JOURNAL OF MASS SPECTROMETRY J. Mass Spectrom. 2006; 41: 1073–1085 Published online 3 August 2006 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/jms.1066 Diastereomeric differentiation of peptides with CuII and FeII complexation in an ion trap mass spectrometer M´ elanie Lagarrigue, 1 Anne Boss ´ ee, 1 Carlos Afonso, 2 Fran ¸ coise Fournier, 2 Bruno Bellier 1 and Jean-Claude Tabet 2* 1 Centre d’Etudes du Bouchet, D ´ epartement Analyse Chimique, Vert-le-Petit, France 2 Laboratoire Synth ` ese, Structure et Fonction de Mol ´ ecules Bioactives (UMR 7613), Universit ´ e Pierre et Marie Curie, Paris Cedex05, France Received 14 February 2006; Accepted 22 May 2006 Complexation by transition metal ions (Cu II and Fe II ) was successfully used to differentiate the diastereomeric YAGFL, Y D AGFL and Y D AGF D L pentapeptides by electrospray ionization-ion trap mass spectrometry in the positive ion mode using low-energy collision conditions. This distinction was allowed by the stereochemical effects due to the .D/ Leu/ .L/ Leu and the .D/ Ala/ .L/ Ala residues yielding various steric interactions which direct relative dissociation rate constants of the binary [.M - H/ + Me II ] + complexes (Me = Cu or Fe) subjected to low-energy, collision-induced dissociation processes. The interpretation of the collision-induced dissociation spectra obtained from the diastereomeric cationized peptides allowed the location of the deprotonated site(s), leading to the postulation of ion structures and fragmentation pathways for both the [.M - H/ + Cu II ] + and [.M - H/ + Fe II ] + complexes, which differed significantly. With Cu II , consecutive fragmentations, initiated by the decarboxylation at C-terminus, were favored relative to sequence product ions. On the other hand, with Fe II , competitive fragmentations resulting in abundant sequence product ions and significant internal losses were preferred. This could be explained by different localizations of the negative charge, which directs the orientation of both the [.M - H/ + Cu II ] + and [.M - H/ + Fe II ] + binary complexes fragmentations. Indeed, the free negative charge of the [.M - H/ + Cu II ] + ions was mainly located at one oxygen atom: either at the C-terminal carboxylic group or, to a minor extent, at the Tyr phenol group (i.e. zwitterionic forms). On the other hand, the negative charge of the [.M - H/ + Fe II ] + ions was mainly located at one of the nitrogen atoms of the peptide backbone and coordinated to Fe II (i.e. salt non-zwitterionic form). Moreover, this study reveals the particular behavior of Cu II reduced to Cu I , which promotes radical losses not observed from the peptide – Fe II complexes. Finally, this study shows the analytical potentialities of the complexation of transition metal ions with peptides providing structural information complementary to that obtained from low-energy, collision-induced dissociation processes of protonated or deprotonated peptides. Copyright  2006 John Wiley & Sons, Ltd. KEYWORDS: diastereomeric differentiation; peptides; ion trap mass spectrometry; transition-metal ion complexation; conformation INTRODUCTION The development of infectious and bacterial resistance to conventional antibiotics, due to their extensive use, has resulted in an increased interest in antibacterial peptides as new targets for antibiotics. 1 Most of the native antibacterial peptides are composed of at least 60 L-amino acid residues. However, diastereomeric peptidic antibiotics (containing D- and L-amino acids) have great potential to be developed for therapeutic use, because they can present several advantages over all-L amino acid antibacterial peptides. 2 Indeed, D-amino acid incorporation into antibacterial peptides can strongly modify their activity (selectivity, antagonist properties, etc.). L Correspondence to: Jean-Claude Tabet, Centre d’Etudes du Bouchet, D´ epartement Analyse Chimique, Vert-le-Petit, France. E-mail: tabet@ccr.jussieu.fr Consequently, it is indispensable to develop analytical techniques that allow diastereomeric peptide differentiation. Mass spectrometry represents a method of choice by its ability to provide important structural information. 3,4 Therefore, mass spectrometry has been widely used for the differentiation of isomeric peptides or carbohydrates, 5–8 particularly using metal ion complexation, which appears to be very efficient for such purposes. Indeed, the par- ticular reactivity of the metal–peptide complexes can be very useful in obtaining precise structural information and has been the subject of numerous studies. First, interactions between peptides and alkali-metal cations, 9 alkaline-earth-metal cations 10 or transition-metal cations 11 – 13 were studied under high-energy collision conditions, using fast-atom-bombardment (FAB) ionization or sector mass spectrometers. 14 Nowadays, soft ionization methods such as Copyright  2006 John Wiley & Sons, Ltd.