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Asparagine-linked glycosylation is a common post-translational modification of proteins in eukaryotes. Mutations in the human ALG3 gene cause changed levels and altered glycan structures on mature glycoproteins and are the cause of a severe congenital disorder of glycosylation (CDG-Id). Diverse glycoproteins are also under-glycosylated in Saccharomyces cerevisae alg3 mutants. Here we analyzed site-specific glycosylation occupancy in this yeast model system using peptide-N-glycosidase F to label glycosylation sites with an asparagine-aspartate conversion that creates a new endoproteinase AspN cleavage site, followed by proteolytic digestion, and detection of peptides and glycopeptides by LC-ESI-MS/MS. We used this analytical method to identify and measure site-specific glycosylation occupancy in alg3 mutant and wild type yeast strains. We found decreased site-specific N-glycosylation occupancy in the alg3 knockout strain preferentially at Asn-Xaa-Ser sequences located in secondary structural elements, features previously associated with poor glycosylation efficiency. Furthermore, we identified 26 previously experimentally unverified glycosylation sites. Our results provide insights into the underlying mechanisms of disease in CDG-Id, and our methodology will be useful in site-specific glycosylation analysis in many model systems and clinical applications.

Efficient separation of mucins (200 kDa-2 MDa) was demonstrated using gradient SDS agarose/polyacrylamide composite gel electrophoresis (SDS-AgPAGE). Inclusion of urea (SDS-UAgPAGE) in the gels casting were shown to have no effect on the migration of mucins in the gel and allowed casting of gel at room temperature. This simplified the procedure for multiple casting of agarose polyacrylamide gradients and increased reproducibility of these gels. Hence, the implementation of urea makes the technique applicable for high throughput isolation and screening of mucin oligosaccharides by LC-MS after releasing the oligosaccharides from isolated, blotted mucin subpopulations. It was also shown that the urea addition had no effect on other supporting applications such as western and lectin blotting. In addition, identification of the mucin protein after tryptic digestion and LC-MS was possible and no protein carbamylation due to the presence of urea in the gel was detected. LC-MS software developed for metabolomic analysis was used for O-linked oligosaccharide detection and differential display of various mucin samples. Using this method, heterogeneous glycosylation of mucins and mucin-type molecules isolated by SDS-AgPAGE and SDS-UAgPAGE was shown to consist of more than 80 different components in a single band, and in the extreme cases, up to 300-500 components (MUC5B/AC from saliva and sputum and). Metabolomic software was also used to show that the migration of mucin isoforms within the gel is due to heterogeneous size distribution of the oligosaccharides, with the slower migrating bands enriched in high-molecular-weight oligosaccharides., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Asparagine-linked glycosylation is a common and vital co- and post-translocational modification of diverse secretory and membrane proteins in eukaryotes that is catalyzed by the multiprotein complex oligosaccharyltransferase (OTase). Two isoforms of OTase are present in Saccharomyces cerevisiae, defined by the presence of either of the homologous proteins Ost3p or Ost6p, which possess different protein substrate specificities at the level of individual glycosylation sites. Here we present in vitro characterization of the polypeptide binding activity of these two subunits of the yeast enzyme, and show that the peptide-binding grooves in these proteins can transiently bind stretches of polypeptide with amino acid characteristics complementary to the characteristics of the grooves. We show that Ost6p, which has a peptide-binding groove with a strongly hydrophobic base lined by neutral and basic residues, binds peptides enriched in hydrophobic and acidic amino acids. Further, by introducing basic residues in place of the wild type neutral residues lining the peptide-binding groove of Ost3p, we engineer binding of a hydrophobic and acidic peptide. Our data supports a model of Ost3/6p function in which they transiently bind stretches of nascent polypeptide substrate to inhibit protein folding, thereby increasing glycosylation efficiency at nearby asparagine residues., (Copyright © 2011 The Protein Society.)

