Although the importance of glycosylation has been thoroughly recognized in association with a number of biological processes, efficient assessments of glycans have been hampered by both the limited size of specimens and lengthy sample preparations, particularly in clinical settings. Here we report a simple preparative method for N-glycan analyses. It involves only short one-step chloroform-methanol extraction in presence or absence of water prior to PNGase F deglycosylation. The procedure was successfully applied to the investigation of N-glycans obtained from small numbers of in vitro cultured cancer cells (≤1 × 10(5)) and to tumor tissues, including patient biopsies of small size. MALDI-MS analysis confirmed the efficient release of all N-glycan types including complex forms with poly-N-acetyllactosamine chains. In addition, nonaqueous extraction of specimens from several established cancer cell lines, as well as patient tumor tissues, yielded high-mannose glycans with one GlcNAc moiety (Man3-9GlcNAc), strongly suggesting preservation of enzymatic activity analogous to Endo H enzyme. In summary, the method is both a step toward the practical use of glycan profiling and a way to detect Endo H-like activity in cancer specimens.

Central European Institute for Technology Masaryk University Kamenice 5 625 00 Brno Czech Republic

Department of Respiratory Diseases and Tuberculosis University Hospital Brno Medical Faculty Masaryk University 625 00 Brno Czech Republic

National Centre for Biomolecular Research Faculty of Science Masaryk University Kamenice 5 625 00 Brno Czech Republic

The Institute of Human Virology University of Maryland School of Medicine 725 West Lombard Street Baltimore Maryland 21201 United States

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