Jeanette Adams

Structure determination of soybean and wheat glucosylceramides by tandem mass spectrometry.

Glucosylceramide (GluCer) is a major sphingolipid of plant tissue and, thus, abundant in nature and in dietary food sources. The lipid backbones of mammalian GluCer (sphingosine, d18:1(delta4), and ceramide) induce cell death (apoptosis) and inhibit colon carcinogenesis, it is critical to know the structures of GluCer present in plants as a first step toward understanding this potential link between diet and cancer. This study characterized the molecular species of GluCer from soybean and wheat by low-resolution, high-resolution and tandem mass spectrometry. Soybean GluCer was comprised primarily (>95%) of ceramide with 4,8-sphingadiene (d18:2(delta4,delta8)) and alpha-hydroxypalmitic acid (h16:0); the remainder had the same backbone with h18:0, h20:0, h22:0 and h24:0 fatty acids. Wheat GluCer had three major ceramide, d18:2(delta4,delta8) with h16:0, d18:1(delta8) with h16:0 and d18: 2(delta4,delta8) with h20:0, and smaller amounts of other homologs. These backbones differ from those of mammalian sphingolipids, which often have a delta4-double bond (but rarely a delta8-double bond), and have alpha-hydroxy fatty acids in only some cases. Previously unexplained fragmentations that were diagnostic for the type of sphingoid base backbone (i.e. by homolytic cleavage of the doubly allylic C-6-C-7 bond to yield a stable distonic allylic radical cation and an allylic radical neutral) were also identified. Hence this method should be useful in the identification of double bonds in sphingolipids, and structure-function relationships between sphingolipids and colon carcinogenesis.

Structure determination of ceramides and neutral glycosphingolipids by collisional activation of [M + Li]+ ions

N eutral glycosphingolipids are a general class of biological molecules that are important components of cell membranes. Their basic structure (1) comprises one or more neutral sugar residues attached to a ceramide residue. The ceramide group (R = H) is a long-chain sphingoid base, such as (4E)-sphingenine [R’ = CH=CH(CH,),CH,] or sphinganine [R’ = (CH,),CHJ, that is substituted at the amino group by a fatty acyl group (R” = alkyl chain). If the R group were a simple sugar, the glycosphingolipid would be called a cerebroside or a 1-@-glycosylceramide [ 11. The structure of the glycosyl group can vary, as can the structures of both the sphingoid base and N-a@ chain. Thus, easy but complete structural elucidation of neutral glycosphingolipids has been a challenge in biochemistry, and mass spectrometry has played an important role. Derivatization and gas chromatography/mass spectrometry have been used to separately characterize the sugar, ceramide, sphingoid base, and fatty acyl components [2, 31. Egge and Peter-KataliniC [4] reviewed the many uses of fast atom bombardment (FAB) of [M + HI+, [M + Na]+, and [M HIions for structure elucidation of different types of glycosphingolipids. Of more recent application is the combination of collision-induced dissociation (CID) of FAB-desorbed [M + HI+, [M HI-, and their fragment ions 15-71. Costello and Vath [6] have contributed significantly in this latter area and have recently reviewed their work. As an alternative to using CID of [M + H]+ and [M HIions, we have been exploring the use of alkali [&lo], and alkaline earth [ll], metal ions as adducts to enhance structural determination upon CID. Here, we report our initial results of applying CID of [M + Li]+ ions to the structural determination of six ceramides and 10 neutral glycosphingolipids. All spectra were acquired by using a VG Instruments (Manchester, UK) 70-S forward geometry mass spectrometer and B/E linked scans. Precursor ions were

A charge-remote allylic cleavage reaction: Mechanistic possibilities

The collision-induced allylic cleavage reactions of deuterium-labeled [M − H + 2Li)+ and [M − H]- ions of monounsaturated fatty acids were investigated. Three concerted mechanistic possibilities were considered for this process: a l,4-elimination of a vinylic H, a retro-ene reaction, and a l,4-conjugate elimination. A fourth mechanistic possibility, a two-step radical version of the retro-ene and l,4-conjugate elimination reactions, was also considered. The radical reactions are in accord with the isotopic labeling results and offer certain mechanistic consistencies for cleavage of both C-C allyl bonds; they are expected, however, to have large activation energies. The lower-energy concerted alternatives, the retro-ene reaction for cleavage of the proximal and the l,4-conjugate elimination for cleavage of the distal C-C allyl bond, are also consistent with experimental results. The alternative of two different concerted mechanisms for cleavage of the two allyl bonds, however, is at odds with the charge-remote concept.

Gas-phase fragmentations of anionic complexes of serine- and threonine-containing peptides

Abstract The (M + Ca − 3H) − and (M − H) − anions of 11 tetra- and larger peptides that contain either Ser or Thr are studied by using fast atom bombardment (FAB) and collision-induced decomposition (CID). The Ser- and Thr-containing negative ions decompose to lose either 30 u (Ser) or 44 u (Thr) in the ion source, metastably, and upon CID. The CID of the FAB-formed (M + Ca − 3H − X) − or (M − H − X) − fragment ions, in which X = 30 or 44 u, is the same as the CID of (M + Ca − 3H) − or (M − H) − anions of peptides in which the Ser and Thr residues are replaced with Gly. Two possible mechanisms for the decompositions are discussed, and theoretical molecular mechanical calculations provide further evidence regarding the mechanisms.

