Selective cyanylation of cysteinyl residues as an approach for the mass spectrometric determination of protein structures

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Eur. J. Mass Spectrom. 5, 123 - 131 (1999)

Selective cyanylation of cysteinyl residues as an approach for the mass spectrometric determination of protein structures

Stefanie Barbirz, H. Peter Happersberger, Michael Przybylski and Michael O. Glocker^*
Faculty of Chemistry, University Konstanz, PO Box M732, D-78457 Konstanz, . E-mail:

ABSTRACT:

The cyanylation reaction of cysteinethiol groups in two model peptides Arg⁸-Vasopressin (AVP) and ATPase117 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.

Keywords: selective mono-thiol modification, cyanylation, 1-cyano-4-dimethylamino-pyridinium tetrafluoroborate, disulfide bonds, ESI-MS, MALDI-MS

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