A METHOD FOR COUNTING DISULFIDE BRIDGES IN SMALL PROTEINS BY REDUCTION WITH MERCAPTOETHANOL AND ELECTROSPRAY MASS-SPECTROMETRY

A simple and rapid method for counting the number of internal disulfid e bridges in a protein by incubation with 2-mercaptoethanol and electr ospray mass spectrometry analysis of the products was developed. 2-Mer captoethanol yields intermediate mixed disulfides during reduction of a protein. This results in a molecular weight increase of the protein by 78 Da per disulfide bond, which can easily be determined by electro spray mass spectrometry (ESMS). The number of mercaptoethanol adducts observed by ESMS reveals the number of disulfide bridges in the peptid e or protein. Since the protein-mercaptoethanol-disulfide bonds are th emselves further reduced by excess mercaptoethanol, the course of the reaction has to be followed in order to detect the maximum number of i ntermediates. Owing to the volatility of mercaptoethanol, samples can be taken out of the reaction solution for MS analysis without prior pu rification. Successful experiments were carried out using proteins wit h one, two, four or six S-S-bonds, covering a mass range from about 1 to over 23 kDa.