycopeptides from Hydrazide Beads by Hydroxylamine Assisted PNGase F Deglycosylation for N-Glycoproteome Analysis.

Selective enrichment of glycopeptides from complex sample followed by cleavage of N-glycans by PNGase F to expose an easily detectable mark on the former glycosylation sites has become the popular protocol for comprehensive glycoproteome analysis. On account of the high enrichment specificity, hydrazide chemistry based solid-phase extraction of N-linked glycopeptides technique has sparked numerous interests. However, the enzymatic release of glycopeptides captured by hydrazide beads through direct incubation of the beads with PNGase F is not efficient due to the inherent steric hindrance effect. In this study, we developed a hydroxylamine assisted PNGase F deglycosylation (HAPD) method using the hydroxylamine to release glycopeptides captured on the hydrazide beads through the cleavage of hydrazone bonds by transamination followed with the PNGase F deglycosylation of the released glycopeptides in the free solution. Because of the homogeneous condition for the deglycosylation, the recovery of deglycosylated peptides (deglycopeptides) was improved significantly. It was found that 27% more N-glycosylation sites were identified by the HAPD strategy compared with the conventional method. Moreover, the ratio of identified N-terminal glycosylated peptides was improved over 5-fold.

Capture and Dimethyl Labeling of Glycopeptides on Hydrazide Beads for Quantitative Glycoproteomics Analysis

Incorporation of isotopic tag onto peptides via chemical labeling is a popular approach for quantitative proteomics. Chemical labeling via solution based methods usually lead to a tedious process and sample loss because several sample preparation steps including buffer exchange and desalting are performed. In this study, a solid phase based labeling approach by integration of glycopeptide enrichment and stable isotope labeling on hydrazide beads was developed for relative quantification of protein glycosylation, by which enrichment, washing, labeling, and release of the glycopeptides were all performed on the hydrazide beads sequentially. This approach was proved to be accurate in quantitative glycoproteome analysis and have good linearity range with 2 orders of magnitude for quantification of glycopeptides. Compared with dimethyl labeling conventionally performed in solution, the developed approach has better enrichment recovery (10-330% improvement) and high detection sensitivity in which 42% of annotated glycosites (vs 26%) still can be quantified using only 10 μg of four standard glycoprotein mixtures and 400 μg of bovine serum album interference as starting sample. The applicability of the approach for quantitative glycopeptide profiling was also explored by differential analysis of glycoproteome between human normal serum and liver cancer serum.

Glycan Analysis by Reversible Reaction to Hydrazide Beads and Mass Spectrometry

Investigation into glycoproteins and their associated glycans is the key to understanding the function of glycoproteins in biological pathways and disease development. Current methods for glycan analysis are generally based on multiple preparation processes to separate glycans from proteins and other molecules prior to analysis. During the multistep purification processes, glycans are continuously lost and the procedure increases the difficulty for accurate quantitative analysis of glycans. Here we describe the development of a novel technique, which uses hydrazide beads to capture glycans. It is based on the conjugation of glycans to hydrazide beads through the formation of reversible hydrazone, washing out unbound nonglycans, then releasing captured glycans by acids. The results showed that the glycans were able to be isolated from concatenate peptides by using hydrazide beads. This technique was also applied to the analysis of glycans from sera sample. The integrated capture-release on the solid-phase simplifies the procedure for glycan preparation from a complex mixture and can be a powerful tool for glycan analysis.

Glycoproteome Quantification of Human Lung Cancer Cells Exposed to Amorphous Silica Nanoparticles

Engineered nanopartilces (NPs) are beginning to be used in many areas as their unique properties. Recent years, the safety concerns on engineered NPs have been increased as the absence of the systemic understanding of biological systems exposed to NPs. Although amorphous silica NPs are gradually used in cosmetics, foods and medicinal products, the potential biological effects of them exposed to biological systems are still not comprehensively elaborated. Herein, we studied the biological effects of silica NPs to human lung cancer cells by quantitative glycoproteomic strategy. A549 cells exposed to silica NPs were incubated with sodium periodate oxidation buffer to oxidize glycoproteins and then the cells were collected and cell lysis was carried out with the aid of ultrasonication in ice bath. The protein concentration was determined with Commasie brilliant blue stain. For control experiment, normal A549 cells were disposed as above mentioned method. Then the same amount of proteins extracted from A549 cells exposed to silica NPs or not were incubated with hydrazide beads to capture glycosylated proteins, respectively. The captured glycoproteins were digested with trypsin and PNGase F sequentially and both the non-glycopeptides and glycopeptides were collected, respectively. The obtained glycopeptides were used for the identification of the captured glycoproteins in both groups. And the collected non-glycopeptides were used for quantification of the captured glycoproteins through stable isotope dimethyl labeling. Therefore, the identification and quantification of glycoproteins was more reliable and sensitive for collecting both the information from non-glycopeptides and glycopeptides over only collecting the glycopeptides because of the quantification of glycoproteins with a large number of non-glycoproteins. The quantification results showed that the expression of several critical proteins relating to cellular carbohydrate metabolism, cell cycle, cell-cell signaling and interaction, cellular movement etc. have been changed significantly. Some of the differently expressed proteins are membrane proteins, which may relate with the cellular uptake of silica NPs. The proteomic platform is showing a promising power to quantitatively investigate the biological response of a biological system to NPs exposure.

