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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. [ABSTRACT FROM AUTHOR]