The mucosal tissues of the gastrointestinal, respiratory, reproductive, and urinary tracts, and the surface of the eye present an enormous surface area to the exterior environment. All of these tissues are covered with resident microbial flora, which vary considerably in composition and complexity. Mucosal tissues represent the site of infection or route of access for the majority of viruses, bacteria, yeast, protozoa, and multicellular parasites that cause human disease. Mucin glycoproteins are secreted in large quantities by mucosal epithelia, and cell surface mucins are a prominent feature of the apical glycocalyx of all mucosal epithelia. In this review, we highlight the central role played by mucins in accommodating the resident commensal flora and limiting infectious disease, interplay between underlying innate and adaptive immunity and mucins, and the strategies used by successful mucosal pathogens to subvert or avoid the mucin barrier, with a particular focus on bacteria.

This protocol shows how to obtain a detailed glycan compositional and structural profile from purified glycoproteins or protein mixtures, and it can be used to distinguish different isobaric glycan isomers. Glycoproteins are immobilized on PVDF membranes before the N-glycans are enzymatically released by PNGase F, isolated and reduced. Subsequently, O-glycans are chemically released from the same protein spot by reductive β-elimination. After desalting with cation exchange microcolumns, the glycans are separated and analyzed by porous graphitized carbon liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Optionally, the glycans can be treated with sialidases or other specific exoglycosidases to yield more detailed structural information. The sample preparation takes approximately 4 d, with a heavier workload on days 2 and 3, and a lighter load on days 1 and 4. The time for data interpretation depends on the complexity of the samples analyzed. This method can be used in conjunction with the analysis of enriched glycopeptides by capillary/nanoLC-ESI-MS/MS, which together provide detailed information regarding the site heterogeneity of glycosylation.

Mass spectrometry (MS) of glycoproteins is an emerging field in proteomics, poised to meet the technical demand for elucidation of the structural complexity and functions of the oligosaccharide components of molecules. Considering the divergence of the mass spectrometric methods employed for oligosaccharide analysis in recent publications, it is necessary to establish technical standards and demonstrate capabilities. In the present study of the Human Proteome Organisation (HUPO) Human Disease Glycomics/Proteome Initiative (HGPI), the same samples of transferrin and immunoglobulin-G were analyzed for N-linked oligosaccharides and their relative abundances in 20 laboratories, and the chromatographic and mass spectrometric analysis results were evaluated. In general, matrix-assisted laser desorption/ionization (MALDI) time-of-flight MS of permethylated oligosaccharide mixtures carried out in six laboratories yielded good quantitation, and the results can be correlated to those of chromatography of reductive amination derivatives. For underivatized oligosaccharide alditols, graphitized carbon-liquid chromatography (LC)/electrospray ionization (ESI) MS detecting deprotonated molecules in the negative ion mode provided acceptable quantitation. The variance of the results among these three methods was small. Detailed analyses of tryptic glycopeptides employing either nano LC/ESI MS/MS or MALDI MS demonstrated excellent capability to determine site-specific or subclass-specific glycan profiles in these samples. Taking into account the variety of MS technologies and options for distinct protocols used in this study, the results of this multi-institutional study indicate that MS-based analysis appears as the efficient method for identification and quantitation of oligosaccharides in glycomic studies and endorse the power of MS for glycopeptide characterization with high sensitivity in proteomic programs.

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.

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 Ϫ H] Ϫ 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/GlcNAc␤1-3GalNAcol or Gal/GlcNAc␤1-3(GlcNAc␤1-6)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 Ϫ 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 (Gal␤1-3/ 4GlcNAc␤1-) 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. I dentification and understanding of biological processes involving glycoconjugates is a scientific area of increasing interest. This increase in interest has worked hand in hand with the development of more sensitive and specific analytical techniques in this field. Mass spectrometry has been the detection and characterization tool most frequently used in recent years for analysis of glycoproteins, glycopeptides, glycolipids, or free oligosaccharides (some reviews in [1][2][3][4][5]). In principle, mass spectrometry suffers from the fact that the monomeric components (the monosaccharides) of oligosaccharide chains consist of isomeric building blocks, and that linkage configuration and position are difficult to obtain with the technique. Even so, researchers have applied mass spectrometric techniques to answer biological questions (some examples in [6 -9]).Characterization of released or free oligosaccharides by mass spectrometry using electrospray ionization in negative mode has recently been shown to give both sequence and linkage information [10,11]. Negative ion mode has been shown to be applicable to both negatively charged oligosaccharides and for neutral oligosaccharides [12][13][14], and offers high sensitivity of detection without requiring derivatization or adduct formation to aid ionization. Electrospray is also easily coupled to with high resolution liquid chromatography, an important factor in resolving the degeneracy of structural isomers found i...