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

Abstract

Abstract: We have assigned the disulfide structure of Md‐65 agouti‐related protein (Md65‐AGRP) using differential reduction and alkylation followed by direct sequencing analysis. The mature human AGRP is a single polypeptide chain of 112 amino acid residues, consisting of an N‐terminal acidic region and a unique C‐terminal cysteine‐rich domain. The C‐terminal domain, a 48 amino acid peptide named Md65‐AGRP, was expressed in Escherichia coli cells and refolded under different conditions from the mature recombinant protein. The disulfide bonds in the cystine knot structure of Md65‐AGRP were partially reduced using tris(2‐carboxyethyl) phosphine (TCEP) under acidic conditions, followed by alkylation with N ‐ethylmaleimide (NEM). The procedure generated several 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 bonds. 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 [Bures, E. J., Hui, J. O., Young, Y. et al. (1998) Biochemistry 37 , 12172–12177].