Efficient Procedure for N-Glycan Analyses and Detection of Endo H-Like Activity in Human Tumor Specimens.
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.
Erika
E
Lattová
The Institute of Human Virology, University of Maryland School of Medicine , 725 West Lombard Street, Baltimore, Maryland 21201, United States.
Joseph
J
Bryant
Department of Respiratory Diseases and Tuberculosis, University Hospital Brno, Medical Faculty, Masaryk University , 625 00 Brno, Czech Republic.
Jana
J
Skřičková
Central European Institute for Technology, Masaryk University , Kamenice 5, 625 00 Brno, Czech Republic.|National Centre for Biomolecular Research, Faculty of Science, Masaryk University , Kamenice 5, 625 00 Brno, Czech Republic.
Zbyněk
Z
Zdráhal
The Institute of Human Virology, University of Maryland School of Medicine , 725 West Lombard Street, Baltimore, Maryland 21201, United States.
Mikuláš
M
Popovič
D006031:Glycosylation; D006801:Humans; D008358:Mannose; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism*; D009369:Neoplasms / Q000737:chemistry / Q000473:pathology*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D011134:Polysaccharides / Q000032:analysis*; D018401:Sample Size; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014407:Tumor Cells, Cultured
D011134:Polysaccharides; C037199:poly-N-acetyllactosamine; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D008358:Mannose
Biopsy; EndoH; cancer cells; glycans; glycosylation; mass spectrometry; tumor tissue