Structural analysis of leukotriene C4 isomers using collisional activation and 157 nm photodissociation

Arugadoss Devakumar 0 1 3 David K. O'Dell 0 1 2 J. Michael Walker 0 1 2 James P. Reilly 0 1 3 0 Address reprint requests to Dr. J. P. Reilly, Department of Chemistry, Indiana University , Bloomington, IN 47405, USA 1 Published online October 9, 2007 Received July 17, 2007 Revised October 1, 2007 Accepted October 2, 2007 2 Gill Center for Biomolecular Science, Indiana University , Bloomington, Indiana, USA 3 Department of Chemistry, Indiana University , Bloomington, Indiana, USA The fragmentation of 5-hydroxy-6-glutathionyl-7,9,11,14-eicosatetraenoic acid [leukotriene C4 or LTC4 (5, 6)] and its isomeric counterpart LTC4 (14, 15) were studied by low and high-energy collisional induced dissociation (CID) and 157 nm photofragmentation. For singly charged protonated LTC4 precursors, photodissociation significantly enhances the signal intensities of informative fragment ions that are very important to distinguish the two LTC4 isomers and generates a few additional fragment ions that are not usually observed in CID experiments. The ion trap enables MSn experiments on the fragment ions generated by photodissociation. Photofragmentation is found to be suitable for the structural identification and isomeric differentiation of cysteinyl leukotrienes and is more informative than low or high-energy CID. We describe for the first time the structural characterization of the LTC4 (14, 15) isomer by mass spectrometry using CID and 157 nm light activation methods. (J Am Soc Mass Spectrom 2008, 19, 14 -26) 2008 American Society for Mass Spectrometry - then 5-HPETE into the reactive allylic epoxide leukotriene called A4 (LTA4). The 5-hydroxy-6-glutathionyl7,9,11,14-eicosatetraenoic acid named LTC4 can be produced by the conjugation of LTA4 with tripeptide glutathione in the presence of leukotriene C4 synthases [6]. Other members of the SRS-A family include LTD4 and LTE4 corresponding to the cysteinyl glycine and cysteine adducts of LTA4. LTC4 catabolism involves peptide cleavage reactions involving glutamyl transpeptidase and various dipeptidases to yield LTD4 and LTE4, both of which have biological activity. The cell can chemically transform LTA4 into LTB4 depending on the presence of A4 hydrolases. LTC4, LTD4, and LTE4 are collectively referred to as cysteinyl leukotrienes. Cysteinyl leukotrienes provoke contractions of various smooth muscles and have been implicated as mediators of acute hypersensitivity reactions including asthma [2]. The involvement of cysteinyl leukotrienes in bronchial asthma has been clinically demonstrated [10]. There is great interest currently in drugs that inhibit the leukotriene-dependent bronchial contraction and reduce the intensity of bronchial spasms [1]. Mass spectrometry has been widely used to elucidate the role and structure of cysteinyl-leukotrienes for almost two decades. The first direct analysis of intact native cysteinyl leukotrienes was accomplished using fast atom bombardment ionization (FAB) mass spectrometry by Murphy and coworkers [11]. Both positive and negative ion FAB spectra of cysteinyl leukotrienes show intense molecular ions and weak fragment ions due to the loss of the cysteine-containing moiety. Although FAB proEsaturated fatty acids, mainly arachidonic acid icosanoids are oxygenated metabolites of polyunwith 20 carbon atoms. Prostaglandins, thromboxanes, leukotrienes, and other hydroxy and epoxy fatty acids belong to this diverse family of biologically active metabolites. They are enzymatically formed by cyclooxygenase or by specific lipoxygenases and epoxygenases [1 4]. Their role in a broad range of diseases, such as atherosclerosis, immunologic-allergic reactions, and inflammatory disorders, is widely acknowledged [2, 57]. These molecules are synthesized within cells in response to various stimuli. Following their release, they interact with specific G-protein coupled receptors [4, 5]. Thus, these molecules serve an important role as chemical communicators of cellular activation. Among the various products of the lipoxygenase (LOX) pathway of arachidonic acid metabolism, the class of compounds called slow reacting substance of anaphylaxis (SRS-A) has received the most attention because of their potent biological activities [5]. SRS-A consists of varying amounts of the cysteinyl leukotrienes LTC4, LTD4, and LTE4, all derived from a common biological precursor LTA4. The leukotrienes are derived from the 5-lipoxygenase pathway of metabolism typically by inflammatory cells such as eosinophils, monocytes, macrophages, and mast cells [5, 8, 9]. 5-Lipoxygenase activating protein sequentially converts arachidonic acid into 5-hydroperoxyeicosatetra-enoic acid (5-HPETE), and vides information on the molecular ion, the mass spectra contain little structural information. The introduction of matrix-assisted laser desorption-ionization (MALDI) mass spectrometry dramatically changed the status of biomolecular analysis. The simple addition of matrix to the sample preparation provides soft and efficient ionization of various fragile, nonvolatile samples. Despite its advantages, MALDI has not been extensively used for the characterization of low molecular weight compounds. In MALDI mass spectrometric analysis, the alkali metal ions in the sample promote the formation of matrix clusters and their alkali ion adducts, suppressing the analyte signals, particularly in the low mass region [12, 13]. As an alternative, electrospray ionization (ESI) has become the ionization method of choice to eliminate matrix chemical noise [14]. More recently, ESI tandem mass spectrometry of eicosanoids has been reviewed by Murphy et al. [15]. Collisions of both positive and negative molecular ion species ([M H] and [M H] ) with neutral atoms at low-pressure can drive unique rearrangement reactions and break carboncarbon bonds to generate abundant ion products. While mass spectrometric studies to define the chemical structure of leukotrienes have been performed, identifying and distinguishing structurally related isomers and isobaric eicosanoids remains challenging. Deuterated derivatives of LTC4 and the structurally related 5-oxo-7glutathionyl-8,11,14-eicosatrienoic acid (FOG7) have been distinguished by MS3 tandem mass spectrometry [16]. The mammalian lipoxygenase enzymes produce 5-, 12-, and 15-hydroperoxides that can in turn give rise to numerous structurally related compounds. Distinguishing these similar molecules is a challenging task and new methods to accomplish this would be very helpful. Little is known about the existence of other cysteinyl leukotrienes arising from either alternative LOX metabolism (12 or 15) or similar compounds arising from glutathione ring opening of epoxides produced by epoxygenase metabolism [1719]. The biological significance of leukotriene14, 15 analogs presented by Reddanna et al. and Sala et al. leads to an increasing need to characterize the structures of these compounds [17, 19]. We recently reported that 157 nm laser ph (...truncated)