Electron detachment dissociation for top-down mass spectrometry of acidic proteins.

Barbara Ganisl,Markus Hartl,Klaus Bister,Monika Taucher,Taras Valovka,Kathrin Breuker

doi:10.1002/chem.201003709

Barbara Ganisl, Markus Hartl + Show 4 more

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https://doi.org/10.1002/chem.201003709

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Abstract

Electron detachment dissociation (EDD) is an emerging mass spectrometry (MS) technique for the primary structure analysis of peptides, carbohydrates, and oligonucleotides. Herein, we explore the potential of EDD for sequencing of proteins of up to 147 amino acid residues by using top-down MS. Sequence coverage ranged from 72 % for Melittin, which lacks carboxylic acid functionalities, to 19 % for an acidic 147-residue protein, to 12 % for Ferredoxin, which showed unusual backbone fragmentation next to cysteine residues. A limiting factor for protein sequencing by EDD is the facile loss of small molecules from amino acid side chains, in particular CO2. Based on the types of fragments observed and fragmentation patterns found, we propose detailed mechanisms for protein backbone cleavage and side chain dissociation in EDD. The insights from this study should further the development of EDD for top-down MS of acidic proteins.

Highlights

The unique radical-ion chemistry involved in electron capture dissociation (ECD)[1] produces fragment ions from protein backbone cleavage that provide detailed sequence information in top-down mass spectrometry (MS) experiments,[2] including the identification and localization of post-translational modifications (PTMs).[2b,3] ECD requires precursor ions carrying multiple positive charges, typically [M+n H]n+ ions formed by electrospray ionization (ESI).[4]
We have studied here the unimolecular dissociation by Electron detachment dissociation (EDD) of highly charged [MÀn H]nÀ ions of Melittin, Ubiquitin, Ferredoxin, and an acidic 147 amino acid residue construct (BASP1ACHTUNGRE(D121–216)) derived from BASP1, with calculated pI values[15] of 12.02, 6.56, 3.88, and 4.61, respectively
The radical [MÀn H](nÀm)ÀmC ions formed by detachment of up to three electrons from molecular ions constituted 34 % of all EDD products

Summary

Introduction

The unique radical-ion chemistry involved in electron capture dissociation (ECD)[1] produces fragment ions from protein backbone cleavage that provide detailed sequence information in top-down mass spectrometry (MS) experiments,[2] including the identification and localization of post-translational modifications (PTMs).[2b,3] ECD requires precursor ions carrying multiple positive charges, typically [M+n H]n+ ions formed by electrospray ionization (ESI).[4] Acidic proteins, are more readily ionized under ESI operated in negative ion mode, which generally produces [MÀn H]nÀ ions.[5] Electron detachment dissociation (EDD),[6] introduced by Zubarev and co-workers, involves radical ion chemistry and can be applied to [MÀn H]nÀ ions. It has been shown that EDD provides information on the primary struc-.

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