A cross-linked polymer possessing a high density of hydrazide groups: high-throughput glycan purification and labeling for high-performance liquid chromatography analysis

Previously, we communicated that rapid and efficient purification of carbohydrates can be achieved by employing sugar-specific chemical ligation with hydrazide-functionalized polymer beads; we termed this process ‘glycoblotting’. The polymer beads are designed to recover carbohydrates by an imine exchange reaction in which hydrazone bonds between the beads and carbohydrates are transferred to oxime bonds between the aminooxy probe and the carbohydrates. To apply our concept to reductive amination with common fluorescent dyes, such as 2-aminobenzamide, the method for releasing the carbohydrates from the beads was examined, and we found that heating the beads with several percentages of acetic acid was efficient. Additionally, we obtained fundamental data on our novel method, such as the recovery ratio, the quantitative capability and the reproducibility. From the results, we concluded that rapid and accurate glycan analysis can be achieved with this novel method. Overall, these novel technologies represent a significant advance toward more efficient glycan analyses, especially by using high-performance liquid chromatography. Intact glycans were recovered and labeled with common fluorescent dyes by using polymer beads possessing a high density of hydrazide groups. To achieve that, the method for releasing glycans from the beads was examined and optimized. Additionally, fundamental data on our novel method such as recovery ratio, quantitative capability, reproducibility and so forth were obtained. Our hydrazide beads hold the promise for a future standard method due to its high throughput, reproducibility and accuracy of glycan analysis.

Simultaneous Analysis of Heparan Sulfate, Chondroitin/Dermatan Sulfates, and Hyaluronan Disaccharides by Glycoblotting-Assisted Sample Preparation Followed by Single-Step Zwitter-Ionic-Hydrophilic Interaction Chromatography

Glycosaminoglycans (GAGs) play important roles in cell adhesion and growth, maintenance of extracellular matrix (ECM) integrity, and signal transduction. To fully understand the biological functions of GAGs, there is a growing need for sensitive, rapid, and quantitative analysis of GAGs. The present work describes a novel analytical technique that enables high throughput cellular/tissue glycosaminoglycomics for all three families of uronic acid-containing GAGs, hyaluronan (HA), chondroitin sulfate (CS)/dermatan sulfate (DS), and heparan sulfate (HS). A one-pot purification and labeling procedure for GAG Δ-disaccharides was established by chemo-selective ligation of disaccharides onto high density hydrazide beads (glycoblotting) and subsequent labeling by fluorescence. The 17 most common disaccharides (eight comprising HS, eight CS/DS, and one comprising HA) could be separated with a single chromatography for the first time by employing a zwitter-ionic type of hydrophilic-interaction chromatography column. These novel analytical techniques were able to precisely characterize the glycosaminoglycome in various cell types including embryonal carcinoma cells and ocular epithelial tissues (cornea, conjunctiva, and limbus).

Enrichment of glycopeptides for glycan structure and attachment site identification

We present a method to enrich for glycoproteins from proteomic samples. Sialylated glycoproteins were selectively periodate-oxidized, captured on hydrazide beads, trypsinized and released by acid hydrolysis of sialic acid glycosidic bonds. Mass spectrometric fragment analysis allowed identification of glycan structures, and additional fragmentation of deglycosylated ions yielded peptide sequence information, which allowed glycan attachment site and protein identification. We identified 36 N-linked and 44 O-linked glycosylation sites on glycoproteins from human cerebrospinal fluid.

