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

Lisanne J M Kempkes,Giel Berden,Jonathan Martens,Jos Oomens

doi:10.1002/jms.4298

Lisanne J M Kempkes, Giel Berden + Show 2 more

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https://doi.org/10.1002/jms.4298

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Abstract

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.

Highlights

We observe that neutral loss of a thiol radical (SH) occurs only for z‐ions that contain a cysteine residue at the cleavage site; these fragments are commonly referred to as w‐type ions
For the longer z‐ions, which do contain a Cys residue but not at the cleavage site; SH loss is not observed upon electron transfer dissociation (ETD)
Spectroscopic probing of the structure of these fragment ions is challenging because of the more involved ETD/CID MSn scheme required to produce them, and the inherent lower ion yields as compared with the w‐type ions; such experiments are reserved for future studies

Summary

Introduction

Cyclic u‐type fragments may form as a result of bond formation between the N‐terminal Cα atom and the Cβ atom of the adjacent residue.[16,18,21,26] A possible mechanism for the loss of the entire side chain involves H‐atom abstraction from the Cγ and subsequent α‐cleavage and expulsion of the side chain as an even‐electron neutral species, leaving the radical on the peptide backbone.[21] We use infrared multiple‐photon dissociation (IRMPD) spectroscopy[41,42,43,44,45,46,47,48] to experimentally confirm their structure for two z‐33 ETD fragments from Cys containing peptides.

Results

Conclusion