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 ofthe sialyl Lewis(x) antigen on an N-linked glycoprotein. High performanceliquid chromatography-electrospray mass spectrometry (HPLC-ESI/MS) was usedto identify which of the five N-linked glycosylation sites of human plasmaalpha1-acid-glycoprotein (orosomucoid, OMD) contain the sialyl Lewis(x)antigen. OMD was digested with proteolytic enzymes and analyzed by reversedphase chromatography coupled with on-line ESI/MS. A tandem massspectrometry experiment was designed to detect the presence of the sialylLewis(x) antigen based on the observation of an 803 mass to charge ratio (m/z ) ion produced in the intermediate pressure region of the ESIinterface. The ESI/MS signal at m/z 803 is consistent with an oxonium ionfor a glycan structure containing NeuAc, Gal, GlcNAc, and Fuc. The identityof the m/z 803 ion was confirmed by ESI/MS/MS analysis of the m/z 803fragment ion and comparison with a sialyl Lewis(x) standard. Thestereochemistry and linkage positions were assigned using previous NMRanalysis 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 abilityto monitor sialyl Lewis(x) expression at each of the five sites is ofinterest in the study of OMD's role in inflammatory diseases.