Resolvin D1, protectin D1, and related docosahexaenoic acid-derived products: Analysis via electrospray/low energy tandem mass spectrometry based on spectra and fragmentation mechanisms

Song Hong 0 Yan Lu 0 Rong Yang 0 Katherine H. Gotlinger 0 Nicos A. Petasis 0 Charles N. Serhan 0 0 Analytical Core, Center for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women's Hospital, Department of Oral Medicine, Infection and Immunity, Harvard School of Dental Medicine, Harvard Medical School , Boston, Massachusetts, USA Resolvin D1 (RvD1) and protectin D1 (Neuroprotectin D1, PD1/NPD1) are newly identified anti-inflammatory lipid mediators biosynthesized from docosahexaenoic acid (DHA). In this report, the spectra-structure correlations and fragmentation mechanisms were studied using electrospray low-energy collision-induced dissociation tandem mass spectrometry (MS/MS) for biogenic RvD1 and PD1, as well as mono-hydroxy-DHA and related hydroperoxy-DHA. The loss of H2O and CO2 in the spectra indicates the number of functional group(s). Chain-cut ions are the signature of the positions and numbers of functional groups and double bonds. The observed chain-cut ion is equivalent to a hypothetical homolytic-segment (cc, cm, mc, or mm) with addition or extraction of up to 2 protons (H). The -cleavage ions are equivalent to: [cc H], with H from the hydroxyl through a -ene or -ene rearrangement; [cm 2H], with 2H from hydroxyls of PD1 through a -ene rearrangement, or 1H from the hydroxyl and the other H from the -carbon of mono-HDHA through an -H- -ene rearrangement; [mc H], with H from hydroxyl through a -ene or -ene rearrangement, or from the -carbon through an -H- -ene rearrangement; or [mm] through charge-direct fragmentations. The -ene or -ene facilitates the H shift to position and -cleavage. Deuterium labeling confirmed the assignment of MS/MS ions and the fragmentation mechanisms. Based on the MS/MS spectra and fragmentation mechanisms, we identified RvD1, PD1, and mono-hydroxy-DHA products in human neutrophils and blood, trout head-kidney, and stroke-injury murine braintissue. (J Am Soc Mass Spectrom 2007, 18, 128 -144) 2007 American Society for Mass Spectrometry - Nfects of fish oil on human diseases such as arthriumerous reports demonstrate the beneficial eftis, Alzheimers disease, lung fibrosis, and inflammatory bowel diseases [13]. As an essential -3 polyunsaturated fatty acid, docosahexaenoic acid (DHA) is a major component of fish oil [4]. Novel bioactive oxygenated DHA products biosynthesized in resolving inflammatory exudates and tissues were recently identified, and their structures and bioactions were elucidated [5 8]. They were named as the D series of resolvins (resolution phase interaction products) and protectins (protecting brain and other organs against inflammatory diseases) because their biosynthetic pathways display potent anti-inflammatory and immunoregulatory properties (Scheme 1)[59]. As illustrated in Scheme 1, DHA is initially converted to 17S-hydroperoxy-DHA (HpDHA), then further enzymatically transferred to resolvin D1(RvD1) and protectin D1 (neuroprotectin D1, PD1/ NPD1) via epoxide intermediates. With aspirin treatment, the aspirin-acetylated cyclooxygenase type II (COX-2) converts DHA into 17R-HpDHA, which is further converted to 17R-RvD1 and 17R-PD1 [5]. To obtain the temporal and spatial profiles and regulation of biosynthesis of RvD1 and PD1 as well as other DHA products during physiopathological processes, their identification needs to be accurate, sensitive, and fast. One suitable approach is liquid chromatography coupled to an ultraviolet spectrometer and a Scheme 1. Biosynthetic pathways for Resolvin D1 (RvD1) and Protectin D1 (PD1). low collision-energy tandem mass spectrometer (LCUV-MS/MS) with atmospheric pressure ionization, specifically electrospray ionization (ESI) [5 8]; this is used on full product ion scan bases for structure elucidation and identification of resolvins and protectins at low nanogram levels [5 8]. Authentic standards of resolvins and protectin D1 prepared via total organic synthesis are used for confirmation during the identification. The low levels (under a few nanograms/sample) of these mediators present in vivo make other instrumentation, such as nuclear magnetic resonance or optical spectrometers, inappropriate for the analysis. In comparison, different types of mass spectrometry were used in the earlier studies of DHA-derived products. These included gas chromatography-MS with electronimpact ionization used by Van Rollins and Murphy and coworkers to analyze the structures of mono-hydroxyDHA products after silanization and methylation [9]. Kim and Salem conducted preparation and structure analysis of HDHAs and HpDHAs using LC-positive ion thermospray MS/MS [10]. They also developed an analytical method and conducted stereochemistry studies of all the HDHAs produced by human platelets and rat brain homogenates using chiral LC-thermospray-MS and GC/MS (electron-impact ionization) [11]. Low-energy ionization primarily generates molecular (or pseudo-molecular) ions for collision-induced dissociation (CID) MS/MS analysis, through which the MS/MS spectra obtained are used widely to identify and elucidate the structures of lipid mediators derived from polyunsaturated fatty acids [1220]. The lowenergy CID of eicosanoids includes charge-remote and charge-directed fragmentations [12, 21], many of which occur through -hydroxy- -ene like rearrangement as referred to by Murphy, i.e., -cleavage of the carbonOcarbon bond ( position to hydroxy group), facilitated by a double bond (ene) in the position [22]. For polyunsaturated fatty acids and their derivatives, ions formed in low-energy CID MS/MS via the loss of only H2O and CO2 are usually much more abundant than ions formed through cleavage of the carbon chain. In this report, the former are called peripheral-cut ions and the latter are called chain-cut ions. Peripheral-cut ions provide information about the number of hydroxys and carboxylates in a compound; chaincut ions are the signatures for the positions of hydroxys and double bonds. The ions formed via a combination of chain-cut and peripheral-cut processes are called chainplus-peripheral-cut ions [23]. The structure of a compound is deduced from the integration of the structures assigned to its MS/MS ions. Thus, the fragmentation mechanisms for the formation of MS/MS ions are very important for the identification using the MS/MS ions of a novel and/or known molecular structure. These mechanisms form the basis for lipidomic databases, which consist of lipid mass spectra and other analytical data, and search algorithms [23]. Here, we report the analysis of resolvin D1 and protectin D1, as well as other DHA-derived products without derivatization, using low-collision-energy tandem mass spectra acquired on anions generated from electrospray ionization of molecules eluted from LC. For structure elucidation and identification, the ion structures and MS/MS fragmentation mechanisms are proposed and confirmed via deuterium-label (...truncated)