Abstract
AbstractA simple and rapid method for counting the number of internal disulfide bridges in a protein by incubation with 2‐mercaptoethanol and electrospray mass spectrometry analysis of the products was developed. 2‐Mercaptoethanol 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 electrospray mass spectrometry (ESMS). The number of mercaptoethanol adducts observed by ESMS reveals the number of disulfide bridges in the peptide or protein. Since the protein–mercaptoethanol–disulfide bonds are themselves further reduced by excess mercaptoethanol, the course of the reaction has to be followed in order to detect the maximum number of intermediates. Owing to the volatility of mercaptoethanol, samples can be taken out of the reaction solution for MS analysis without prior purification. Successful experiments were carried out using proteins with one, two, four or six SS‐bonds, covering a mass range from about 1 to over 23 kDa.
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