We present an atomic force microscopy (AFM) and ellipsometry study of ultrathin smectic films on silicon substrates. By controlling the amount of the liquid-crystal material that is spin coated on the substrate, we are able to prepare films consisting of a defined small number (ranging from 1 to 4) of smectic layers. AFM measurements show that the films possess a specific surface structure with a lateral feature size of a few microns and steplike height variations of 3.3 nm. The height of the steps corresponds to the smectic layer spacing of the material used, indicating that the surface structure is the result of a partial formation of the topmost smectic layer of these films. The pattern of the surface structure either corresponds to isolated islands (regions in which the film thickness is enhanced by one smectic layer) or consists of pores (film thickness decreased by one layer). A smooth surface is only obtained if the amount of the liquid-crystal material is precisely tuned to certain values, indicating the formation of a complete smectic top layer. A well-defined relation exists between the liquid crystal concentration in the spin-coating solution and the obtained structure, enabling the controlled generation of island structures, pore structures, or smooth surfaces. The two-dimensional island or pore structure is stable on the time scale of a few days. Preliminary results concerning the thermal stability are reported. Our study highlights the usefulness of AFM measurements for the study of smectic liquid-crystal surfaces., (©2011 American Physical Society)

Non-optimal codons are generally characterised by a low concentration of isoaccepting tRNA and a slower translation rate compared to optimal codons. In a previous study, we reported a 20-fold reduction in maltose binding protein (MBP) level when the non-optimal codons in the signal sequence were optimised. In this study, we report that the 20-fold reduction is rescued when MBP is expressed at 28 degrees C instead of 37 degrees C, suggesting that the signal sequence optimised MBP protein (MBP-opt) may be misfolded, and is being degraded at 37 degrees C. Consistent with this idea, transient induction of the heat shock proteases prior to MBP expression at 28 degrees C restores the 20-fold difference, demonstrating that the difference in production levels is due to post-translational degradation of MBP-opt by the heat-shock proteases. Analysis of the structure of purified MBP-wt and MBP-opt grown at 28 degrees C showed that although they have similar secondary structure content, MBP-opt is more resistant to thermal unfolding than is MBP-wt. The two proteins also exhibit different tryptic fragment profiles, further confirming that they are folded into conformationally different states. This is the first study to demonstrate that signal sequence non-optimal codons can influence the folding of the mature exported protein.

We describe a new method for producing homogeneous eukaryotic N-glycoproteins. The method involves the engineering and functional transfer of the Campylobacter jejuni glycosylation machinery in Escherichia coli to express glycosylated proteins with the key GlcNAc-Asn linkage. The bacterial glycans were then trimmed and remodeled in vitro by enzymatic transglycosylation to fulfill a eukaryotic N-glycosylation. It provides a potentially general platform for producing eukaryotic N-glycoproteins.

Asparagine-linked glycosylation is catalysed by oligosaccharyltransferase (OTase). In Trypanosoma brucei OTase activity is catalysed by single-subunit enzymes encoded by three paralogous genes of which TbSTT3B and TbSTT3C can complement a yeast Deltastt3 mutant. The two enzymes have overlapping but distinct peptide acceptor specificities, with TbSTT3C displaying an enhanced ability to glycosylate sites flanked by acidic residues. TbSTT3A and TbSTT3B, but not TbSTT3C, are transcribed in the bloodstream and procyclic life cycle stages of T. brucei. Selective knockdown and analysis of parasite protein N-glycosylation showed that TbSTT3A selectively transfers biantennary Man(5)GlcNAc(2) to specific glycosylation sites whereas TbSTT3B selectively transfers triantennary Man(9)GlcNAc(2) to others. Analysis of T. brucei glycosylation site occupancy showed that TbSTT3A and TbSTT3B glycosylate sites in acidic to neutral and neutral to basic regions of polypeptide, respectively. This embodiment of distinct specificities in single-subunit OTases may have implications for recombinant glycoprotein engineering. TbSTT3A and TbSTT3B could be knocked down individually, but not collectively, in tissue culture. However, both were independently essential for parasite growth in mice, suggesting that inhibiting protein N-glycosylation could have therapeutic potential against trypanosomiasis.

