Most methods for the analysis of oligosaccharides from biological sources require a glycan derivatization step: glycans may be derivatized to introduce a chromophore or fluorophore, facilitating detection after chromatographic or electrophoretic separation. Derivatization can also be applied to link charged or hydrophobic groups at the reducing end to enhance glycan separation and mass-spectrometric detection. Moreover, derivatization steps such as permethylation aim at stabilizing sialic acid residues, enhancing mass-spectrometric sensitivity, and supporting detailed structural characterization by (tandem) mass spectrometry. Finally, many glycan labels serve as a linker for oligosaccharide attachment to surfaces or carrier proteins, thereby allowing interaction studies with carbohydrate-binding proteins. In this review, various aspects of glycan labeling, separation, and detection strategies are discussed.FigureMALDI-FTICR-MS of 2AA-labeled total plasma N-glycans

Staphylococcus aureus is a major human pathogen associated with high mortality. The emergence of antibiotic resistance and the inability of antibiotics to counteract bacterial cytotoxins involved in the pathogenesis of S. aureus call for novel therapeutic approaches, such as passive immunization with monoclonal antibodies (mAbs). The complexity of staphylococcal pathogenesis and past failures with single mAb products represent considerable barriers for antibody-based therapeutics. Over the past few years, efforts have focused on neutralizing α-hemolysin. Recent findings suggest that the concerted actions of several cytotoxins, including the bi-component leukocidins play important roles in staphylococcal pathogenesis. Therefore, we aimed to isolate mAbs that bind to multiple cytolysins by employing high diversity human IgG1 libraries presented on the surface of yeast cells. Here we describe cross-reactive antibodies with picomolar affinity for α-hemolysin and 4 different bi-component leukocidins that share only ∼26% overall amino acid sequence identity. The molecular basis of cross-reactivity is the recognition of a conformational epitope shared by α-hemolysin and F-components of gamma-hemolysin (HlgAB and HlgCB), LukED and LukSF (Panton-Valentine Leukocidin). The amino acids predicted to form the epitope are conserved and known to be important for cytotoxic activity. We found that a single cross-reactive antibody prevented lysis of human phagocytes, epithelial and red blood cells induced by α-hemolysin and leukocidins in vitro, and therefore had superior effectiveness compared to α-hemolysin specific antibodies to protect from the combined cytolytic effect of secreted S. aureus toxins. Such mAb afforded high levels of protection in murine models of pneumonia and sepsis.

Antibody glycosylation has been shown to change with various processes. This review presents mass spectrometric approaches for antibody glycosylation analysis at the level of released glycans, glycopeptides, and intact protein. With regard to IgG fragment crystallizable glycosylation, mass spectrometry has shown its potential for subclass-specific, high-throughput analysis. In contrast, because of the vast heterogeneity of peptide moieties, fragment antigen binding glycosylation analysis of polyclonal IgG relies entirely on glycan release. Next to IgG, IgA has gained some attention, and studies of its O- and N-glycosylation have revealed disease-associated glycosylation changes. Glycoproteomic analyses of IgM and IgE are lagging behind but should complete our picture of glycosylation's influence on antibody function.

This review presents recent progress in employing hydrophilic interaction liquid chromatography (HILIC) for glycan and glycopeptides analysis. After an introduction of this technique, the following themes are addressed: (i) implementation of HILIC in large-scale studies for analyzing the human plasma N-glycome; (ii) the use of HILIC UPLC (ultrahigh pressure liquid chromatography) for fast high-resolution runs and its successful application with online MS for glycan and glycopeptide analysis; (iii) high-throughput profiling using HILIC solid-phase extraction in combination with MS detection; (iv) HILIC sample preparation for CE and CGE; (v) the latest glycoproteomic approaches implementing HILIC separation; (vi) future perspectives of HILIC including its use in large-scale glycoproteomics studies such as the analysis of entire glycoproteomes at the glycopeptide level.

Variations in the formal electrochemical potential (E0) and electron‐transfer rates (k0) of the blue copper protein azurin have been directly observed. A new method, fluorescent cyclic voltammetry (FCV), was used to resolve the properties of 100–1000 proteins. On this scale, the presence of large variations in the values of both E0 and k0 could be established and several forms of heterogeneity were differentiated.