Selective Cyanylation of Cysteinyl Residues as an Approach for the Mass Spectrometric Determination of Protein Structures

The cyanylation reaction of cysteine–thiol groups in two model peptides Arg⁸-Vasopressin (AVP) and ATPase1–17 with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) was studied by UV spectroscopy, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and electrospray ionization mass spectrometry (ESI-MS). Cyanylation of the peptides in citrate buffer at pH 3 using a 5-fold molar excess of CDAP led to incompletely cyanylated products only. Monocyanylated AVP showed characteristic matrix adducts with the applied MALDI-MS sample preparation procedure. By contrast, the desired, fully cyanylated products were formed nearly quantitatively in phosphate buffer at pH 4 using a 10-fold molar excess of CDAP over free-thiol content. Surprisingly, we found that the cyano group was removed enzymatically during proteolytic digestion at pH 8. Cyanylation exclusively modified thiol groups in peptides as was shown by ESI tandem mass spectrometric sequencing of the mono- and biscyanylated AVP derivatives.