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Total Glycomics Methods

The last few years have seen the publication of many methods for the structural analysis of glycans from their parent glycoproteins incorporating several of the techniques discussed earlier. Most rely on preliminary purification of the glycoprotein, site analysis, and then removal of the glycans for further analysis by ESI or MALDI MS and/or HPLC. The nature of the monosaccharide constituents is further investigated by HPLC or GC/MS after suitable derivatiza- tion or by exoglycosidase digestions. As a representative example, Morelle and Michalski [358] have published full practical details involving PNGase F release of the glycans from SDS-PAGE gel-separated glycoproteins followed by cleanup with porous graphitized carbon. O-glycans were then released by reductive elimination and cleaned with Dowex beads. Exoglycosidase digestion and methylation analysis by GC/MS was performed to identify the monosaccharides, and glycan profiles and glycan compositions were obtained by MALDI- TOF MS. Finally, nano-MS/MS was performed to obtain sequence and linkage information.

In another method where practical details were reported [359], PNGase F was again used to release the glycans, which were then labeled with 2-AA. This procedure lends itself to analysis by HPLC and capillary electrophoresis (CE) as well as to analysis by MALDI-TOF. Glycans were examined from human plasma glycoproteins and glycan cleanup employed hydrophilic interaction

chromatography, which allowed the separation of the labeled glycans from an excess of labeling reagent, proteins, lipids, and salts. High throughput was achieved by conducting the sample preparation in 96-well plate.

Full experimental details have also been published of a method for the analysis of human plasma glycoproteins and their attached glycans [360]. The target low-abundance proteins were concentrated by immunoprecipitation and removal of albumin, IgG, IgA, transferrin, haptoglobin, and a-1-antitrypsin and then separated by 1D anion-exchange chromatography with an eight-step salt elution. Fractions from each elution steps were transferred onto a 2D reversed- phase HPLC column, and the eluted compounds were digested with trypsin. MALDI MS was performed with a quadrupole ion trap (QIT)-TOF and LC/MS employed an Orbitrap. Finally, Wuhrer et al. [361, 362] have described a nanoscale LC/MS technique using NP LC/MS that is capable of producing fragmentation data from small N-linked glycans at the low femtomole level.

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