COLLISION-INDUCED DISSOCIATIONS AND B/E LINKED SCANS FOR STRUCTURAL DETERMINATION OF MODIFIED FATTY ACID ESTERS

Abstract Structures of modified fatty acid esters are determined by collisionally activating [M + Li]+ ions desorptively ionized by fast atom bombardment (FAB). Collision-induced dissociations (CIDs) that occur in the first field-free region of a normal geometry mass spectrometer are observed by using B/E linked scans. The CID spectra provide immediate information about chain length, location and type of substituent in saturated, unsaturated, methyl-branched and hydroxy esters. New spectra of more unusual ethyl esters that contain longer alkyl branches and keto substituents also indicate the location and type of substituent. Novel charge-remote reactions that are directly analogous to thermolytic hydro-acyloxy eliminations and heteroatom analogs of the retro-ene reaction are suggested to explain the formation of some fragment ions from the alkyl and keto esters. Deuterium labeling provides evidence for mechanisms for structurally informative charge-mediated fragmentations of the hydroxy esters. Individual components of complex mixtures can be determined, and the detection limit for the structural determination of methyl palmitate is 25 ng. Methods also are described for optimizing the practical use of FAB in conjunction with B/E linked scans, which can be hampered by poor precursor ion resolution.

The importance of charge-separation reactions in tandem mass spectrometry of doubly protonated angiotensin II formed by electrospray ionization: Experimental considerations and structural implications.

The occurrence of charge-separation reactions in tandem mass spectrometry of doubly protonated angiotensin II is demonstrated by the use of mass-analyzed ion kinetic energy spectrometry (MIKES) and kinetic energy release distributions (KERDs). Linked scans at a constant B/E severely discriminate against product ions formed by charge-separation reactions. Although the products are significantly more abundant in MIKES experiments, instrumental discrimination still makes quantitation of relative product ion abundances highly inaccurate. The most probable KERs (Tm. p.) and the average KERs (Tave.) of the reactions are determined from the KERDs, and these values are compared to the KERs determined from the peak widths at half-height (T0. 5). The measurement of T0. 5 is a poor approximation to Tm. p. and Tave.. The Tm. p. is used to calculate a most probable intercharge distance, which is compared to results from molecular dynamics calculations. The results provide evidence with regard to the mechanisms of fragmentation of multiply charged ions and the location of the charge site in relation to the decomposition reactions.

Mechanisms of fragmentation of cationic peptide ions

Abstract Fragmentation mechanisms for formation of several commonly occurring product ions in high-energy collision-induced induced decomposition spectra of either (M + Cat 2+ − H) + ions of peptides cationized with alkaline earth metal ions, (M + Ca + ) + ions cationized with alkali metal ions, or (M + H) + ions are evaluated by using deuterium-labelled peptides. The different sources of hydrogen transferred in the reactions are identified. Our study supports some previously proposed mechanisms but also provides evidence for others.

On the use of scans at a constant ratio of B/E for studying decompositions of peptide metal(II)-ion complexes formed by electrospray ionization.

The use of sector mass spectrometers to study metastable ion decompositions of peptide metal-ion complexes formed by electrospray ionization is discussed. Products that are formed by charge-separation reactions are characterized by large kinetic energy release distributions. This causes scans at a constant B/E to give incorrect product ion abundances and possibly incorrect mass assignments. Two instrumental methods exist that can be used either to detect the ions or to estimate relative ion abundances: a floated collision cell or mass-analyzed ion kinetic energy spectrometry (MIKES) scans. The floated collision cell, by virtue of an altered B/E scan law, however, discriminates against important metastable ion reactions that occur outside the cell. MIKES scans provide a clearer estimate of product ions that arise by metastable ion charge-separation reactions. Problems with pseudotandem (first field-free region) experiments are also discussed.

Gas-Phase Chemistry of Alkali Adducts of Simple and Complex Molecules

Gas-phase interactions between alkali metal ions and organic molecules have been of interest since the early 1970’s. Initial studies were conducted to determine metal ion affinities of simple monofunctional compounds.(1–6) Thermionic emitters were used as sources of metal ions, and Fourier-transform ion cyclotron resonance (FTICR) mass spectrometers were used for mass analysis. Consequently, alkali metal ions were exploited as chemical ionization reagents.(2d, 6, 7) The role of the metal ion as a reagent has changed as the methods for forming ions and types of mass spectrometers used for analysis have evolved.

How to measure the kinetic energy distribution of the precursor ion main beam in mass-analyzed ion kinetic energy spectrometry experiments

Scans of the electrostatic analyzer (ESA) across the precursor ion beam in reverse-geometry (BE) mass spectrometers that are operated under double-focusing conditions do not measure the “energy resolution of the main beam”: They only measure double-focusing resolution. The only way that ESA scans can measure the kinetic energy distribution of the main beam is to operate the instrument so that angular (directional) focusing is not achieved. Thus, the mass spectrometer is no longer double-focusing. Under double-focusing conditions, however, scans of the accelerating voltage while the magnetic field and ESA are held constant can be used to measure either the kinetic energy distribution of the main beam that enters the magnet or the energy-resolving power of the instrument. Scans at a constant ratio of B2/E can be used similarly. The energy-resolving power of any ESA is defined by its dispersion and the widths of the energy-resolving object and image slits that immediately precede and follow the ESA, respectively. The use of BE, EB, and triple-sector instruments to measure energy-resolving power and the kinetic energy distribution of the precursor ion main beam is compared and discussed.