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

A multitude of monoclonal IgG antibodies directed against a variety of therapeutic targets is currently being developed and produced by biotechnological companies. The biological activity of IgGs is modulated by the N-glycans attached to the fragment crystallizable (Fc) part. For example, lack of core-fucoses on these N-glycans may lead to a drastic enhancement of antibody-mediated cellular cytotoxicity. Moreover, sialylation of Fc N-glycans determines the immunosuppressive properties of polyclonal IgG from human blood, which stimulates research into Fc glycosylation of human plasma IgG in various disease settings. This review presents and evaluates the different approaches which are used for IgG glycosylation analysis: N-glycans may be enzymatically or chemically released from purified IgG, prior to chromatographic or mass spectrometric analysis. Moreover, IgGs may be treated with endoproteinases such as trypsin, followed by glycosylation analysis at the glycopeptide level, which is generally accomplished by HPLC with ESI-MS. Alternatively, intact IgGs or fragments thereof obtained by enzymatic cleavages in the hinge region and by reduction may be analyzed by a large number of analytical techniques, including MS and chromatography or CE.

Many diseases are associated with changes in the glycosylation of plasma proteins. To search for glycan biomarkers, large sample sets have to be investigated for which high-throughput sample preparation and analysis methods are required. We here describe a 96 well plate-based high-throughput procedure for the rapid preparation of 2-aminobenzoic acid (2-AA) labeled N-glycans from 10 microL of human plasma. During this procedure, N-glycans are released from glycoproteins and subsequently labeled with 2-AA without prior purification. A hydrophilic interaction chromatography (HILIC)-based solid phase extraction method is then applied to isolate the 2-AA labeled N-glycans, which can be analyzed by MALDI-TOF-MS, HPLC with fluorescence detection, and CE-MS. The relative standard deviation for the intrabatch repeatability and the interbatch repeatability of the sample preparation method remained below 7% and below 9%, respectively, for all peaks observed by HPLC. Similar results were obtained with MALDI-TOF-MS, where 47 N-glycans could be measured consistently. The 2-AA labeled N-glycans were additionally analyzed by a CE-ESI-Q-TOF-MS method, which featured high resolution and mass accuracy, allowing the unambiguous determination of the N-glycan compositions. Up to four times, 96 human plasma samples can be handled in parallel, which, together with the versatility of the 2-AA label, makes this procedure very attractive for glycomics analysis of larger sample cohorts.

Over the last two decades, coupled capillary electrophoresis (CE)-mass spectrometry (MS) has developed into a generally accepted technique with a wide applicability. A growing number of CE-MS applications make use of capillaries where the internal wall is modified with surface coating agents. In CE-MS, capillary coatings are used to prevent analyte adsorption and to provide appropriate conditions for CE-MS interfacing. This paper gives an overview of the various capillary coating strategies used in CE-MS. The main attention is devoted to the way coatings can contribute to a proper CE-MS operation. The foremost capillary coating methods are discussed with emphasis on their compatibility with MS detection. The role of capillary coatings in the control of the electroosmotic flow and the consequences for CE-MS coupling are treated. Subsequently, an overview of reported applications of CE-MS employing different coating principles is presented. Selected examples are given to illustrate the usefulness of the coatings and the overall applicability of the CE-MS systems. It is concluded that capillary coatings can enhance the performance and stability of CE-MS systems, yielding a highly valuable and reproducible analytical tool.

High-throughput methods for oligosaccharide analysis are required when searching for glycan-based biomarkers. Next to mass spectrometry-based methods, which allow fast and reproducible analysis of such compounds, further separation-based techniques are needed, which allow for quantitative analysis. Here, an optimized sample preparation method for N-glycan-profiling by multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF) was developed, enabling high-throughput glycosylation analysis. First, glycans are released enzymatically from denatured plasma glycoproteins. Second, glycans are labeled with APTS using 2-picoline borane as a nontoxic and efficient reducing agent. Reaction conditions are optimized for a high labeling efficiency, short handling times, and only limited loss of sialic acids. Third, samples are subjected to hydrophilic interaction chromatography (HILIC) purification at the 96-well plate format. Subsequently, purified APTS-labeled N-glycans are analyzed by CGE-LIF using a 48-capillary DNA sequencer. The method was found to be robust and suitable for high-throughput glycan analysis. Even though the method comprises two overnight incubations, 96 samples can be analyzed with an overall labor allocation time of 2.5 h. The method was applied to serum samples from a pregnant woman, which were sampled during first, second, and third trimesters of pregnancy, as well as 6 weeks, 3 months, and 6 months postpartum. Alterations in the glycosylation patterns were observed with gestation and time after delivery.