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Glycosylation is a diverse but critically important posttranslational modification that modulates the physical, chemical and biological properties of proteins. Alterations in glycosylation have been noted in a number of diseases including cancer. The discovery of alterations in the glycosylation of serum glycoproteins which may offer potential as biomarkers is attracting considerable research interest. In the current study, the significant improvements in efficiency, selectivity, and analysis speed offered by ultra performance liquid chromatography (UPLC) profiling of fluorescently labeled N-linked oligosaccharides on a recently introduced sub-2 [micro]m hydrophilic interaction (HILIC) based stationary phase are demonstrated to identify cancer associated alterations in the serum N-glycome of patients bearing stomach adenocarcinoma. The contribution of the glycosylation present on four highly abundant serum proteins namely, IgG, hapteglobin, transferrin, and [alpha]1-acid glycoprotein was evaluated. Alterations in the glycosylation present on these four proteins isolated from the pathologically staged cancer serum using either affinity purification or two-dimensional electrophoresis were then investigated as possible markers for stomach cancer progression. In agreement with previous reports, an increase in sialylation was observed on haptoglobin, transferrin, and [alpha]1-acid glycoprotein in the cancerous state. Increased levels of core fucosylated biantennary glycans and decreased levels of monogalactosylated core fucosylated biantennary glycans were present on IgG with increasing disease pro. gression. The speed and selectivity offered by the sub-2 [micro]m HILIC phase make it ideal for rapid yet highly efficient separation of complex oligosaccharide mixtures such as that present in the serum N-glycome. 10.1021/ac102860w

A major constituent in the deposit of the brain in a patient with Alzheimer's disease (AD) is the aggregates/fibrils of amyloid-[beta] (A[beta]) peptides containing 39-43 amino acids. The total A[beta] levels and the concentration ratio between the most abundant A[beta](1-40) peptide and the more aggregation-prone A[beta](1-42) in body fluids (e.g., cerebrospinal fluid or CSF) have been suggested as possible criteria for early diagnosis of AD. By immobilizing capture antibodies specific to the two peptides in separate fluidic channels, surface plasmon resonance (SPR) has been used to quantify A[beta](1-40) and A[beta](1-42) present in CSF samples collected from AD patients and healthy donors. With signal amplification by streptavidin conjugated to an antibody that is selective to the common N-terminus of the A[beta] peptides, concentrations as low as 20 pM can be readily measured. The range of A[beta] peptide concentrations measurable by this method spans 4 orders of magnitude. The ability of regenerating the sensor surface for repeated measurements not only improves the reproducibility but also enhances the sample throughput. Our data reveal that the ratio of A[beta](1-40) concentration versus A[beta](1-42) concentration in CSF samples from AD patients is almost twice as high as that from healthy persons. In contrast to the commonly used enzyme-linked immunosorbent assay (ELISA), SPR obviates the need of a more expensive and less stable enzyme conjugate and the use of carcinogenic substrate for the signal detection and allows the binding events to be monitored in real time. 10.1021/ac102257m

A novel application of capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was proposed to efficiently detect and monitor the interaction between polymeric nanoparticles and the [beta]-Amyloid peptide (A[[beta].sub.1-42]), a biomarker for Alzheimer's Disease (AD), at concentrations close to physiological conditions. The CE-LIF method allowed the interaction between PEGylated poly(alkyl cyanoacrylate) nanoparticles (NPs) and the soluble A[[beta].sub.1-42] peptide monomers to be highlighted. These results were confirmed by surface plasmon resonance (SPR) and confocal laser scanning microscopy (CLSM). Whereas SPR showed an interaction between the NPs and the A[[beta].sub.1-42] peptide, CLSM allowed the formation of large aggregates/ assemblies at high NP and peptide concentrations to be visualized. All these results suggested that these nanoparticles could bind the A[[beta].sub.1-42] peptide and influence its aggregation kinetics. Interestingly, the non-PEGylated poly(alkyl cyanoacrylate) NPs did not alter the aggregation kinetics of the A[[beta].sub.1-42] peptide, thus emphasizing the high level of discrimination of the CELIF method with respect to NPs. 10.1021/ac102045x

Glycoblotting, high throughput method for N-glycan enrichment analysis based on the specific chemical ligation between aminooxy/hydrazide-polymers/solids and reducing N-glycans released from whole serum and cellular glycoproteins, was proved to be feasible for selective enrichment analysis of O-glycans of common (mucin) glycoproteins. We established a standard protocol of glycoblotting-based O-glycomics in combination with nonenzymatic chemical treatment to release reducing Oglycans predominantly from various glycoprotein samples. It was demonstrated that the nonreductive condition employing a simple ammonium salt, ammonium carbamate, made glycoblotting-based enrichment analysis of O-glycans possible without significant loss or unfavorable side reactions. A general workflow of glycoblotting using a hydrazide bead (BlotGlyco H), on-bead chemical manipulations, and subsequent mass spectrometry allowed for rapid O-glycomics of human milk osteopontin (OPN) and urinary MUC1 glycoproteins purified from healthy donors in a quantitative manner. It was revealed that structures of O-glycans in human milk OPN were varied with habitual fucosylation and N-acetyllactosamine units. It was also suggested that purified human urinary MUC1 was modified preferentially by sialylated O-glycans (94% of total) with 7:3 ratio of core 1 to core 2 type O-glycans. Versatility of the present strategy is evident because this method was proved to be suited for the enrichment analysis of general biological and clinical samples such as human serum and urine, cultured human cancer cells, and formalin-fixed paraffin-embedded tissue sections. It is our belief that the present protocols would greatly accelerate discovery of disease-relevant O-glycans as potential biomarkers. 10.1021/ac101599p

Amyloid-like fibrils from a number of small peptides that are unrelated by sequence adopt a cross-[beta]-spine in which the two sheets fully interdigitate to create a dry interface. Formation of such a dry interface is usually associated with self-assembly of extended hydrophobic surfaces. Here we investigate how a dry interface is created in the process of protofilament formation in vastly different sequences using two amyloidogenic peptides, one a polar sequence from the N terminus of the yeast prion Sup35 and the other a predominantly hydrophobic sequence from the C terminus of A[beta]-peptide. Using molecular dynamics simulations with three force fields we show that spontaneous formation of two ordered one-dimensional water wires in the pore between the two sheets of the Sup35 protofilaments results in long-lived structures, which are stabilized by a network of hydrogen bonds between the water molecules in the wires and the polar side chains in the [beta]-sheet. Upon decreasing the stability of the metastable structures, water molecules are expelled resulting in a helically twisted protofilament in which side chains from a pair of [beta]-strands in each sheet pack perfectly resulting in a dry interface. Although drying in hydrophobically dominated interfaces is abrupt, resembling a liquid to vapor transition, we find that discrete transitions between the liquid to one-dimensional ordered water in the nanopore enclosed by the two [beta]-sheets to dry interface formation characterizes protofilament assembly in the yeast prions. Indeed, as the two sheets of the hydrophobic A[beta]-sequence approach each other, fibril formation and expulsion of water molecules occur rapidly and nearly simultaneously. amyloid fibrils | dewetting transition | electrostatic interactions | prion diseases doi/ 10.1073/pnas.1008616107

