Localization of Labile Posttranslational Modifications by Electron Capture Dissociation: The Case of γ-Carboxyglutamic Acid Neil L. Kelleher, § Roman A. Zubarev, ‡ Kristine Bush, † Bruce Furie, † Barbara C. Furie, † Fred W. McLafferty,* ,‡ and Christopher T. Walsh § Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, Research East, Beth Deaconess Israel Hospital, Boston, Massachusetts 02115, and Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14850-1301 Tandem mass spectrometry (MS/ MS) of 28 residue pep- tides harboring γ-carboxylated glutamic acid residues, a posttranslational modification of several proenzymes of the blood coagulation cascade, using either collisions or infrared photons results in complete ejection of the γ-CO 2 moieties (-44 Da) before cleavage of peptide-backbone bonds. However, MS/ MS using electron capture dissocia- tion (ECD) in a Fourier transform mass spectrometer cleaves backbone bonds without ejecting CO 2 , allowing direct localization of this labile modification. Sulfated side chains are also retained in ECD backbone fragmentations of a 21-mer peptide, although CAD causes extensive SO 3 loss. ECD thus is a unique complement to conventional methods for MS/ MS, causing less undesirable loss of side-chain functionalities as well as more desirable back- bone cleavages. INTRODUCTION Despite the development of several methods for tandem mass spectrometry (MS/ MS), 1 all methods generate similar types of fragment ions by adding energy to the precursor ions to induce threshold dissociation. Thus, while detection of ever more unstable modifications to biomolecules is possible using methods such as electrospray ionization (ESI), 2 further microcharacterization of these ionized species with such energetic methods can be difficult as a result of ejection of the modification before backbone bond cleavage. 3,4 An example of such labile species is generated by posttranslational modification of zymogens of serine proteases in the blood coagulation cascade 5,6 by the vitamin K-dependent γ-glutamyl carboxylase. The carboxylase uses reduced vitamin K, O 2 , and CO 2 to introduce a CO 2 moiety at the γ carbon of glutamic acid residues at the N termini of its protein substrates. These zymogens contain up to 12 γ-carboxyglutamic acid (Gla) moieties within 45 residues. 7 Dissection of enzymatic timing and regioselectivity of modification requires cleavage of bonds between these sites in partially carboxylated intermediates; problems with this include the high clustering of such sites and the tendency for decarboxylation in solution. This latter property of Gla residues was utilized in a method using DCl to introduce two γ-deuterium atoms for localization of Gla residues in peptides, 8,9 with more direct attempts for this using MS/ MS with low- or high-energy collisions resulting in complete decarboxylation before backbone fragmentation. 3 Recently, the new MS/ MS method of electron capture dis- sociation (ECD) 10,11 for electrosprayed ions cleaves peptide backbones primarily at the C R -N bond rather than at the amide linkage as with collisionally activated dissociation (CAD) or infrared photodissociation (Scheme 1). An early indication that ECD may bypass normal threshold dissociation channels (either by nonergodic fragmentation or a weakened backbone bond in the odd electron ion) came from fragmentation of D 2 O reacted cytochrome c ions; ECD apparently reduced deuterium atom scrambling, which was extensive with CAD. 12 Paralleling this * Corresponding author. Tel.: 607-255-4699. Fax: 607-255-7880. E-mail: fredwmcl@ aol.com. † Beth Deaconess Israel Hospital. ‡ Cornell University. § Harvard Medical School. (1) Threshold methods for MS/ MS: (a) Gauthier, J. W.; Trautman, T. R.; Jacobson, D. B. Sustained Off-Resonance Irradiation. Anal. Chim. Acta 1991 , 246, 211-225. (b) Lee, S. A.; Jiao, C. Q.; Huang, Y.; Freiser, B. S. Multiple Excitation Collisional Activation. Rapid Commun. Mass Spectrom. 1993 , 7, 819-821. (c) Chorush, R. A.; Little, D. P.; Beu, S. C.; Wood, T. D.; McLafferty, F. W. Surface-Induced Dissociation. Anal. Chem. 1995 , 67, 1042-1046. (d) Little, D. P.; Speir, J. P.; Senko, M. 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