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.
- Klíčová slova
- Biopsy, EndoH, cancer cells, glycans, glycosylation, mass spectrometry, tumor tissue,
- MeSH
- glykopeptidasa metabolismus MeSH
- glykosylace MeSH
- lidé MeSH
- mannosa MeSH
- mannosyl-glykoprotein endo-beta-N-acetylglukosaminidasa metabolismus MeSH
- nádorové buňky kultivované MeSH
- nádory chemie patologie MeSH
- polysacharidy analýza MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- velikost vzorku MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- glykopeptidasa MeSH
- mannosa MeSH
- mannosyl-glykoprotein endo-beta-N-acetylglukosaminidasa MeSH
- poly-N-acetyllactosamine MeSH Prohlížeč
- polysacharidy MeSH
Investigation of protein glycosylation is an important area in biomarker discovery and biopharmaceutical research. Alterations in protein N-glycosylation can be an indication of changes in pathological conditions in the medical field or production parameters of biotherapeutics. Rapid development of these disciplines calls for fast, high-throughput, and reproducible methods to analyze protein N-glycosylation. Currently used methods require either long deglycosylation times or large excess of enzymes. In this paper, we report on the use of PNGase F immobilization onto the surface of magnetic microparticles and their use in rapid and efficient removal of N-glycans from glycoproteins. The use of immobilized PNGase F also allowed reusability of the enzyme-coated beads as the magnetic microparticles can be readily partitioned from the sample by a magnet after each deglycosylation reaction. The efficiency and activity of the PNGase F coated magnetic beads was compared with in-solution enzyme reactions using standard glycoproteins possessing the major N-glycan types of neutral, high mannose, and highly sialylated carbohydrates. The PNGase F coated magnetic beads offered comparable deglycosylation level to the conventional in-solution based method in 10-min reaction times for the model glycoproteins of immunoglobulin G (mostly neutral carbohydrates), ribonuclease B (high mannose type sugars), and fetuin (highly sialylated oligosaccharides) with the special features of easy removal of the enzyme from the reaction mixture and reusability.
- Klíčová slova
- Deglycosylation, Enzyme immobilization, Magnetic microparticles, PNGase F,
- MeSH
- elektroforéza MeSH
- enzymy imobilizované metabolismus MeSH
- glykopeptidasa metabolismus MeSH
- glykoproteiny chemie metabolismus MeSH
- glykosylace MeSH
- imunoglobulin G chemie metabolismus MeSH
- mikrosféry * MeSH
- oligosacharidy metabolismus MeSH
- polysacharidy metabolismus MeSH
- ribonukleasy chemie metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- enzymy imobilizované MeSH
- glykopeptidasa MeSH
- glykoproteiny MeSH
- imunoglobulin G MeSH
- oligosacharidy MeSH
- polysacharidy MeSH
- ribonuclease B MeSH Prohlížeč
- ribonukleasy MeSH
BACKGROUND: Hemojuvelin (HJV) is one of essential components for expression of hepcidin, a hormone which regulates iron transport. HJV is mainly expressed in muscle and liver, and processing of HJV in both tissues is similar. However, hepcidin is expressed in liver but not in muscle and the role of the muscle HJV is yet to be established. Our preliminary analyses of mouse tissue HJV showed that the apparent molecular masses of HJV peptides are different in liver (50 kDa monomer and 35 and 20 kDa heterodimer fragments) and in muscle (55 kDa monomer and a 34 kDa possible large fragment of heterodimer). One possible explanation is glycosylation which could lead to difference in molecular mass. RESULTS: We investigated glycosylation of HJV in both liver and muscle tissue from mice. PNGase F treatment revealed that the HJV large fragments of liver and muscle were digested to peptides with similar masses, 30 and 31 kDa, respectively, and the liver 20 kDa small fragment of heterodimer was digested to 16 kDa, while the 50 kDa liver and 55 kDa muscle monomers were reduced to 42 and 48 kDa, respectively. Endo H treatment produced distinct digestion profiles of the large fragment: a small fraction of the 35 kDa peptide was reduced to 33 kDa in liver, while the majority of the 34 kDa peptide was digested to 33 kDa and a very small fraction to 31 kDa in muscle. In addition, liver HJV was found to be neuraminidase-sensitive but its muscle counterpart was neuraminidase-resistant. CONCLUSIONS: Our results indicate that different oligosaccharides are attached to liver and muscle HJV peptides, which may contribute to different functions of HJV in the two tissues.
- MeSH
- extracelulární prostor metabolismus MeSH
- genový knockout MeSH
- glykopeptidasa metabolismus MeSH
- glykosylace MeSH
- GPI-vázané proteiny MeSH
- játra cytologie metabolismus MeSH
- membránové proteiny nedostatek genetika izolace a purifikace metabolismus MeSH
- myši MeSH
- neuraminidasa metabolismus MeSH
- orgánová specificita MeSH
- protein hemochromatózy MeSH
- svaly cytologie metabolismus MeSH
- transport proteinů MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- glykopeptidasa MeSH
- GPI-vázané proteiny MeSH
- HJV protein, mouse MeSH Prohlížeč
- membránové proteiny MeSH
- neuraminidasa MeSH
- protein hemochromatózy MeSH