A combined mass spectrometry (MS) and tandem mass spectrometry (MS/MS) approach implemented with matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI FTICR MS) in the negative ion mode is described for enhanced glycopeptide detection and MS/MS analysis. Positive ion mode MS analysis is widely used for glycopeptide characterization, but the analyses are hampered by potential charge-induced fragmentation of the glycopeptides and poor detection of the glycopeptides harboring sialic acids. Furthermore, tandem MS analysis (MS/MS) via collision-induced dissociation (CID) of glycopeptides in the positive ion mode predominantly yields glycan fragmentation with minimal information to verify the connecting peptide moiety. In this study, glycoproteins such as, bovine lactoferrin (b-LF) for N-glycosylation and kappa casein (k-CN) for O-glycosylation were analyzed in both the positive- and negative ion modes after digestion with bead-immobilized Pronase. For the b-LF analysis, 44 potential N-linked glycopeptides were detected in the positive ion mode while 61 potential N-linked glycopeptides were detected in the negative ion mode. By the same token, more O-linked glycopeptides mainly harboring sialic acids from k-CN were detected in the negative ion mode. The enhanced glycopeptide detection allowed improved site-specific analysis of protein glycosylation and superior to positive ion mode detection. Overall, the negative ion mode approach is aimed toward enhanced N- and O-linked glycopeptide detection and to serve as a complementary tool to positive ion mode MS/MS analysis. 10.1021/ac101856r

Ammonium-based alkali-catalyzed [beta]-elimination under nonreducing conditions was investigated in detail for the stability of the released mucin-type O-glycan chains with [beta]1,3-1inked cores. In contrast to the previously studied [beta]1,4-linkage of the N-glycan-type, which was shown to be stable under the ammonium-based alkaline conditions, the [beta]l,3-linkaga is labile toward alkaline treatment and considerable peeling was observed with both model heptasaccharides and standard glycoproteins. The former include eight reducing glucoheptasaccharides with different and commonly occurring linkages ([alpha]l,2-, [beta]1,2-, [alpha]l,3-,[beta]1,3-, [alpha]l,4-, [beta]1,4-, [alpha]1,6-, and [beta]l,6-linkages), and the latter include mucin-type bovine submaxillary mucin and bovine fetuin, which contains both O-and N-glycans. The results indicated that complete prevention of peeling under nonreducing alkali-catalyzed hydrolysis conditions remains difficult. The yields of released O-and N-glycans were also assessed by use of the two glycoproteins as models. Compared with conventional procedures, Carlson degradation for O-glyean release and PNGase F digestion for N-glycan release, the nonreducing ammonium-based alkaline hydrolysis gave lower yields. Great care has to be taken when employing such nonreducing alkaline conditions in glycomic analysis and in obtaining glycoprotein glycans for functional studies. 10.1021/ac102300r

The accuracy in quantitative analysis of N-linked glycopeptides and glycosylation site mapping in cancer is critical to the fundamental question of whether the aberration is due to changes in the total concentration of glycoproteins or variations in the type of glycosylation of proteins. Toward this goal, we developed a lectin-directed tandem labeling (LTL) quantitative proteomics strategy in which we enriched sialylated glycopeptides by SNA, labeled them at the N-terminus by acetic anhydride ([sup.1][H.sub.6]/ [sup.2][D.sub.6]) reagents, enzymatically deglycosylated the differentially labeled peptides in the presence of heavy water ([H.sub.2][sup.18]O), and performed LC/MS/MS analysis to identify glycopeptides. We successfully used fetuin as a model protein to test the feasibility of this LTL strategy not only to find true positive glycosylation sites but also to obtain accurate quantitative results on the glycosylation changes. Further, we implemented this method to investigate the sialylation changes in prostate cancer serum samples as compared to healthy controls. Herein, we report a total of 45 sialylated glycopeptides and an increase of sialylation in most of the glycoproteins identified in prostate cancer serum samples. Further quantitation of nonglycosylated peptides revealed that sialylation is increased in most of the glycoproteins, whereas the protein concentrations remain unchanged. Thus, LTL quantitative technique is potentially an useful method for obtaining simultaneous unambiguous identification and reliable quantification of N-linked glycopeptides. 10.1021/ac101486d

Reported here are analyses of the interactions between a select group of solution-phase glycoproteins and a unique boronic acid capture surface. The boronic acid derivative, 4-[(2-aminoethyl)carbamoyl]phenylboronic acid, AECPBA, was synthesized and then immobilized on carboxymethyl dextran surfaces using simple coupling methods. From surface plasmon resonance spectroscopy responses, it is found that model glycoprotcins interact strongly with the AECPBA surface and subsequently can be readily released from the AECPBA surface using borate buffer. A striking difference between the glycoprotcins fetuin and asialofetuin (desialylated fetuin), in terms of glycoprotein binding to the AECPBA surface, indicates that the interaction of glycoproteins with the immobilized AECPBA is dictated by the terminal saccharide of the heteroglycan chain. Surprisingly, secondary interactions of glycosylated and nonglycosylated proteins with the carboxymethyl dextran hydrogel matrix are observed. Importantly, it is demonstrated that use of tris(hydroxymethyl)aminomethane buffer allows for decreased secondary interactions of nonglycosylated proteins on the AECPBA/dextran surface, as noted with the model protein ExtrAvidin. 10.1021/ac101911a