Cytochromes of the c-type function on the outer side of the cytoplasmic membrane in bacteria where they also are assembled from apo-cytochrome polypeptide and haem. Two distinctly different systems for cytochrome c maturation are found in bacteria. System I present in Escherichia coli has eight to nine different Ccm proteins. System II is found in Bacillus subtilis and comprises four proteins: CcdA, ResA, ResB and ResC. ResB and ResC are poorly understood polytopic membrane proteins required for cytochrome c synthesis. We have analysed these two B. subtilis proteins produced in E. coli and in the native organism. ResB is shown to bind protohaem IX and haem is found covalently bound to residue Cys-138. Results in B. subtilis suggest that also ResC can bind haem. Our results complement recent findings made with Helicobacter CcsBA supporting the hypothesis that ResBC as a complex translocates haem by attaching it to ResB on the cytoplasmic side of the membrane and then transferring it to an extra-cytoplasmic location in ResC, from where it is made available to the apo-cytochromes.

Asparagine-linked glycosylation is a common posttranslational modification of diverse secretory and membrane proteins in eukaryotes, where it is catalyzed by the multiprotein complex oligosaccharyltransferase. The functions of the protein subunits of oligoasccharyltransferase, apart from the catalytic Stt3p, are ill defined. Here we describe functional and structural investigations of the Ost3/6p components of the yeast enzyme. Genetic, biochemical and structural analyses of the lumenal domain of Ost6p revealed oxidoreductase activity mediated by a thioredoxin-like fold with a distinctive active-site loop that changed conformation with redox state. We found that mutation of the active-site cysteine residues of Ost6p and its paralogue Ost3p affected the glycosylation efficiency of a subset of glycosylation sites. Our results show that eukaryotic oligosaccharyltransferase is a multifunctional enzyme that acts at the crossroads of protein modification and protein folding.

Increasing evidence suggests a central role for oxidative stress in the pathology of prion diseases, a group of fatal neurodegenerative disorders associated with structural conversion of the prion protein (PrP). Because UV-light-induced protein damage is mediated by direct photo-oxidation and radical reactions, we investigated the structural consequences of UVB radiation on recombinant murine and human prion proteins at pH 7.4 and pH 5.0. As revealed by circular dichroism and dynamic light scattering measurements, the observed PrP aggregation follows two independent pathways: (i) complete unfolding of the protein structure associated with rapid precipitation or (ii) specific structural conversion into distinct soluble beta-oligomers. The choice of pathway was directly attributed to the chromophoric properties of the PrP species and the susceptibility to oxidation. Regarding size, the oligomers characterized in this study share a high degree of identity with oligomeric species formed after structural destabilization induced by other triggers, which significantly strengthens the theory that partly unfolded intermediates represent initial precursor molecules directing the pathway of PrP aggregation. Moreover, we identified the first suitable photo-trigger capable of inducing refolding of PrP, which has an important biotechnological impact in terms of analyzing the conversion process on small time scales.

Asparagine-linked glycosylation is the most common post-translational modification of proteins catalyzed in eukaryotes by the multiprotein complex oligosaccharyltransferase. Apart from the catalytic Stt3p, the roles of the subunits are ill defined. Here we describe functional investigations of the Ost3/6p components of the yeast enzyme. We developed novel analytical tools to quantify glycosylation site occupancy by enriching glycoproteins bound to the yeast polysaccharide cell wall, tagging glycosylated asparagines using endoglycosidase H glycan release, and detecting peptides and glycopeptides with LC-ESI-MS/MS. We found that the paralogues Ost3p and Ost6p were required for efficient glycosylation of distinct defined glycosylation sites. Our results describe a novel method for relative quantification of glycosylation occupancy in the genetically tractable yeast system and show that eukaryotic oligosaccharyltransferase isoforms have different activities toward protein substrates at the level of individual glycosylation sites.

