Efficient Procedure for N-Glycan Analyses and Detection of Endo H-Like Activity in Human Tumor Specimens
Language English Country United States Media print-electronic
Document type Journal Article, Research Support, Non-U.S. Gov't
- Keywords
- Biopsy, EndoH, cancer cells, glycans, glycosylation, mass spectrometry, tumor tissue,
- MeSH
- Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase metabolism MeSH
- Glycosylation MeSH
- Humans MeSH
- Mannose MeSH
- Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase metabolism MeSH
- Tumor Cells, Cultured MeSH
- Neoplasms chemistry pathology MeSH
- Polysaccharides analysis MeSH
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization MeSH
- Sample Size MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase MeSH
- Mannose MeSH
- Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase MeSH
- poly-N-acetyllactosamine MeSH Browser
- Polysaccharides MeSH
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.
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