Glycoproteomics holds the promise of new advances in medical technology. However, mass spectrometry has limitations for the structural determination of glycosylated peptides because the hydrophilic nature of the oligosaccharide moiety in glycopeptides is disadvantageous for ionization, and glycopeptides ionize much less readily than nonglycosylated peptides. Therefore, conventional proteomics tools cannot detect altered glycosylation on proteins. Here, we describe an on-plate pyrene derivatization method using 1-pyrenyldiazomethane for highly sensitive matrix-assisted laser/desorption ionization-tandem mass spectrometry ([MALDI-MS.sup.n]) of glycopeptides in amounts of less than 100 fmol. This derivatization is unique, as the pyrene groups are easily released from glycopeptides during ionization when 2,5-dihydroxybenzoic acid is used as a matrix. As a result, most ions are observed as the underivatized form on the spectra. At the same time, pyrene derivatization dramatically reduces the ionization of peptides. Thus, for glycopeptides in a mixture of abundant peptides, we could obtain MS spectra in which the signals of glycopeptides were intense enough for subjection to [MS.sup.n] in order to determine the structures of both glycan and peptide. Finally, we show that the glycopeptides derived from as little as 1 ng of prostate specific antigen can be detected by this method. 10.1021/ac101555a

Aggregation of amyloid-[beta] (A[beta]) peptides is causatively linked to Alzheimer's disease (AD); thus, suppression of this process by small molecule inhibitors is a widely accepted therapeutic and preventive strategy for AD. Screening of the inhibitors of A[beta] aggregation deserves much attention; however, despite intensive efforts, there are only a few high-throughput screening methods available, all of them having drawbacks related to the application of external fluorescent probes or artificial A[beta] derivatives. We have developed a label-free MALDI MS-based screening test for inhibitors of A[[beta].sub.42] fibrillization that exhibits high sensitivity, speed, and automation possibilities suitable for high-throughput screening. The test was evaluated by transmission electron microscopy and compared with a fluorimetric thioflavin-based assay, where interference of a number of tested compounds with thioflavin T binding and/or fluorescence caused false-positive results. The MALDI MS-based method can significantly speed up in vitro screening of compound libraries for inhibitors of A[[beta].sub.42] fibrillization. 10.1021/ac101583q

Neutralization of carboxylic acid is an important means to avoid sialic acid dissociation when sialylated glycans are analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). In this paper, we describe a simple and rapid method to modify the sialic acids of sialylated glycans in the presence of methylamine and (7-azabenzotriazol-1-yloxy) trispyrrolidinophosphonium hexafluorophosphate (PyAOP). After methylamidation, sialylated glycans can be analyzed by MALDI-MS without loss of the sialic acid moiety. The electrospray ionization mass spectrometry (ESI-MS) and MALDI-MS analysis of both 3'- and 6'-sialyllactose derivatives indicated that the quantitative conversion of sialic acids was achieved, regardless of their linkage types. This derivatization strategy was further validated with the N-glycans released from three standard glycoproteins (fetuin, human acid glycoprotein, and bovine acid glycoprotein) containing different types of complex glycans. Most importantly, this derivatization method enabled the successful characterization of N-glycans of sera from different species (human, mouse, and rat) by MALDI-MS. Because of the mild reaction conditions, the modification in sialic acid residues can be retained. This improvement makes it possible to detect sialylated glycans containing O-acetylated sialic acid moieties using MALDI-MS in positive-ion mode. 10.1021/ac101831t

The small molecule thioflavin T (ThT) is a defining probe for the identification and mechanistic study of amyloid fiber formation. As such, ThT is fundamental to investigations of serious diseases such as Alzheimer's disease, Parkinson disease, and type II diabetes. For each disease, a different protein undergoes conformational conversion to a [beta]-sheet rich fiber. The fluorescence of ThT exhibits an increase in quantum yield upon binding these fibers. Despite its widespread use, the structural basis for binding specificity and for the changes to the photophysical properties of ThT remain poorly understood. Here, we report the co-crystal structures of ThT with two alternative states of [beta]-2 microglobulin ([beta]2m); one monomeric, the other an amyloid-like oligomer. In the latter, the dye intercalates between [beta]-sheets orthogonal to the [beta]-strands. Importantly, the fluorophore is bound in such a manner that a photophysically relevant torsion is limited to a range of angles generally associated with low, not high, quantum yield. Quantum mechanical assessment of the fluorophore shows the electronic distribution to be strongly stabilized by aromatic interactions with the protein. Monomeric [beta]2m gives little increase in ThT fluorescence despite showing three fluorophores, at two binding sites, in configurations generally associated with high quantum yield. Our efforts fundamentally extend existing understanding about the origins of amyloid-induced photophysical changes. Specifically, the p-sheet interface that characterizes amyloid acts both sterically and electronically to stabilize the fluorophore's ground state electronic distribution. By preventing the fluorophore from adopting its preferred excited state configuration, nonradiative relaxation pathways are minimized and quantum yield is increased. Alzheimer's | beta-2 microglobulin | Parkinson | TICT | photophysics doi/ 10.1073/pnas.1002867107

Global analysis of glycoproteins shows great promise for the discovery of therapeutic targets and clinical biomarkers. Selective capture of glycopeptides by hydrazide resin followed by mass spectrometric identification of the peptides released by PNGaseF treatment has been most widely used. However, the majority of the reports using this approach focus on global profiling, rather than relative quantitation of glycoprotein alternations in pathological states. We describe an integrated strategy allowing for relative quantitstian of glycoproteins in complex biological mixtures using this approach. The strategy includes periodate oxidation of tryptic digests, solid-phase enrichment of glycopeptides via hydrazide-eoupled magnetic beads, in conjunction with [sup.18]O stable isotope labeling catalyzed by both trypsin and PNGaseF, and subsequent identification and quantitation by LC-MS/MS analysis. Three [sup.18]O atoms ([sup.18][O.sub.3]) are incorporated into N-linked glycopeptides for samples treated in [sup.18]O-water, two at the carboxyl terminus by trypsin during hydrazide coupling and the third at the N-glycosylation site through PNGaseF-mediated deglycosylation. Thus, mass shifts of 6 and 8 Da are indicative of singly and doubly glycosylated peptides, respectively. Experimental conditions were optimized to promote the trypsin-mediated [sup.18][O.sub.2] incorporation and prevent backbone exchange. The accuracy, reproducibility, and linearity of relative quuntitation were evaluated by using 15 glycoproteins spiked into mouse serum at different concentration ratios. Using this approach, we were able to identify and quantitate 224 N-glycopeptides representing 130 unique glycoproteins from 20 [micro]L of the undepleted mouse serum samples. The strategy can be easily adapted to the analysis of glycoproteins in tissues, cell lines, and other sample origins. 10.1021/ac101564t