The transfer of lipid-linked oligosaccharide to asparagine residues of polypeptide chains is catalyzed by oligosaccharyltransferase (OTase). In most eukaryotes, OTase is a hetero-oligomeric complex composed of eight different proteins, in which the STT3 component is believed to be the catalytic subunit. In the parasitic protozoa Leishmania major, four STT3 paralogues, but no homologues to the other OTase components seem to be encoded in the genome. We expressed each of the four L. major STT3 proteins individually in Saccharomyces cerevisiae and found that three of them, LmSTT3A, LmSTT3B, and LmSTT3D, were able to complement a deletion of the yeast STT3 locus. Furthermore, LmSTT3D expression suppressed the lethal phenotype of single and double deletions in genes encoding other essential OTase subunits. LmSTT3 proteins did not incorporate into the yeast OTase complex but formed a homodimeric enzyme, capable of replacing the endogenous, multimeric enzyme of the yeast cell. Therefore, these protozoan OTases resemble the prokaryotic enzymes with respect to their architecture, but they used substrates typical for eukaryotic cells: N-X-S/T sequons in proteins and dolicholpyrophosphate-linked high mannose oligosaccharides.

The homochirality of biomolecules is a prerequisite for the origin and evolution of terrestrial life. The unique selection of D-monosaccharides, in particular, D-ribose in RNA and D-deoxyribose in DNA, leads to the construction of proteins by L-amino acids. This points to the exclusive role of stereoselectivity in the most important physiological processes. So far, there is no experimental confirmation for the theoretical calculations of the energy differences between enantiomers used for the explanation of the stereoselection of biomolecules. Therefore, the question of why nature prefers one configuration over the other still lacks a definitive answer. Here, we present the first experimental evidence that the D-enantiomer of RNA has a different electronic structure compared to the corresponding L-enantiomer. When varying the incident photon energy of the ultraviolet Raman probe across 5 eV, D- and L-isomers of the RNA duplex with the sequence [r(CUGGGCGG).r(CCGCCUGG)] show differences in the intensity of the vibrational modes with energies of 124.0 meV to 210.8 meV. The intensity difference of these vibrational modes can be traced back to energy differences in the electronic levels of D- and L-RNA leading to the preferential stabilization of the naturally occurring D-configuration of RNA over the L-configuration.

Cystic fibrosis (CF) is characterized by chronic lung infection and inflammation, with periods of acute exacerbation causing severe and irreversible lung tissue damage. We used protein and glycosylation analysis of high-molecular mass proteins in saline-induced sputum from CF adults with and without an acute exacerbation, CF children with stable disease and preserved lung function, and healthy non-CF adult and child controls to identify potential biomarkers of lung condition. While the main high-molecular mass proteins in the sputum from all subjects were the mucins MUC5B and MUC5AC, these appeared degraded in CF adults with an exacerbation. The glycosylation of these mucins also showed reduced sulfation, increased sialylation, and reduced fucosylation in CF adults compared with controls. Despite improvements in pulmonary function after hospitalization, these differences remained. Two CF children showed glycoprotein profiles similar to those of CF adults with exacerbations and also presented with pulmonary flares shortly after sampling, while the remaining CF children had profiles indistinguishable from those of healthy non-CF controls. Sputum mucin glycosylation and degradation are therefore not inherently different in CF, and may also be useful predictive biomarkers of lung condition.

The purpose of this review is to provide a concise overview of developments over the last 15 years in the field of laboratory tests in human medicine that are based on the detection of alterations in the glycan part of glycoconjugates. We show how glycosylation-based diagnostic testing is widespread in the current clinical practice, in different formats. To provide the necessary focus in this extremely broad field, we have only included assays that are either in actual clinical use or that are under active development towards clinical use, with some bias towards assays that were recently developed. The fields included are: cancer, infectious disease, genetic defects of glycoconjugate biosynthesis and catabolism, auto-immunity, drug abuse and liver disease. To conclude this review, we provide a viewpoint on the future of the glyco-diagnostics field in terms of novel technologies, especially with regard to the discovery and clinical implementation of biomarkers that are based on pathologically altered endogenous glycotopes.

We study the behavior of optical properties of human skin across its layers. We describe ellipsometric measurements for spectrally resolved in vivo investigations of biological tissue. We show in vivo measurements on human skin of the ellipsometric parameters Psi and Delta, which describe the change in polarization of light on reflection on a sample. A tape-stripping study reveals the depth profile of the ellipsometric parameters into the stratum corneum. The depth profile shows an increase in both quantities with increased depth. Analyzing the development of Psi on the number of strips using an exponential function shows a steady state after approximately 2 microm. The evolution of Psi and Delta can be described using a morphological model containing an effective medium approximation accounting for the water content of the skin, surface roughnesses of the corneocytes, as well as the alternating cell and lipid layers.

