Disulfide assignment of the C-terminal cysteine knot of agouti-related protein (AGRP) by direct sequencing analysis

We have assigned the disulfide structure of Md-65 agouti-related protein (M d65-AGRP) using differential reduction and alkylation followed by direct se quencing analysis. The mature human AGRP is a single polypeptide chain of 1 12 amino acid residues, consisting of an M-Terminal acidic region and a uni que C-terminal cysteine-rich domain. The C-terminal domain, a 48 amino acid peptide named Md65-AGRP, was expressed in Escherichia coli cells and refol ded under different conditions from the mature recombinant protein. The dis ulfide bonds in the cystine knot structure of Md65-AGRP were partially redu ced using tris(2-carboxyethyl) phosphine (TCEP) under acidic conditions, fo llowed by alkylation with N-ethylmaleimide (NEM). The procedure generated s everal isoforms with varying degrees of NEM alkylation. The multiple forms of Md65-AGRP generated by partial reduction and NEM modification were then completely reduced and carboxymethylated to identify unreactive disulfide b onds. Differentially labeled Md65-AGRP were directly sequenced and analyzed by MALDI mass spectrometry. The results confirmed that Md65-AGRP contained the same disulfide structure as that of Md5-AGRP reported previously [Bure s, J., Hui, J.O., Young, Y. et al.(1988) Biochemistry 37, 12172-12177].