Detection and location of disulfide bonds in proteins by fast atom bombardment mass spectrometry

Yiping Sun, Purdue University

Abstract

In this research, new approaches which combine performic acid oxidation, HPLC-electrochemical detection and FABMS analysis have been developed for rapidly and selectively identifying disulfide-containing peptides in proteins and other biological materials. The peptides were treated with performic acid to modify certain residues and thereby cause a characteristic change in the peptide molecular weight. This change in molecular weight, determined by FABMS, was used to identify the peptides. The molecular weight of a cysteinyl peptide increases by 48 mu for each cysteinyl residue present. This approach was used to identify cystinyl residues in a peptic digest of bovine insulin, and was extended to the analysis of a tryptic digest of CNBr-treated ribonuclease A. A new approach, which combines HPLC-EC with a dual electrode cell and FABMS, has been developed to selectively identify thiol- and disulfide-containing peptides in proteins and other biological materials. The utility of this method was illustrated with a tryptic digest of lysozyme and tissue extracts from bovine pituitary and catfish pancreas. A new EC detector cell that includes three Hg/Au electrodes has been developed and used to distinguish between thiol- and disulfide-containing peptides in a single chromatographic analysis. The performance of three-electrode detector was demonstrated by analyzing a chymotryptic digest of $\alpha$A-crystallin and a peptic digest of partially reduced insulin. The methods developed above were also used to investigate the structure of the sulfhydryl protease, papain. Electrospray ionization mass spectrometry was used to determine the molecular weight of papain. FABMS was used to map the sequence from proteolytic digests of the protein. Glutamic acid residues at positions 47, 118, and 135 were found instead of glutamine residues; an asparagine residue at position 169 was found instead of glycine; and the order of residues 86-87 was found as Tyr-Pro instead of Pro-Tyr. These data support the recently published cDNA sequence rather than the original protein sequence and x-ray structure. In addition, disulfide bonds in the unactivated papin were assigned as Cys 22/Cys 25, Cys 56/Cys 95, and Cys 153/Cys 200, with a free thiol group on the Cys 63.

Degree

Ph.D.

Advisors

Smith, Purdue University.

Subject Area

Analytical chemistry|Biochemistry

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