A method for detecting O-glycanase in biological samples using a combination of MALDI-TOF mass spectrometry and time-resolved fluorimetry.
O-Glycans (mucin type oligosaccharides) are ubiquitously found in various glycoproteins such as mucin-type glycoproteins on the surface of various digestive organs. In the present paper, we propose a method for detecting O-glycanase which catalyzes the hydrolysis of the O-glycan linkage between oligosaccharides and serine/threonine residues of mucin-type glycoprotein. As the substrate for O-glycanases, we chose glycopeptides containing O-glycans derived from bovine fetuin. The present method is divided into two parts. At the initial stage, the presence of O-glycanase was confirmed by observing characteristic ions due to O-glycans and peptides released from the glycopeptide by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. In the second step, europium labeled O-glycosylated peptide permits more detailed analysis such as enzyme kinetics. We demonstrated the usefulness of the present method using O-glycanase (Streptococcus pneumoniae) as model enzyme. The present approach can easily confirm the presence of O-glycanase by detecting both deglycosylated peptides and O-glycans, even if contaminating peptides or glycosidases are present in crude biological samples.
Akai
Ryoko
R
Faculty of Pharmaceutical Sciences, Kinki University, Kowakae 3-4-1, Higashi-osaka 577-8502, Japan.
Kinoshita
Mitsuhiro
M
Kakehi
Kazuaki
K
Lee
Yuan C
YC
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D005470:Fluorometry / Q000379:methods; D006596:Hexosaminidases / Q000032:analysis*; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000032:analysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D000509:alpha-Fetoproteins / Q000032:analysis / Q000235:genetics
D011134:Polysaccharides; D000509:alpha-Fetoproteins; D006596:Hexosaminidases; C060638:glycopeptide alpha-N-acetylgalactosaminidase