MICROSEQUENCE AND MASS-SPECTRAL ANALYSIS OF NONSPECIFIC CROSS-REACTING ANTIGEN-160, A CD15-POSITIVE NEUTROPHIL MEMBRANE GLYCOPROTEIN - DEMONSTRATION OF IDENTITY WITH BILIARY GLYCOPROTEIN-I

Sequence information was obtained from low picomole amounts of nonspec ific cross-reacting antigen (NCA) 160 (M(r) 160,000), a granulocyte me mbrane glycoprotein. Following affinity purification and SDS-polyacryl amide gel electrophoresis, the protein was electrotransferred to nitro cellulose, digested with trypsin, and the peptides were isolated using capillary reversed-phase liquid chromatography. Analysis of these pep tides by Edman microsequencing and mass spectrometry established that NCA-160 was identical to biliary glycoprotein I, a protein that we pre viously cloned from a human colon library (1). NCA-160 from human gran ulocytes is a CD15-positive glycoprotein belonging to the carcinoembry onic antigen family and possesses putative transmembrane and cytoplasm ic domains. Previous efforts to characterize this antigen at the prote in level were hampered by a blocked NH2 terminus. In this study, we co nfirmed 20% of the deduced amino acid sequence starting with approxima tely 50 pmol of sample. Carbohydrate structural data is also presented on a single N-linked oligosaccharide moiety located in the A' domain. The capillary high performance liquid chromatography techniques used here, as well as mass spectrometry, were essential for high sensitivit y analysis of the blotted, digested glycoprotein.