Correct mass and charge assignment for large highly heterogeneous macromolecular ions (e.g., large glycoproteins with significant carbohydrate content) presents a great challenge in native electrospray ionization mass spectrometry (ESI MS). A new approach to this problem combines complexity reduction (mass-selection of a narrow distribution of ionic species from a heterogeneous mixture) and gas-phase ion chemistry (electron-transfer reactions) to induce partial reduction of the ionic charge. The resulting spectra are devoid of complexity and are easy to interpret, leading to correct mass assignment. The new method is tested using several glycoproteins and their complexes, for which standard deconvointion approaches do not work. 10.1021/ac101848z

We developed an automatic apparatus for the release of O-glycans from mucin-type glycoproteins and proteoglycans (Matsuno, Y.-k.; Yamada, K.; Tanabe, A.; Kinoshita, M.; Maruyama, S.-z.; Osaka, Y.-s.; Masuko, T.; Kakehi, K. Anal. Biochem. 2007, 363, 245-257. Yamada, K.; Hyodo, S.; Matsuno, Y. K.; Kinoshita, M.; Maruyama, S. Z.; Osaka, Y. S.; Casal, E.; Lee, Y. C.; Kakehi, K. Anal. Biochem. 2007, 371, 52-61). The method allows rapid release of O-glycans as the reducing form within l0 min. In the present study, we connected the device to a MALDI-TOF MS spotter and achieved routine analysis of O-glycans in biological samples for clinical use after in situ derivatization of the released O-glycans with phenylhydrazine. We applied the method to the analysis of O-glycans expressed on MKN45 cells derived from human stomach cancer cells and found that MKN45 cells expressed characteristic trisialo-polylactosamine-type glycans as reported previously (Yamada, K.; Kinoshita, M.; Hayakawa, T.; Nakaya, S.; Kakehi, K. J. Proteome Res. 2009, 8, 521-537). We also applied the method to the analysis of O-glycans in serum samples. The present technique is the first attempt to use MS measurement for routine clinical diagnostic works. 10.1021/ac101581n

We developed a 3 h procedure for preparing serum N-glycans and labeling them with 8-aminopyrene- 1,3,6trisulfonic acid (APTS) by sequential addition of reagents to the serum and incubation in a polymerase chain reaction (PCR) thermocycler. Moreover, we succeeded in analyzing these samples by capillary electrophoresis on three commercial microfluidics-based platforms: the MCE202 MultiNA, the 2100 Bioanalyzer, and a modified prototype of the eGene system which were originally designed for nucleic acid separation and detection. Although these instruments use short separation channels, our technical improvements made it possible to reliably measure the N-glycans constituting GlycoHepatoTest. This test comprises a panel of biomarkers that allows follow-up of liver fibrosis patients starting from the early stage. In this way and for the first time, we demonstrate a clinical glycomics assay on an affordable, robust platform so that clinical chemistry laboratories can exploit glycomics in the diagnosis and monitoring of chronic liver disease. Another potential application is the rapid screening of the N-glycosylation of recombinant glycoproteins produced for pharmaceutical use. 10.1021/ac101560a

A scanning electrochemical microscopic (SECM) method for in situ imaging of four types of membrane glycan motifs on single adherent cells was proposed using BGC-823 human gastric carcinoma (BGC) cells as the model. These adherent cells were first micropatterned in the microwell of poly(dimethylsiloxane) membrane for precisely controlling the localized surface interaction, and the membrane glycans were then specifically recognized with corresponding lectins labeled with horseradish peroxidase (HRP). On the basis of the enzymatic oxidization of ferrocenylmethanol (FMA) by [H.sub.2][O.sub.2] to yield FMA', the glyean expression level was detected by the reduction current of FMA at the SECM tip. The cell-surface glycans could, thus, be in situ imaged by SECM at a single-cell level without peeling the cells from culture dish. Under the optimized conditions, four types of membrane glycan motifs showed statistically distinguishable expression levels. The SECM results for different glycan motifs on adherent single cells were consistent with those estimated by flow cytometric assay. This work provides a reliable approach for in situ evaluation of the characteristic glycopattern of single living cells and can be applied in cell biologic study based on cell surface carbohydrate expression. 10.1021/ac101688p

Membrane glycoproteins play vital roles in many fundamental physiological and pathophysiological processes in the central nervous system and represent important targets for pharmaceuticals and biomarker discovery. However, their isolation and characterization have been greatly limited. Lectin affinity chromatography (LAC) has evolved as a powerful method to enrich glycoproteins in biofluid and cell/tissue lysate. However, its use in the hydrophobic fraction of the samples has rarely been explored. In this study, we have conducted a systematic investigation on the lectin binding efficiency in the presence of four commonly used detergents. We have found that, under certain concentrations, detergents can minimize nonspecific binding and facilitate the elution of hydrophobic glycoproteins. With the detergent assisted lectin affinity chromatography (DALAC), a total of 1491 proteins were identified with low numbers of false positives from two lectins. Proteins (699) were identified with at least two unique poptides, of which 219 are membrane glycopreteins. Compared to the traditional methods, the DALAC approach significantly increased the recovery of plasma membrane and glycoproteins. NP-40 is recommended as a well rounded detergent for DALAC, but the conditions for enriching certain target proteins need to be empirically determined. This study represents the first global identification of the murine brain glycoproteome. 10.1021/ac1004844

A new strategy for assessing cell surface carbohydrates and P-glycoprotein (P-gp) expression status and quantifying the cell numbers with an electrochemical immunoassay was designed. In order to construct the base of the cytosensor, a novel 3-D architecture was initially fabricated by combining nitrogen-doped carbon nanotubes, thionine, and gold nanoparticles via a simple layer-by-layer method. The formed architecture provided an effective matrix for concanavalin A (Con A) binding and made the immobilized Con A hold high stability and bioactivity. On the basis of the specific recognition of cell surface mannosyl groups to Con A, the Con A/3-D architecture interface showed a predominant capability for cell capture. With another coupled signal amplification based on a enzymatic catalytic reaction of HRP toward the oxidation of thionine by the [H.sub.2][O.sub.2], which was induced by two-step immunoreactions, the proposed cytosensor showed an excellent analytical performance for the detection of HeLa cells ranging from 8.0 x [10.sup.2] to 2.0 x [l0.sup.7] cells [mL.sup.-1] with a limit of detection of 500 cells [mL.sup.-1]. Moreover, with the use of preblocking procedures, the mannosyl groups and P-gp on single HeLa cell could be further detected to be (4 [+ or -] 2) x [10.sup.10] molecules of mannose moieties and 8.47 x [10.sup.6] molecules of P-gp. This strategy offers great promise for sensitive detection of cancer cells and cell surface receptors and thus may help improve cancer diagnosis and treatment. 10.1021/ac9026127

