w-Type ions formed by electron transfer dissociation of Cys-containing peptides investigated by infrared ion spectroscopy.

In mass spectrometry-based peptide sequencing, electron transfer dissociation (ETD) and electron capture dissociation (ECD) have become well-established fragmentation methods complementary to collision-induced dissociation. The dominant fragmentation pathways during ETD and ECD primarily involve the formation of c- and z• -type ions by cleavage of the peptide backbone at the N─Cα bond, although neutral losses from amino acid side chains have also been observed. Residue-specific neutral side chain losses provide useful information when conducting database searching and de novo sequencing. Here, we use a combination of infrared ion spectroscopy and quantum-chemical calculations to assign the structures of two ETD-generated w-type fragment ions. These ions are spontaneously formed from ETD-generated z• -type fragments by neutral loss of 33 Da in peptides containing a cysteine residue. Analysis of the infrared ion spectra confirms that these z• -ions expel a thiol radical (SH• ) and that a vinyl C═C group is formed at the cleavage site. z• -type fragments containing a Cys residue but not at the cleavage site do not spontaneously expel a thiol radical, but only upon additional collisional activation after ETD.