Use of an Immunoaffinity-Mass Spectrometry-based Approach for the Quantification of Protein Biomarkers from Serum Samples of Lung Cancer Patients

It is a challenging task to verify and quantify potential biomarkers expressed at elevated levels in sera from cancer patients. An immunoaffinity-mass spectrometry-based approach has been developed using antibodies to enrich proteins of interest from sera followed by mass spectrometry-based quantification. Antibodies specific to the protein of interest were immobilized to hydrazide resin via the carbohydrate moiety on the Fc region of the antibody. Captured proteins were eluted, reduced, alkylated, and digested with trypsin. Peptides were analyzed by LC coupled with multiple reaction monitoring approach, and quantification was achieved by the addition of stable isotope-labeled (heavy) standard peptides. Using this methodology, we were able to achieve a linear response from 15 to 250 ng/ml for carcinoembryonic antigen (CEA), a known tumor biomarker. Moreover we observed elevated levels of CEA in sera samples from lung cancer patients that to our knowledge is the first time that circulating CEA has been detected by mass spectrometry-based analysis. This approach was further applied to potential protein biomarkers discovered from tumor cell lines and tumor tissues. A linear response was obtained from a multiplex spiking experiment in normal human sera for secretory leukocyte peptidase inhibitor (4-500 ng/ml), tissue factor pathway inhibitor (TFPI) (42-1000 ng/ml), tissue factor pathway inhibitor 2 (TFPI2) (2-250 ng/ml), and metalloproteinase inhibitor 1 (TIMP1) (430-1000 ng/ml). A replicate experiment for a single concentration value yielded a relative coefficient of variation better than 11% for TFPI, secretory leukocyte peptidase inhibitor, and TFPI2. The expression level of the proteins in lung cancer patient sera was assayed by an immunoaffinity-multiple reaction monitoring method, and the results were comparable with those obtained from ELISA. This immunoaffinity-mass spectrometry-based quantification approach thus provides a specific and accurate assay for verifying the expression of potential biomarkers in patient serum samples especially for those proteins for which the necessary reagents for ELISA development are unavailable.

High Sensitivity Detection of Plasma Proteins by Multiple Reaction Monitoring of N-Glycosites

The detection and quantification of plasma (serum) proteins at or below the ng/ml concentration range are of critical importance for the discovery and evaluation of new protein biomarkers. This has been achieved either by the development of high sensitivity ELISA or other immunoassays for specific proteins or by the extensive fractionation of the plasma proteome followed by the mass spectrometric analysis of the resulting fractions. The first approach is limited by the high cost and time investment for assay development and the requirement of a validated target. The second, although reasonably comprehensive and unbiased, is limited by sample throughput. Here we describe a method for the detection of plasma proteins at concentrations in the ng/ml or sub-ng/ml range and their accurate quantification over 5 orders of magnitude. The method is based on the selective isolation of N-glycosites from the plasma proteome and the detection and quantification of targeted peptides in a quadrupole linear ion trap instrument operated in the multiple reaction monitoring (MRM) mode. The unprecedented sensitivity of the mass spectrometric analysis of minimally fractionated plasma samples is the result of the significantly reduced sample complexity of the isolated N-glycosites compared with whole plasma proteome digests and the selectivity of the MRM process. Precise quantification was achieved via stable isotope dilution by adding (13)C- and/or (15)N-labeled reference analytes. We also demonstrate the possibility of significantly expanding the number of MRM measurements during one single LC-MS run without compromising sensitivity by including elution time constraints for the targeted transitions, thus allowing quantification of large sets of peptides in a single analysis.

Selective Enrichment of Tryptophan-Containing Peptides from Protein Digests Employing a Reversible Derivatization with Malondialdehyde and Solid-Phase Capture on Hydrazide Beads

A method for the selective enrichment of tryptophan-containing peptides from complex peptide mixtures such as protein digests is presented. It is based on the reversible reaction of tryptophan with malondialdehyde and trapping of the derivatized Trp-peptides on hydrazide beads via the free aldehyde group of the modified peptides. The peptides are subsequently recovered in their native form by specific cleavage reactions for further (mass spectrometric) analysis. The method was optimized and evaluated using a tryptic digest of a mixture of 10 model proteins, demonstrating a significant reduction in sample complexity while still allowing the identification of all proteins. The applicability of the tryptophan-specific enrichment procedure to complex biological samples is demonstrated for a total yeast cell lysate. Analysis of the processed fraction by 1D-LC-MS/MS confirms the specificity of the enrichment procedure, as more than 85% of the peptides recovered from the enrichment step contained tryptophan. The reduction in sample complexity also resulted in the identification of additional proteins in comparison to the untreated lysate.

Elucidation of N-Glycosylation Sites on Human Platelet Proteins

Among known platelet proteins, a prominent and functionally important group is represented by glycoprotein isoforms. They account e.g. for secretory proteins and plasma membrane receptors including integrins and glycoprotein VI as well as intracellular components of cytosol and organelles including storage proteins (multimerin 1 etc.). Although many of those proteins have been studied for some time with regard to their function, little attention has been paid with respect to their glycosylation sites. Here we report the analysis of N-glycosylation sites of human platelet proteins. For the enrichment of glycopeptides, lectin affinity chromatography as well as chemical trapping of protein bound oligosaccharides was used. Therefore, concanavalin A was used for specific interaction with carbohydrate species along with periodic acid oxidation and hydrazide bead trapping of glycosylated proteins. Derivatization by peptide:N-glycosidase F yielded deglycosylated peptides, which provided the basis for the elucidation of proteins and their sites of modification. Using both methods in combination with nano-LC-ESI-MS/MS analysis 70 different glycosylation sites within 41 different proteins were identified. Comparison with the Swiss-Prot database established that the majority of these 70 sites have not been specifically determined by previous research projects. With this approach including hydrazide bead affinity trapping, the immunoglobulin receptor G6f, which is known to couple to the Ras-mitogen-activated protein kinase pathway in the immune system, was shown here for the first time to be present in human platelets.