The standard, well-established sample preparation protocol to release N-linked glycans from glycoproteins for downstream analysis requires relatively long deglycosylation times (from several hours to overnight) and relatively high endoglycosidase concentration (from 1:250 to 1:500 enzyme:substrate molar ratio). In this paper, we significantly improve this standard protocol by the use of pressure-cycling technology (PCT) to increase the speed and decrease the relative amount of PNGase F during the release of N-linked glycans from denatured glycoproteins. With the application of pressure cycling from atmospheric to as high as 30 kpsi, >95% release of the asparagine-linked glycans from bovine ribonuclease B, human transferrin, and polyclonal human immunoglobulin was rapidly achieved in a few minutes using as low as 1:2500 enzyme: substrate molar ratio. The deglycosylation rate was first examined by SDS-PAGE at the protein level. The released glycans were then quantitated by capillary electrophoresis with laser induced fluorescence detection (CE-LIF). This new sample preparation protocol readily supports large-scale glycan analysis of biopharmaceuticals with rapid deglyeosylation times. 10.1021/ac100098e

Metabolic labeling of glycans with synthetic sugar analogs has emerged as an attractive means for introducing nonnatural chemical functionality into glycoproteins. However, the complexities of glycan biosynthesis prevent the installation of nonnatural moieties at defined, predictable locations within glycoproteins at high levels of incorporation. Here, we demonstrate that the conserved N-acetyglucosamine (GIcNAc) residues within chitobiose cores of N-glycans in the model organism Saccharomyces cerevisiae can be specifically targeted for metabolic replacement by unnatural sugars. We introduced an exogenous GIcNAc salvage pathway into yeast, allowing cells to metabolize GIcNAc provided as a supplement to the culture medium. We then rendered the yeast auxotrophic for production of the donor nucleotide-sugar uridine-diphosphate-GIcNAc (UDP-GIcNAc) by deletion of the essential gene GNA1. We demonstrate that gna1[DELTA] strains require a GIcNAc supplement and that expression plasmids containing both exogenous components of the salvage pathway, GIcNAc transporter NGT1 from Candida albicans and GIcNAc kinase NAGK from Homo sapiens, are required for rescue in this context. Further, we show that cells successfully incorporate synthetic GIcNAc analogs N-azidoacetyglucosamine (GIcNAz) and N-(4-pentynoyl)-glucosamine (GIcNAI) into cell-surface glycans and secreted glycoproteins. To verify incorporation of the nonnatural sugars at N-glycan core positions, endoglycosidase H (endoH)-digested peptides from a purified secretory glycoprotein, Ygp1, were analyzed by mass spectrometry. Multiple Ygpl N-glycosylation sites bearing GIcNAc, isotopically labeled GIcNAc, or GIcNAz were identified; these modifications were dependent on the supplement added to the culture medium. This system enables the production of glycoproteins that are functionalized for specific chemical modifications at their glycosylation sites. click chemistry | GIcNAc | metabolic engineering | N-glycosylation | GNA1 doi/10.1073/pnas.0911247107

Bacillus anthracis spores are enclosed by an exosporium comprised of a basal layer and an external hair-like nap. The filaments of the nap are composed of trimers of the collagen-like glycoprotein Bc1A. The attachment of essentially all Bc1A trimers to the exosporium requires the basal layer protein BxpB, and both proteins are included in stable high-molecular-mass exosporium complexes. Bc1A contains a proteolytically processed 38-residue amino-terminal domain (NTD) that is essential for basaMayer attachment. In this report, we identify three NTD submotifs (SM1a, SM1b, and SM2, located within residues 21 to 33) that are important for Bc1A attachment and demonstrate that residue A20, the amino-terminal residue of processed Bc1A, is not required for attachment. We show that the shortest NTD of BclA--or of a recombinant protein--sufficient for high-level basal-layer attachment is a 10-residue motif consisting of an initiating methionine, an apparently arbitrary second residue, SM1a or SM1b, and SM2. We also demonstrate that cleavage of the Bc1A NTD is necessary for efficient attachment to the basal layer and that the site of cleavage is somewhat flexible, at least in certain mutant NTDs. Finally, we propose a mechanism for BclA attachment and discuss the possibility that analogous mechanisms are involved in the attachment of many different collagen-like proteins of B. anthracis and closely related Bacillus species. doi: 10.1128/JB.01003-09

The imaging capability and high detection sensitivity of time-of-flight secondary ion mass spectrometry (ToFSIMS) makes it a potentially attractive complement to other mass spectrometry methods, such as ESI and MALDI, for the analysis of proteins and peptides. We have explored this possibility by performing a systematic analysis of synthetic opioid and amyloid pepfides with ToF-SIMS using [Bi.sub.3.sup.+] ~ and [Au.sub.3.sup.+] primary ions. In the low mass region of the spectra, a number of single amino acid ion peaks were detected, providing information about the amino acid content in each peptide. In the medium and high mass range of the spectra, peaks corresponding to multiple amino acid ions (backbone cleavage ions) as well as molecular ions were detected, allowing for the determination of the amino acid sequence and the molecular mass of the entire peptide, respectively. Detection efficiencies were determined for the molecular ions of some of the peptides, indicating detection limits in the attomole range. The fragmentation patterns observed in the ToF-SIMS analysis of opioid and amyloid peptides showed interesting similarities with collision-induced dissociation (CID) studies using other mass spectrometry methods. The present work provides important progress toward ToFSIMS proteomics. 10.1021/ac902712f

The viral spike of HIV-1 is composed of three gp120 envelope glycoproteins attached noncovalently to three gp41 transmembrane molecules. Viral entry is initiated by binding to the CD4 receptor on the cell surface, which induces large conformational changes in gp120. These changes not only provide a model for receptor-triggered entry, but affect spike sensitivity to drug- and antibody-mediated neutralization. Although some of the details of the CD4-induced conformational change have been visualized by crystal structures and cryoelectron tomograms, the critical gp41-interactive region of gp120 was missing from previous atomic-level characterizations. Here we determine the crystal structure of an HIV-1 gp120 core with intact gp41-interactive region in its CD4-bound state, compare this structure to unliganded and antibody-bound forms to identify structurally invariant and plastic components, and use ligand-oriented cryoelectron tomograms to define component mobility in the viral spike context. Newly defined gp120 elements proximal to the gp41 interface complete a 7-stranded [beta]-sandwich, which appeared invariant in conformation. Loop excursions emanating from the sandwich form three topologically separate--and structurally plastic--layers, topped off by the highly glycosylated gp120 outer domain. Crystal structures, cryoelectron tomograms, and interlayer chemistry were consistent with a mechanism in which the layers act as a shape-changing spacer, facilitating movement between outer domain and gp41-associated [beta]-sandwich and providing for conformational diversity used in immune evasion. A 'layered' gp120 architecture thus allows movement among alternative glycoprotein conformations required for virus entry and immune evasion, whereas a [beta]-sandwich clamp maintains gp120-gp41 interaction and regulates gp41 transitions. HIV-1 viral spike | molecular motion | protein architecture | receptor-triggered entry | type 1 fusion protein doi/10.1073/pnas.0911004107

