THE ROLE OF ELECTRON CAPTURE DISSOCIATION IN BIOMOLECULAR ANALYSIS Helen J. Cooper, 1 KristinaHa˚kansson, 2 and Alan G. Marshall 3,4 * 1 School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom 2 Department of Chemistry, University of Michigan, 930 North University Avenue, Ann Arbor, Michigan 48109-1055 3 Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, 1800 East Paul Dirac Drive, Tallahassee, Florida 32310-4005 4 Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32310 Received 29 July 2003; received (revised) 8 December 2003; accepted 9 December 2003 Published online in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/mas.20014 The introduction of electron capture dissociation (ECD) to electrospray (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) constitutes a significant ad- vance in the structural analysis of biomolecules. The funda- mental features and benefits of ECD are discussed in this review. ECD is currently unique to FT-ICR MS and the funda- mentals of that technique are outlined. The advantages and complementarity of ECD in relation to other tandem mass spectrometry (MS/MS) techniques, such as infrared multiphoton dissociation (IRMPD) and sustained off-resonance collision- induced dissociation (SORI-CID), are discussed. The instru- mental considerations associated with implementation of ECD, including activated ion techniques and coupling to on-line separation techniques, are covered, as are the allied processes electronic excitation dissociation (EED), electron detachment dissociation (EDD), and hot electron capture (HECD). A major theme of this review is the role of ECD in proteomics, partic- ularly for characterization of post-translational modifications (phosphorylation, glycosylation, carboxyglutamic acid, sulfa- tion, acylation, and methionine oxidation) and the top-down approach to protein identification. The application of ECD to the analysis of polymers, peptide nucleic acids, and oligo- nucleotides is also discussed. # 2004 Wiley Periodicals, Inc., Mass Spec Rev 24:201–222, 2005 Keywords: electron capture dissociation; ECD; FTICR; FT- ICR; FT-MS; proteomics; post-translational modifications I. INTRODUCTION Determination of biomolecular structure is of paramount importance in understanding molecular function. In 2002, the Nobel Prize for Chemistry was awarded to John Fenn and Koichi Tanaka for ‘their development of soft desorption ionization methods for mass spectrometric analyses of biological macro- molecules.’ Fenn’s contribution was the development of electro- spray ionization (ESI) (Fenn et al., 1989). The combination of ESI with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) (Henry et al., 1989; Hendrickson & Emmett, 1999; Lorenz, Maziarz, & Wood, 1999) is a powerful tool for biomolecular analysis. ESI allows the analysis of ions of more than 100,000 Da in mass (Chen et al., 1995) while retaining optimum FT-ICR performance because of the typically observed low (below 4,000) mass-to-charge ratios. Tandem mass spectrometry (MS/MS) (McLafferty, 1983), in which a precursor ion is characterized according to its frag- ments, is a valuable technique for obtaining structural informa- tion. Fragmentation of ions in an ICR trapped-ion cell can be induced by any of several ‘‘slow-heating’’ methods (McLuckey & Goeringer, 1997): infrared multiphoton dissociation (IRMPD) (Woodlin, Bomse, & Beauchamp, 1978; Little et al., 1994), sustained off-resonance irradiation collision-induced dissocia- tion (SORI-CID) (Gauthier, Trautman, & Jacobson, 1991), blackbody infrared radiative dissociation (BIRD) (Price, Schnier, & Williams, 1996; Dunbar & McMahon, 1998), and surface induced dissociation (SID) (McCormack, Jones, & Wysocki, 1992; Chorush et al., 1995); or by ‘‘non-ergodic’’ electron capture dissociation (ECD) (Zubarev, Kelleher, & McLafferty, 1998; McLafferty et al., 2000; Zubarev et al., 2000), the subject of this review. ECD has so far been implemented only for FT-ICR mass analyzers. Since its introduction in 1998 (Zubarev, Kelleher, & McLafferty, 1998), ECD has been shown to display several unique features compared to other fragmentation tech- niques. In this review, we discuss those features and the many advantages and applications of ECD. We provide an overview of the fundamentals of ECD, including a discussion of the types of processes observed following electron capture, instrumental considerations, and allied processes. The mechanism of ECD is a matter of some debate (Zubarev et al., 2002). Although we outline the proposed mechanisms, a full treatment of that area is beyond the scope of this article. We discuss the role of ECD in proteomics, particularly for characterization of post-translational modifications and the top-down approach to protein identifica- tion, as well as ECD of other types of macromolecules. The Mass Spectrometry Reviews, 2005, 24, 201– 222 # 2004 by Wiley Periodicals, Inc. ———— Contract grant sponsor: National High Magnetic Field Laboratory, Tallahassee, FL; Contract grant sponsor: Florida State University; Contract grant sponsor: NSF National High Field FT-ICR Facility; Contract grant number: CHE-99-09502. *Correspondence to: Alan G. Marshall, Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, 1800 East Paul Dirac Drive, Tallahassee, FL 32310-4005. E-mail: marshall@magnet.fsu.edu