Novel Dimeric Acetylcholinesterase Inhibitor Bis(7)-tacrine, but Not Donepezil, Prevents Glutamate-induced Neuronal Apoptosis by Blocking N-Methyl-d-aspartate Receptors

The neuroprotective properties of bis(7)-tacrine, a novel dimeric acetylcholinesterase (AChE) inhibitor, on glutamate-induced excitotoxicity were investigated in primary cultured cerebellar granule neurons (CGNs). Exposure of CGNs to 75 mum glutamate resulted in neuronal apoptosis as demonstrated by Hoechst staining, TUNEL, and DNA fragmentation assays. The bis(7)-tacrine treatment (0.01-1 mum) on CGNs markedly reduced glutamate-induced apoptosis in dose- and time-dependent manners. However, donepezil and other AChE inhibitors, even at concentrations of inhibiting AChE to the similar extents as 1 mum bis(7)-tacrine, failed to prevent glutamate-induced excitotoxicity in CGNs; moreover, both atropine and dihydro-beta-erythroidine, the cholinoreceptor antagonists, did not affect the anti-apoptotic properties of bis(7)-tacrine, suggesting that the neuroprotection of bis(7)-tacrine appears to be independent of inhibiting AChE and cholinergic transmission. In addition, ERK1/2 and p38 pathways, downstream signals of N-methyl-d-aspartate (NMDA) receptors, were rapidly activated after the exposure of glutamate to CGNs. Bis(7)-tacrine inhibited the apoptosis and the activation of these two signals with the same efficacy as the coapplication of PD98059 and SB203580. Furthermore, using fluorescence Ca(2+) imaging, patch clamp, and receptor-ligand binding techniques, bis(7)-tacrine was found effectively to buffer the intracellular Ca(2+) increase triggered by glutamate, to reduce NMDA-activated currents and to compete with [(3)H]MK-801 with an IC(50) value of 0.763 mum in rat cerebellar cortex membranes. These findings strongly suggest that bis(7)-tacrine prevents glutamate-induced neuronal apoptosis through directly blocking NMDA receptors at the MK-801-binding site, which offers a new and clinically significant modality as to how the agent exerts neuroprotective effects.

Glycodelin A, not glycodelin S, is apoptotically active. Relevance of sialic acid modification.

Glycodelin, previously known as PP14 (placental protein-14), is a kernel lipocalin secreted by the glandular epithelium of the endometrium upon progesterone stimulation and by the seminal vesicles. The isoform of the protein present in female reproductive tissue, glycodelin A (GdA), and the male counterpart, glycodelin S (GdS), have identical amino acid sequences, but strikingly different N-linked glycans. It is well documented in literature that GdA is an immunosuppressive protein, and we have shown that this activity is due to its ability to induce apoptosis in activated T cells. The precise role of GdS in seminal plasma is not known. In this study, we report that GdS is not apoptotically active. We observe that the apoptotic activity requires the presence of sialic acid residues on the complex glycans, as in the case of GdA; however, complex glycans of GdS are non-sialylated. We have expressed the wild-type protein in Pichia pastoris, which does not add sialic acid to the secreted proteins, and confirmed our observations that the protein is apoptotically inactive in the non-sialylated form. Our results indicate that differential glycosylation modulates the function of the different glycodelin isoforms.

Preparation of spherical polymer beads of maleic anhydride–styrene–divinylbenzene and metal sorption of its derivatives

AbstractCompounds having acid anhydride moiety have been used for starting materials for many useful derivatives. Spherical crosslinked polymer beads of porous maleic anhydride–styrene–divinylbenzene copolymer are obtained by suspension polymerization. Glycerol is found to be a preferable dispersant to make spherical beads. The beads contained 93% of the anhydride and 7% of the free carboxyls. As one of applications, the beads were hydrolyzed or reacted with anhydrous hydrazine, and the metal sorption behavior of them was examined. The hydrolyzed beads show a similar sorption manner as a conventional cation exchange resin having carboxylic acid groups, but the hydrazide beads sorbed mercury (II) selectively over a wide pH range. This investigation suggests a simple preparative method for the insoluble spherical porous beads of the maleic anhydride copolymer.