N-linked protein glycosylation is found in all domains of life. In eukaryotes, it is the most abundant protein modification of secretory and membrane proteins, and the process is coupled to protein translocation and folding. We found that in bacteria, N-glycosylation can occur independently of the protein translocation machinery. In an in vitro assay, bacterial oligosaccharyltransferase glycosylated a folded endogenous substrate protein with high efficiency and folded bovine ribonuclease A with low efficiency. Unfolding the eukaryotic substrate greatly increased glycosylation. We propose that in the bacterial system, glycosylation sites are located in flexible parts of folded proteins, whereas the eukaryotic cotranslational glycosylation evolved to a mechanism presenting the substrate in a flexible form before folding.

The Campylobacter jejuni pgl locus encodes an N-linked protein glycosylation machinery that can be functionally transferred into Escherichia coli. In this system, we analyzed the elements in the C. jejuni N-glycoprotein AcrA required for accepting an N-glycan. We found that the eukaryotic primary consensus sequence for N-glycosylation is N terminally extended to D/E-Y-N-X-S/T (Y, X not equalP) for recognition by the bacterial oligosaccharyltransferase (OST) PglB. However, not all consensus sequences were N-glycosylated when they were either artificially introduced or when they were present in non-C. jejuni proteins. We were able to produce recombinant glycoproteins with engineered N-glycosylation sites and confirmed the requirement for a negatively charged side chain at position -2 in C. jejuni N-glycoproteins. N-glycosylation of AcrA by the eukaryotic OST in Saccharomyces cerevisiae occurred independent of the acidic residue at the -2 position. Thus, bacterial N-glycosylation site selection is more specific than the eukaryotic equivalent with respect to the polypeptide acceptor sequence.

Human hair is a biological layered system composed of two major layers, the cortex and the cuticle. We show spectrally resolved ellipsometry measurements of the ellipsometric parameters Psi and Delta of single human hairs. The spectra reflect the layered nature of hair and the optical anisotropy of the hair's structure. In addition, measurements on strands of human hair show a high reproducibility of the ellipsometric parameters for different hair fiber bundles from the same person. Based on the measurements, we describe a dielectric model of hair that explains the spectra in terms of the dielectric properties of the major parts of hair and their associated layer thicknesses. In addition, surface roughness effects modeled by a roughness layer with a complex refractive index given by an effective medium approach can be seen to have a significant effect on the measurements. We derive values for the parameters of the cuticle surface roughness layer of the thickness d(ACu)=273 to 360 nm and the air inclusion fA=0.6 to 5.7%.

Capillary liquid chromatography-mass spectrometry using graphitised carbon stationary phase and ion trap mass spectrometry was shown to be a powerful technique for analysing glycosaminoglycans digested with endoglycosidases. Commonly found disaccharides from heparin/heparan sulphate digests at sub nanomole levels were found to be separated by mass and/or retention time and detected by negative ion electrospray mass spectrometry predominantly as [M-H]- ions using a standard electrospray interface and flow rate between 6-10 microL/min. Graphitised carbon liquid chromatography-fragmentation mass spectrometry provided sequence data of disaccharides and oligosaccharides. Sequence information was obtained from either collision of the [M-H]- ions (low sulphated disaccharides) or of the [M+Na-2H]- ions (highly sulphated disaccharides). This separation and identification method of endoglycosidase digestion and sample preparation using a combination of cation exchange and graphitised carbon, was used to successfully analyse digests of keratan sulphate (keratanase) and heparin (heparinase) standards, and hyaluronic acid (hyaluronidase) from synovial fluid samples.

