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Amino-terminal Sequences of the Major Tryptic Peptides Obtained from Carcinoembryonic Antigen by Digestion with Trypsin in the Presence of Triton X-100¹

Division of Immunology, City of Hope National Medical Center, Duarte, California 91010

Sequence studies of carcinoembryonic antigen (CEA) were initiated to obtain direct chemical evidence regarding the structure of CEA for comparison with that of CEA cross-reacting antigens. To obtain, purify, and perform amino acid sequencing on mg amounts of CEA (a glycoprotein with a molecular weight of 180,000, composed of 60% carbohydrate by weight), we needed to develop new approaches and refine existing techniques. This report describes the procedures developed during the course of this study and presents initial results. Trypsin digestion in the presence of 0.25% Triton X-100 produced seven major glycopeptide fragments that were separated and purified by high-pressure liquid chromatography on ion-exchange resins followed by Sephadex gel chromatography. NH₂-terminal sequences were determined on 1- to 2-mg amounts of the glycopeptides by high-pressure liquid chromatography for the analysis of phenylthiohydantoin derivatives of amino acids. Four peptides gave sequences through 20 cycles of Edman degradation, one gave sequences through 13 cycles, and two gave sequences through 10 cycles. Three of these peptides also gave sequences of up to 8 to 13 cycles for a minor component. The abrupt halt in Edman degradation for each peptide was interpreted as the failure to sequence beyond a carbohydrate substitution on the polypeptide chain. Since each sequence obtained was unique, the results substantiate the claim that the polypeptide chain of CEA is a definite chemical entity and that the microheterogeneity resides in the carbohydrate portion of the molecule.

¹ Supported in part by National Cancer Institute Grants CA16434 and CA19163 from the National Large Bowel Cancer Program.

² To whom requests for reprints should be addressed, at Division of Immunology, City of Hope National Medical Center, 1500 East Duarte Road, Duarte, Calif. 91010.

³ Present address: S. U. N. Y. at Buffalo, Department of Cell and Molecular Biology, Cary Hall, Buffalo, N. Y. 14214.

Received 1/23/78. Accepted 4/20/78.