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 α1-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.
CITATION STYLE
Dage, J. L., Ackermann, B. L., & Halsall, H. B. (1998). Site localization of sialyl Lewis(x) antigen on α1-acid glycoprotein by high performance liquid chromatography-electrospray mass spectrometry. Glycobiology, 8(8), 755–760. https://doi.org/10.1093/glycob/8.8.755
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