Cryptococcus albidus, isolated from the sediments of Century Pulp and Paper Mill, Lalkuan, Nainital, Uttarakhand, India, produced a copper containing oxidase, laccase, that was capable of degrading environmental pollutants. Bagasse was the most efficient inducer for laccase production. The Taguchi approach was used to optimize the growth media for five factors, i.e., pH, copper sulphate, carbon, nitrogen, and the inducer at four levels using an M-16 orthogonal array. The optimum conditions for laccase production were pH (6), CuS[O.sub.4] (2 mmol/L), meat peptone (0.5%), glucose (0.1%), and bagasse (1.0%). After optimization, laccase production increased seven times from 32 to 219 IU/mg. The inducer (bagasse) had maximum effect on laccase production leading to 52% increase, while pH had minimum effect with 7% increase. Growth media with laccase activity (2 U/mL) was applied for the bioremediation of dyes, effluent, and chemical compounds. These experiments showed that the growth media with laccase activity (2 U/mL) produced by Cryptococcus albidus had good potential for bioremediation of toxic and recalcitrant compounds. Further, the laccase enzyme extracted from the growth media was fractionated by DEAE-cellulose ion-exchange chromatography, and the molecular weight of the enzyme determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was found to be 64 kDa. The activity of laccase was confirmed by native PAGE, in which ABTS was used for staining gel. Key words: bioremediation, dyes, fungal laccase, optimization, purification, Taguchi approach. Cryptococcus albidus, isolee des sediments de l'usine de pates et papiers Century, Lalkuan, Nainital, Uttarakhand, Inde, a produit une oxydase contenant du cuivre et de la laccase capable de degrader les polluants environnementaux. La bagasse etait l'inducteur le plus efficace de production de laccase. L'approche de Taguchi a ete utilisee pour optimiser les supports de croissance pour cinq facteurs, c.-a-d. le pH, le sulfate de cuivre, le carbone, l'azote et un inducteur a quatre niveaux en utilisant un dispositif orthogonal M-16. Les conditions optimales pour la production de laccase etaient : pH 6, CuS[O.sub.4] de 2 mmol, peptone de viande 0,5 %, glucose 0,1 % et bagasse 1,0 %. Apres l'optimisation, la production de laccase a augmente sept fois, de 32 a 219 IU/mg. L'inducteur (bagasse) avait l'effet maximal sur la production de laccase, menant a une augmentation de 52 %, alors que le pH avait un effet minimal, menant a une augmentation de 7 %. Un support de croissance avec activite de laccase (2 U/mL) a ete utilise pour la degradation biologique de teintures, d'effluents et de composes chimiques. Ces experiences ont montre que le support de croissance avec activite de laccase (2 U/mL) produit par le Cryptococcus albidus presentait un bon potentiel de degradation biologique de composes toxiques et recalcitrants. De plus, l'enzyme laccase extraite du support de croissance a ete fractionnee par chromatographie a cellulose echangeuse d'ions; le poids moleculaire de l'enzyme determine par electrophorese de polyacrylamide en presence de sulfate de sodium dodecylique (SDS-PAGE) etait de 64 kDa. L'activite de la laccase a ete confirmee par electrophorese d'echantillons a l'etat natif sur gel de polyacrylamide, dans laquelle de l'ABTS a ete utilise pour colorer le gel. Mots-cles : degradation biologique, teintures, laccase fongique, optimisation, purification, approche de Taguchi. [Traduit par la Redaction], Introduction Laccase (EC 1.10.3.2, p-diphenol:dioxygen oxidoreductase), a copper-containing glycoprotein, has been successfully used for bioremediation of contaminated sites and effluent. It can decolourize a variety of dyes like orange G, [...]

CD137 (also known as 4-1BB) is a surface co-stimulatory glycoprotein originally described as present on activated T lymphocytes. Artificial stimulation of this molecule with monoclonal antibodies or other agonist moieties therapeutically augments the cellular immune response against tumors, regardless of the absence of CD137 on tumor cells. These pharmacological agents, when administered systemically, surpass the immune effects of the membrane-bound natural ligand (CD137 or 4-1BB ligand), the activity of which is confined to cell-to-cell interactions. Greater affinity and broader distribution of the CD137 pharmacological agonists cause much more intense receptor crosslinking and stronger intracellular signals than the natural ligand. Target engagement on a variety of immune cell types such as T, natural killer and dendritic cells and on tumor vessels could switch on multiple mechanisms of action. As an agonist, anti-CD137 monoclonal antibody has entered Phase II clinical trials; elucidation of the mechanisms behind the anti-tumor efficacy requires further research in mice and patients to understand and rationally combine these new treatments.

ERp57 is an oxidoreductase that, in conjunction with calnexin and calreticulin, assists disulfide bond formation in folding glycoproteins. ERp57 also forms a mixed disulfide with the MHC class I-specific chaperone tapasin, and this dimeric conjugate edits the peptide repertoire bound by MHC class I molecules. In cells unable to form the conjugate, because of tapasin mutation in human studies or ERp57 deletion in mouse studies, peptide loading is impeded. Subtle differences between the mouse and human systems have been observed. Here, we address these differences and expand the analysis to investigate the role of ERp57 redox functions in MHC class I peptide loading. We show in human cells that in the absence of conjugate formation MHC class I recruitment and/or stabilization in the MHC class I peptide-loading complex is impaired, similar to observations in mouse cells. However, we found no role for the enzymatic activities of either the a or a' domain redox sites of ERp57 in peptide loading. Our data argue that the function of ERp57 in peptide loading is likely caused by other ERp57 functional domains or a combinatorial feature of the tapasin-ERp57 conjugate. antigen presentation | antigen processing | human | protein folding | quality control

The synthesis and characterization of a series of related foldamers designed to examine the effect of hydrophobic side chain structure on this behavior are described. The results have provided a refined model of the conformational switching of this series of foldamers in which the hydrogel state of foldamer is the result of the highly ordered assembly of alternatively folded molecules.

