Structural identification of electron transfer dissociation products in mass spectrometry using infrared ion spectroscopy.

Jonathan Martens,Giel Berden,Jos Oomens,Josipa Grzetic

doi:10.1038/ncomms11754

Abstract

Tandem mass spectrometry occupies a principle place among modern analytical methods and drives many developments in the ‘omics' sciences. Electron attachment induced dissociation methods, as alternatives for collision-induced dissociation have profoundly influenced the field of proteomics, enabling among others the top-down sequencing of entire proteins and the analysis of post-translational modifications. The technique, however, produces more complex mass spectra and its radical-driven reaction mechanisms remain incompletely understood. Here we demonstrate the facile structural characterization of electron transfer dissociation generated peptide fragments by infrared ion spectroscopy using the tunable free-electron laser FELIX, aiding the elucidation of the underlying dissociation mechanisms. We apply this method to verify and revise previously proposed product ion structures for an often studied model tryptic peptide, [AlaAlaHisAlaArg+2H]2+. Comparing experiment with theory reveals that structures that would be assigned using only theoretical thermodynamic considerations often do not correspond to the experimentally sampled species.

Highlights

Tandem mass spectrometry occupies a principle place among modern analytical methods and drives many developments in the ‘omics’ sciences
We have characterized each of the z -type fragments from the ETD mass spectrometry (MS)/MS spectrum using infrared spectroscopy, providing a comprehensive identification of their structures and conformations
We identify the m/z 368 fragment ion as the z3þ species (z3_I) based on infrared spectral matching. This structure features an alternative hydrogen bonding arrangement in comparison with the open-shell z3 þ and z4 þ fragments described above, most significantly affecting the C terminus and red shifting the carboxyl C 1⁄4 O stretch atfoohnpredengiz-se3so_2hmI8e.le5latwkzr3yJamyoþfofaltrhÀnoedm[1] ozlop4twehþnee-rsfiprhnaoegelslmintzeieo3rnng_tysI.jtsuhAtsartunclcooztsu3e_r1dIe,.8-isC0sh0aedlclcelmufielnqaÀetue1didvaafsoslperzen3ctt_throIaeIf for structure z3_II and c4_I, a low-energy c4þ conformation, are presented in the bottom two panels of Fig. 7 and support the assignment of z3_I. These results demonstrate the first use of Infrared ion spectroscopy (IRIS) to characterize the structures of ETD-generated peptide fragments

Summary

Introduction

Tandem mass spectrometry occupies a principle place among modern analytical methods and drives many developments in the ‘omics’ sciences. ECD in Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS)[1,2,3] and more recently ETD in a much broader range of mass spectrometers[4,5,6,7] constitute the two primary variations of this method These methods have shown impressive improvements over collision-induced dissociation tandem MS, primarily in the sense that labile post-translational modifications are not detached during activation revealing their position along the backbone, and that sequence coverage is increased, making the sequencing of intact proteins in top-down strategies possible[8,9,10,11,12]. ExD product ions are known to undergo hydrogen atom migration reactions that increase/decrease the expected masses of the fragment ions, having direct practical implications for ExD-based sequencing applications[21,22]

Methods

Results

Conclusion