Site localization of sialyl Lewis(x) antigen on alpha1-acid glycoprotein by high performance liquid chromatography-electrospray mass spectrometry.
A simple, fast and sensitive method was developed to verify the presence of the sialyl Lewis(x) antigen on an N-linked glycoprotein. High performance liquid chromatography-electrospray mass spectrometry (HPLC-ESI/MS) was used to identify which of the five N-linked glycosylation sites of human plasma alpha1-acid-glycoprotein (orosomucoid, OMD) contain the sialyl Lewis(x) antigen. OMD was digested with proteolytic enzymes and analyzed by reversed phase chromatography coupled with on-line ESI/MS. A tandem mass spectrometry experiment was designed to detect the presence of the sialyl Lewis(x) antigen based on the observation of an 803 mass to charge ratio ( m/z ) ion produced in the intermediate pressure region of the ESI interface. The ESI/MS signal at m/z 803 is consistent with an oxonium ion for a glycan structure containing NeuAc, Gal, GlcNAc, and Fuc. The identity of the m/z 803 ion was confirmed by ESI/MS/MS analysis of the m/z 803 fragment ion and comparison with a sialyl Lewis(x) standard. The stereochemistry and linkage positions were assigned using previous NMR analysis but could be determined with permethylation analysis if necessary. The analysis of OMD gave a pattern showing signal for the sialyl Lewis(x) antigen coeluting with each of the five N-linked glycopeptides. The ability to monitor sialyl Lewis(x) expression at each of the five sites is of interest in the study of OMD's role in inflammatory diseases.
Dage
J L
JL
Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221-0172, USA, Hoechst Marion Roussel Inc., Cincinnati, OH 45215, USA, and Eli Lilly and Company, Indianapolis, IN 46285, USA.
Ackermann
B L
BL
Halsall
H B
HB
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism*; D009961:Orosomucoid / Q000378:metabolism*; D000080506:Sialyl Lewis X Antigen
D009844:Oligosaccharides; D009961:Orosomucoid; D000080506:Sialyl Lewis X Antigen