The synthesis of [beta]-N-linked glycoaminoacids and glycodipeptides by linking a protected amino sugar moiety to an amino acid or peptide under microwave irradiation is described.

The structural and NMR spectroscopic studies of the [Zn.sup.2} binding sites of human and rat [beta]-amyloid (1-28) fragments in water/sodium dodecyl sulfate micelles is demonstrated.

Atypical antipsychotic drugs are well tolerated and thus often preferred in women of fertile age; yet the information on their placental transfer and use during the prenatal period is limited. The aim of this study was to study the placental transfer of quetiapinem, a widely used atypical antipsychotic, with special reference to the role of the placental transporter protein, P-glycoprotein (P-gp). This was performed in 18 dually perfused placentas, using the well established P-gp inhibitors PSC833 (valspodar) and GG918 to inhibit the function of P-gp. We also aimed to clarify the significance of two potentially functional ABCB1 single nuclear polymorphisms (SNPs), 2677G>T/A and 3435C>T, on the transplacental transfer (TPT) of quetiapine. The placental transfer of quetiapine in the control group as measured by TP[T.sub.AUC] % (absolute fraction of the dose crossing placenta) was 3.7%, which is 29% less than the transfer of the freely diffusible antipyrine, which was 5.2%. The P-gp inhibitors had no significant effect on the transfer of quetiapine as measured by TP[T.sub.AUC] % (P=0.77). No correlation was found between the transplacental transfer of quetiapine (TP[T.sub.AUC]%) and placental P-gp expression (P=0.61). The 3435T allele in exon 26 was associated with significantly higher placental transfer of quetiapine (P=0.04). We conclude that quetiapine passes the human placenta but that the blood-placental barrier partially limits the transplacental transfer of quetiapine. Administration of P-gp inhibiting drugs with quetiapine is not likely to increase fetal exposure to quetiapine, although the ABCB1 C3435T polymorphism may contribute to inter-individual variation in fetal exposure to quetiapine. Keywords P-glycoprotein, placenta, quetiapine, ABCB1 polymorphism, Introduction The treatment of psychosis during pregnancy is still a significant challenge; older antipsychotic drugs, such as chlorpromazine or fluphenazine, have been associated with prenatal complications in animal experiments (Iqbal [...]

A compact biosensor for a label-free, rapid (

Osteopontin (OPN), a multifunctional secreted phosphoglycoprotein, plays diverse roles in bone biology, immune regulation, cell survival, inflammation, and cancer metastasis. Here we show its role in determining lymphocyte homeostasis and body mass in response to hindlimb unloading (HU), a model for evaluating effects of weightlessness on the musculoskeletal and other physiological systems. Using this stress model, we compared OP[N.sup.-/-] mice with OP[N.sup.+/+] mice subjected to HU for 3 days. Whereas OP[N.sup.+/+] mice suffered a marked reduction of body weight and significant spleen and thymus atrophy, OP[N.sup.-/-] mice exhibited minor weight loss and much less spleen and thymus atrophy. The HU-induced lymphoid organ atrophy was the result of dramatically diminished numbers, respectively, ofT and B cells in the spleen and CD[4.sup.+]CD[8.sup.+] double-positive cells in the thymus of OP[N.sup.+/+] mice. Increased levels of corticosterone, which modulates lymphocyte activation responses and apoptosis during stress, were found only in OP[N.sup.+/+] mice. Apoptotic cell death was evident in the spleen and thymus of OP[N.sup.+/+] mice subjected to HU but not in OP[N.sup.-/-] mice. Importantly, lymphocytes from both OP[N.sup.+/+] and OP[N.sup.-/-] mice were equally sensitive to corticosteroid-induced apoptosis. These results reveal that OPN is required for enhanced corticosterone production, immune organ atrophy, and weight loss in mice subjected to HU. apoptosis | immune | stress | lymphocytes | space flight

The capability of 2D IR spectroscopy to elucidate time-evolving structures is enhanced by a programmable mid-IR pulse shaper that greatly improves the ease, speed, and accuracy of data collection. Traditional ways of collecting 2D IR spectra are difficult to implement, cause distorted peak shapes, and result in poor time resolution and/or phase problems. We report on several methods for collecting 2D IR spectra by using a computer-controlled germanium acoustooptic modulator that overcomes the above problems. The accuracy and resolution of each method is evaluated by using model metal carbonyl compounds that have well defined line-shapes. Furthermore, phase cycling can now be employed to largely alleviate background scatter from heterogeneous samples. With these methods in hand, we apply 2D IR spectroscopy to study the structural diversity in amyloid fibers of aggregated human islet amyloid polypeptide (hlAPP), which is involved with type 2 diabetes. The 2D IR spectra reveal that the [beta]-sheet fibers have a large structural distribution, as evidenced by an inhomogeneously broadened [beta]-sheet peak and strong coupling to random coil conformations. Structural diversity is an important characteristic of hlAPP because it may be that partly folded peptides cause the disease. This experiment on hlAPP is an example of how computer generation of 2D IR pulse sequences is a key step toward automating 2D IR spectroscopy, so that new pulse sequences can be implemented quickly and a diverse range of systems can be studied more easily. femtosecond spectroscopy | infrared spectroscopy | pulse shaping | protein structure

Protein and peptide assembly into amyloid has been implicated in functions that range from beneficial epigenetic controls to pathological etiologies. However, the exact structures of the assemblies that regulate biological activity remain poorly defined. We have previously used [Zn.sup.2+] to modulate the assembly kinetics and morphology of congeners of the amyloid [beta] peptide (A[beta]) associated with AIzheimer's disease. We now reveal a correlation among A[beta]-[Cu.sup.2+] coordination, peptide self-assembly, and neuronal viability. By using the central segment of A[beta], HHQKLVFFA or A[beta](13-21), which contains residues H13 and H14 implicated in A[beta]-metal ion binding, we show that [Cu.sup.2+] forms complexes with A[beta](13-21) and its K16A mutant and that the complexes, which do not selfassemble into fibrils, have structures similar to those found for the human prion protein, PrP. N-terminal acetylation and H14A substitution, Ac-A[beta](13-21)H14A, alters metal coordination, allowing [Cu.sup.2+] to accelerate assembly into neurotoxic fibrils. These results establish that the N-terminal region of A[beta] can access different metal-ion-coordination environments and that different complexes can lead to profound changes in A[beta] self-assembly kinetics, morphology, and toxicity. Related metal-ion coordination may be critical to the etiology of other neurodegenerative diseases. copper-binding | neurotoxicity | self-assembly

