Professor Lauc coauthored over 200 research articles that are cited over 7000 times. DNA sequencing.137 However, because it has been used for fetuin = 83).147 The same analytical approach has been used both for IgG = 98),148 demonstrating a potential application of used technology for robust quantification of glycans as noninvasive plasma biomarkers. 5.2.2. Measurement and Data Processing Fluorescently labeled negatively charged glycans are electrokinetically injected into capillaries by applying a low voltage for a short period of time. Injected glycans migrate in the applied electric field through capillaries and are being separated based on their hydrodynamic volumes and their mass-to-charge ratios or, as recently demonstrated for HMOs, based on the secondary equilibrium of the borateCvicinal diol complexation.149 Migration time alignment standards (coinjected bracketing standards) are used to minimize migration time shifts between samples and facilitate glycan identification and quantification, by enabling electropherogram alignment and GU unit assignation. After manual or automated peak integration, total area normalization is usually used to extract glycan amounts as relative %area used for further analysis, again MMV008138 followed by batch correction and statistical analysis. Alternatively, total height normalization can also be used to obtain relative peak height proportions (%rPHP). 5.2.3. Glycan Structure and Characterization Analogous to UHPLC, structures of glycans separated by CGE-LIF are also elucidated by comparison of individual glycan peak glucose unit (GUCGE) with MMV008138 the GUCGE values of specific glycan structures in available databases and utilization of exoglycosidases sequencing.142,150 GUCGE values are assigned based on fluorescently (e.g., APTS) Rabbit Polyclonal to Caspase 14 (p10, Cleaved-Lys222) labeled standard oligosaccharide ladder, usually maltodextrin (homopolymer of 1 1,4-linked glucose), although dextran has also been used. The retention time of each unknown oligosaccharide correlates with the length of the sugar oligomer and is converted to a GUCGE scale used for a database search. It is of utmost importance that the same standard oligosaccharide ladder is used for analysis and the database buildup because CGE migration depends on hydrodynamic volumes affected by the molecular configuration and conformation.151 The development of databases containing CGE-LIF separated glycans has been lagging behind HPLC/UHPLC glycan databases due to more complex structural confirmation of individual glycans caused by difficulties of CGE coupling to MS. However, this is slowly changing, and nowadays several expanding databases, e.g., GUcal152 (recently broadened with the GlycoStore data)152,153 and glyXbase,154 exist (Table 1). Populating these databases with glycans labeled with alternative fluorescent labels and originating from glycoproteins other than human IgG will facilitate the use of CGE-LIF technology for low- and HT glycomic studies. Exoglycosidase sequencing has been used as a complementary approach to assist the MMV008138 glycan structure characterization both for continuum. Typically, this results in ionization biases, a reduction of measurement sensitivity, and issues in peak annotation.181 Third, the sialic acid residue in sialylated glycan species is extremely fragile and prone to both in-source and post-source metastable fragmentation. Partial, as well as full loss of sialic acid residues, will result in loss of biologically relevant information and induce quantitative biases in complex glycan mixtures. Finally, sialylation introduces a large source of (biologically relevant) variation as the sialic acids can be bound to the rest of the glycan moiety through various linkages (i.e., MMV008138 2,3, 2,6, 2,8, and 2,9). Sialylated glycans with multiple sialic acid residues often show linkage heterogeneity, resulting in a large number of potential isomeric glycan compositions. Without the exoglycosidase treatment (which cannot be considered HT), it is impossible to differentiate these in a typical MS1 analysis (which is common when using MALDI-MS), unless using chemical derivatization, which was shown to be feasible in HT fashion, for ethyl esterification by Reiding et al.175 To increase measurement sensitivity, substantial efforts were made to purify and.