This study aimed to characterize human salivary glycoforms and the natural glycosylation variation of the major ABO blood group bearing high molecular weight glycoprotein fraction MG1, which mainly consists of MUC5B mucin. Reduced and alkylated mucins from individuals of blood group A, B, and O were purified by sodium dodecyl sulfate-agarose/polyacrylamide composite gel electrophoresis (SDS-AgPAGE), blotted to polyvinylidene fluoride (PVDF) membranes, and visualized with alcian blue. O-linked oligosaccharides were released from MUC5B glycoform bands by reductive beta-elimination and analyzed by liquid chromatography (LC) electrospray ion trap mass spectrometry (MS). Slow electrophoretically migrating MUC5B components (sm) were found to be dominated by neutral oligosaccharides, and fast-migrating (fm) components were dominated by sulfated oligosaccharides. ABO blood group-specific sequences were found on all glycoforms, and novel oligosaccharides containing blood group A and B type sequences were sequenced. This is the first molecular description of the influence of the blood group ABO system on salivary MUC5B oligosaccharides. Expanding these results from the three A, B, and O individuals into larger population (29 individuals), we found oligosaccharide sequences corresponding to the blood group of the donor on MUC5B from 23 individuals. The remaining six individuals were characterized by a high degree of sialylation. These individuals were assigned as nonsecretors, whereas blood group-expressing individuals were assigned as secretors. Western blot assays with antibodies confirmed increased expression of Sialyl Lewis a (Si-Le(a)) in the nonsecretors. Our results highlight that salivary MUC5B consists of glycoforms with distinct glycosylation that vary extensively between individuals and that some of this variation is owing to blood group and secretor status.

SMG (submucosal gland) secretions are a major component of the airway surface liquid, are associated with innate immunity in the lung, and have been reported to be altered in lung disease. Changes in lung mucosal glycosylation have been reported in CF (cystic fibrosis), which may be responsible for differential bacterial binding to glycosylated components in the lung mucosa and hence increased pre-disposition to pulmonary infection. Glycoproteomic analysis was performed on SMG secretions collected from explanted bronchial tissue of subjects with severe lung disease, with and without CF, and controls without lung disease. Mucins MUC5B and MUC5AC were shown to be the dominant high-molecular-mass glycoprotein components, with a minor non-mucin glycoprotein component, gp-340, also present. Oligosaccharides containing blood-group determinants corresponding to subjects' blood type were abundant on MUC5B/MUC5AC, as were Lewis-type epitopes and their sialylated analogues, which are ligands for pathogens and leucocytes. No significant differences were found in the glycosylation of MUC5B/MUC5AC or gp-340 between CF and non-CF subjects with severe lung disease, implying that CF does not influence SMG secretion mucin glycosylation in end-stage lung disease. There were also no significant differences found in the glycosylation of these components in severe lung disease compared with non-diseased lungs. This suggests that previously reported changes in the glycosylation of respiratory glycoconjugates in CF, and other pulmonary conditions, are not due to the glycosylation of components in SMG secretions, but may involve other secretions, responses or extracellular factors.

The bioinformatic tool GlycosidIQ was developed for computerized interpretation of oligosaccharide mass spectrometric fragmentation based on matching experimental data with theoretically fragmented oligosaccharides generated from the database GlycoSuiteDB. This use of the software for glycofragment mass fingerprinting obviates a large part of the manual, labor intensive, and technically challenging interpretation of oligosaccharide fragmentation. Using 130 negative ion electrospray ionization-tandem mass spectrometry fragment spectra from identified oligosaccharide structures, it was shown that the GlycosidIQ scoring algorithms were able to correctly identify oligosaccharides in the great majority of cases (correct structure top ranked in 78% of the cases and an additional 17% were ranked second highest in the sample set).