Herpes simplex virus glycoprotein B (gB) is one of four glycoproteins essential for viral entry and cell fusion. Recently, an x-ray structure of the nearly full-length trimeric gB ectodomain was determined. Five structural domains and two linker regions were identified in what is probably a postfusion conformation. To identify functional domains of gB, we performed random linker-insertion mutagenesis. Analyses of 81 mutants revealed that only 27 could fold to permit processing and transport of gB to the cell surface. These 27 mutants fell into three categories. Insertions into two regions excluded from the solved structure (the N terminus and the C-terminal cytoplasmic tail) had no negative effect on cell fusion and viral entry activity, identifying regions that can tolerate altered structure without loss of function. Insertions into a disordered region in domain II and the adjacent linker region also permitted partial cell fusion and viral entry activity. Insertions at 16 other positions resulted in loss of cell fusion and viral entry activity, despite detectable levels of cell surface expression. Four of these insertion sites were not included in the solved structure. Two were between residues exposed to a cavity that is too small to accommodate the 5-amino acid insertions, consistent with the solved structure being different from the native prefusion structure. Ten were between residues exposed to the surface of the trimer, identifying regions that may be critical for interactions with other viral proteins or cellular components or for transitions from the prefusion to postfusion state. cell fusion | glycoprotein B | viral entry | mutation

Curli are functional amyloid fibers assembled by enteric bacteria such as Escherichia coli and Salmonella spp. In E. coli, the polymerization of the major curli fiber subunit protein CsgA into an amyloid fiber depends on the minor curli subunit protein, CsgB. The outer membrane-localized CsgB protein shares [approximately equal to] 30% sequence identity with the amyloid-forming protein CsgA, suggesting that CsgB might also have amyloidogenic properties. Here, we characterized the biochemical properties of CsgB and the molecular basis for CsgB-mediated nucleation of CsgA. Deletion analysis revealed that a CsgB molecule missing 19 amino acids from its C terminus ([CsgB.sub.trunc]) was not outer membrane-associated, but secreted away from the cell. [CsgB.sub.trunc] was overexpressed and purified from the extracellular milieu of cells as an SDS-soluble, nonaggregated protein. Soluble [CsgB.sub.trunc] assembled into fibers that bound to the amyloid-specific dyes Congo red and thioflavin-T. [CsgB.sub.trunc] fibers were able to seed soluble CsgA polymerization in vitro. [CsgB.sub.trunc] displayed modest nucleator activity in vivo, as demonstrated by its ability to convert extracellular CsgA into an amyloid fiber. Unlike WT CsgB, [CsgB.sub.trunc] was only able to act as a nucleator when cells were genetically manipulated to secrete higher concentrations of CsgA. This work represents a unique demonstration of functional amyloid nucleation and it suggests an elegant model for how E. coli guides efficient amyloid fiber formation on the cell surface. amyloid | nucleation | aggregation | seeding Congo red

Asn-linked glycans (N-glycans) play important roles in the quality control (QC) of glycoprotein folding in the endoplasmic reticulum (ER) lumen and in ER-associated degradation (ERAD) of proteins by cytosolic proteasomes. A UDP-Glc:glycoprotein glucosyltransferase glucosylates N-glycans of misfolded proteins, which are then bound and refolded by calreticulin and/or calnexin in association with a protein disulfide isomerase. Alternatively, an [alpha]-1,2-mannosidase (Mns1) and mannosidase-like proteins (ER degradation-enhancing [alpha]-mannosidase-like proteins 1, 2, and 3) are part of a process that results in the dislocation of misfolded glycoproteins into the cytosol, where proteins are degraded in the proteasome. Recently we found that numerous protists and fungi contain 0-11 sugars in their N-glycan precursors versus 14 sugars in those of animals, plants, fungi, and Dictyostelium. Our goal here was to determine what effect N-glycan precursor diversity has on N-glycan-dependent QC systems of glycoprotein folding and ERAD. N-glycan-de+pendent QC of folding (UDP-Glc:glycoprotein glucosyltransferase, calreticulin, and/or calnexin) was present and active in some but not all protists containing at least five mannose residues in their N-glycans and was absent in protists lacking Man. In contrast, N-glycan-dependent ERAD appeared to be absent from the majority of protists. However, Trypanosoma and Trichomonas genomes predicted ER degradation-enhancing [alpha]-mannosidase-like protein and Mns1 orthologs, respectively, each of which had [alpha]-mannosidase activity in vitro. Phylogenetic analyses suggested that the diversity of N-glycan-dependent QC of glycoprotein folding (and possibly that of ERAD) was best explained by secondary loss. We conclude that N-glycan precursor length has profound effects on N-glycan-dependent QC of glycoprotein folding and ERAD. protein folding | protists | Trichomonas | Entamoeba

An unknown aspect of protein misfolding and topological organization and symmetry of an amyloid fibril itself which could be helpful to study the polymorphism of protein non-native [beta]-pleated aggregates are described.

Chemoenzymatic synthesis of the respective galactosylated and alpha2,3/6-sialylated N-glycans is performed to address the issue whether core fucosylation and the bisecting N-acetylglucosamine residue modulate ligand properties of complex-type biantennary N-glycans. The study results help in devising new, non-natural modifications to realize the full potential of glycoengineering for diagnostic and therapeutic purposes.

The effect of copper, zinc and calcium on the structure stability of serum amyloid A (SAA)2.2 in aqueous solution is examined using various probes of quaternary, tertiary, and secondary structure. The result shows that the structure and stability of SAA2.2 are differently affected depending on the metal type and concentration.

The microheterogeneity of large sugar chains in glycopeptides from S-layer glycoproteins containing up to 51 monosaccharide residues at a single O-attachment site on a 12 amino acid peptide backbone was investigated by Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). Structural elucidation of glycopeptides with the same amino acid sequence and different glycoforms, having such a high saccharide-to-peptide ratio, was achieved by applying infrared multiphoton dissociation (IRMPD) MS/MS for the first time. A 100% sequence coverage of the glycan chain and a 50% coverage of the peptide backbone fragmentation were obtained. The microheterogeneity of carbohydrate chains at the same glycosylation site, containing largely rhamnose, could have been reliably assessed.

The mutagenesis experiments, designed to understand the nature of the packing constraints within the amyloid motif are reviewed. The plastic nature of amyloid appears to involve the malleability of both side chain interactions and H-bonded secondary structure and at its most basic level could be attributed to an apparently significant stability contribution from the noncovalent multimeric nature of the amyloid fibril.