Tandem mass spectrometry (MS/MS) currently claims a key role in the identification and characterization of proteins [1–7]. Successful mass spectrometric…

Tandem mass spectrometry (MS/MS) currently claims a key role in the identification and characterization of proteins [1–7]. Successful mass spectrometric analysis of peptides and proteins relies on the ability to systematically sever peptide backbone bonds. In conventional collision-activated dissociation (CAD) MS/MS, ion fragmentation is activated by collisions with a buffer gas [8]. However, acquiring structural information through this method becomes significantly more challenging when the peptide is substantially long (more than approximately 20 residues [9]) or contains either labile post-translational modifications or multiple basic residues.

Alternative fragmentation methods, electron capture dissociation (ECD) [10] and electron transfer dissociation (ETD) [11–13], have been recently introduced into the field of proteomics. Unlike the collision-activated process, ECD and ETD do not cleave chemical modifications from the peptide, but rather induce random breakage of the peptide backbones. ECD and ETD can be used as orthogonal (to standard CAD) fragmentation methods to increase the informational content of tandem MS experiments. In spite of the remarkable prospects for proteomics, these techniques have been primarily implemented only in mass spectrometers that have ion traps such as Fourier Transform Ion Cyclotron Resonance (ECD case) and radiofrequency linear ion trap (ETD case) mass spectrometers.

Historically, experiments employing ECD/ETD-type fragmentation have been conducted inside a vacuum system with an operating pressure of no more than 1 mTorr. However, ion/ion and ion/molecule reactions can be very efficient even at atmospheric pressure. For instance, the use of proton-transfer reactions at the essentially atmospheric pressure is, nowadays, the most straightforward route to charge reduction of multiply charged peptides and proteins generated by electrospray. The effectiveness of this approach was first demonstrated by Orgozalek et al in 1992 by coupling the output of an electrospray with an externally housed corona discharge source [14]. Later, methods merging electrosprayed cations with corona discharged anions have been seriously modified and successfully demonstrated [15–18]. Recently, our group has reported the fragmentation of peptides at essentially atmospheric pressure conditions achieved by merging electrosprayed peptide ions with corona generated radicals [19].

In this paper, we further explore the developed ion dissociation technique which is capable of providing efficient fragmentation of peptides and proteins with typical patterns containing fragments specific to both CID and ECD/ETD. In the presented experimental setup, ion dissociation occurs within the flow reactor, located in the atmospheric pressure region between the ion source and the mass spectrometer. The suggested fragmentation mechanism involves the interaction of ESI-generated peptide ions with hydroxyl radicals originating from the corona discharge source.