Neutral O-linked oligosaccharides released from the salivary mucin MUC5B were separated and detected by negative ion LC-MS and LC-MS(2). The resolution of the chromatography and the information obtained from collision induced dissociation of detected [M - H](-) ions were usually sufficient to identify the sequence of individual oligosaccharides, illustrated by the fact that 50 different oligosaccharides ranging from disaccharides to nonasaccharides could be assigned from the sample. Fragmentation was shown to yield mostly reducing end sequence fragments (Z(i) and Y(i)), enabling primary sequence assignment. Specific fragmentation pathways or patterns were also detected giving specific linkage information. The reducing end core (Gal/GlcNAcbeta1-3GalNAcol or Gal/GlcNAcbeta1-3(GlcNAcbeta1-6)GalNAcol) could be deduced from the pronounced glycosidic C-3 cleavage and A(i) type cleavages of the reducing end GalNAcol, together with the non reducing end fragment from the loss of a single substituted GalNAcol. Substitution patterns on GlcNAc residues were also found, indicative for C-4 substitution ((0,2)A(i) - H(2)O cleavage) and disubstitution of C-3 and C-4 (Z(i)/Z(i) cleavages). This kind of fragmentation can be used for assigning the mode of chain elongation (Galbeta1-3/4GlcNAcbeta1-) and identification of Lewis type antigens like Lewis a/x and Lewis b/y on O-linked oligosaccharides. In essence, negative ion LC-MS(2) was able to generate extensive data for understanding the overall glycosylation pattern of a sample, especially when only a limited amount of material is available.

A technique with subpicomolar sensitivity was developed for analyzing O-linked oligosaccharides released from glycoproteins separated by gel electrophoresis. The protocol involves gel electrophoresis, electroblotting to poly-(vinylidene fluoride) membrane, reductive beta-elimination, and analysis of released oligosaccharides by liquid chromatography coupled to negative ion electrospray mass spectrometry. It was also found that N-linked oligosaccharides could be recovered under the same conditions, found both as free oligosaccharides and as distinct glycopeptides created from reductive cleavage of the protein backbone, giving some information on site-specific glycosylation. The method was used to demonstrate that the difference between human alpha-2HS-glycoprotein isoforms separated by 2D-gel electrophoresis was partially due to sialylation of both O-linked and N-linked oligosaccharides. It was also shown that both acidic and neutral oligosaccharides could be recovered and analyzed simultaneously from high molecular mass (200,000-5,000,000 Da) highly glycosylated mucin glycoproteins collected from small intestine and saliva and separated by sodium dodecyl sulfate-agarose/polyacrylamide composite gels. Mass spectrometric data not only gave information about the mass distribution of the heterogeneous mixtures of oligosaccharides from [M - xH](x-) ions but also gave information about the isomeric heterogeneity of the oligosaccharides from their resolution by porous graphitized carbon chromatography. Tandem mass spectrometry was explored as a technique for distinguishing between oligosaccharide isomers with different sequences and also between oligosaccharides with the same sequence but with different linkage configurations.

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

Human open eye tear fluid was separated by low-percentage SDS/PAGE to detect high-molecular-mass protein components. Two bands were found with apparent molecular masses of 330 and 270 kDa respectively. By peptide-mass fingerprinting after tryptic digestion, the proteins were found to be isoforms of the DMBT1 gene product, with over 30% of the predicted protein covered by the tryptic peptides. By using gradient SDS/agarose/polyacrylamide composite gel electrophoresis and staining for glycosylation, it was shown that the two isoforms were the major high-molecular-mass glycoproteins of >200 kDa in human tear fluid. Western blotting showed that the proteins expressed sialyl-Le(a). After the release of oligosaccharides by reductive beta-elimination from protein blotted on to PVDF membrane, it was revealed by liquid chromatography-MS that the O-linked oligosaccharides were comprised mainly of highly sialylated oligosaccharides with up to 16 monosaccharide units. A majority of the oligosaccharides could be described by the formula dHex(0-->2)NeuAc(1-->)(x)Hex(x)HexNAc(x)(-ol), x=1-6, where Hex stands for hexose, dHex for deoxyhexose, HexNAc for N-acetylhexosamine and NeuAc for N-acetylneuraminate. The number of sialic acids in the formula is less than 5. Interpretation of collision-induced fragmentation tandem MS confirmed the presence of sialic acid and suggested the presence of previously undescribed structures carrying the sialyl-Le(a) epitopes. Small amounts of neutral and sulphated species were also present. This is the first time that O-linked oligosaccharides have been detected and described from protein variant of the DMBT1 gene.