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Receptor activity-modifying proteins (RAMPs) are cell surface-anchored coreceptors essential in determining the receptor specificity of a variety of peptide hormones, including calcitonin receptor-like receptor. Determination of the three-dimensional structure of RAMP proteins is critical to understanding their molecular interactions with, and modulation of receptor functioning. The RAMP1 molecule contains four Cysteines that participate in stabilizing the protein's tertiary structure. In this study, we analyzed and determined the disulfide bridge arrangement of the ectodomain of RAMP1. A recombinant RAMP1 construct was digested with trypsin and the resulting peptides were separated by HPLC. Fractions containing disulfide linkages were identified by mass shifts resulting from reductive alkylation. Aliquots of each of the disulfide-linked mixed peptides (prior to alkylation) were sequenced by Edman degradation. Of the four resulting peptide sequences, three were found to be tryptic and one was semi-tryptic, the latter peptide containing phenylalanine at its C-terminus. The co-migration in HPLC of the two disulfide-linked peptide pairs following trypsinization definitively establishes the Cysteine linkage map of the RAMP1 construct.