Identification of abundant alkyl ether glycerophospholipids in the human lens by tandem mass spectrometry techniques

Previous studies have shown that the human lens contains glycerophospholipids with ether linkages. These lipids differ from conventional glycerophospholipids in that the sn-1 substituent is attached to the glycerol backbone via an 1-O-alkyl or an 1-O-alk-1'-enyl ether rather than an ester bond. The present investigation employed a combination of collision-induced dissociation (CID) and ozone-induced dissociation (OzID) to unambiguously distinguish such 1-O-alkyl and 1-O-alk-1'-enyl ethers. Using these methodologies the human lens was found to contain several abundant 1-O-alkyl glycerophos-phoethanolamines, including GPEtn(16:0e/9Z-18:1), GPEtn(11Z-18:1e/9Z-18:1), and GPEtn(18:0e/9Z-18:1), as well as a related series of unusual 1-O-alkyl glycerophosphoserines, including GPSer(16:0e/9Z-18:1), GPSer(11Z-18:1e/9Z-18:1), GPSer(18:0e/9Z-18:1) that to our knowledge have not previously been observed in human tissue. Isomeric 1-O-alk-1'-enyl ethers were absent or in low abundance. Examination of the double bond position within the phospholipids using OzID revealed that several positional isomers were present, including sites of unsaturation at the n-9, n-7, and even n-5 positions. Tandem CID/OzID experiments revealed a preference for double bonds in the n-7 position of 1-O-ether linked chains, while n-9 double bonds predominated in the ester-linked fatty acids [e.g., GPEtn(11Z-18:1e/9Z-18:1) and GPSer(11Z-18:1e/9Z-18:1)]. Different combinations of these double bond positional isomers within chains at the sn-1 and sn-2 positions point to a remarkable molecular diversity of ether-lipids within the human lens.

Differentiation of complex lipid isomers by radical-directed dissociation mass spectrometry

Contemporary lipidomics protocols are dependent on conventional tandem mass spectrometry for lipid identification. This approach is extremely powerful for determining lipid class and identifying the number of carbons and the degree of unsaturation of any acyl-chain substituents. Such analyses are however, blind to isomeric variants arising from different carbon carbon bonding motifs within these chains including double bond position, chain branching, and cyclic structures. This limitation arises from the fact that conventional, low energy collision-induced dissociation of even-electron lipid ions does not give rise to product ions from intrachain fragmentation of the fatty acyl moieties. To overcome this limitation, we have applied radical-directed dissociation (RDD) to the study of lipids for the first time. In this approach, bifunctional molecules that contain a photocaged radical initiator and a lipid-adducting group, such as 4-iodoaniline and 4-iodobenzoic acid, are used to form noncovalent complexes (i.e., adduct ions) with a lipid during electrospray ionization. Laser irradiation of these complexes at UV wavelengths (266 nm) cleaves the carbon iodine bond to liberate a highly reactive phenyl radical. Subsequent activation of the nascent radical ions results in RDD with significant intrachain fragmentation of acyl moieties. This approach provides diagnostic fragments that are associated with the double bond position and the positions of chain branching in glycerophospholipids, sphingomyelins and triacylglycerols and thus can be used to differentiate isomeric lipids differing only in such motifs. RDD is demonstrated for well-defined lipid standards and also reveals lipid structural diversity in olive oil and human very-low density lipoprotein.

The fragmentation pathways of protonated Amiton in the gas phase : towards the structural characterisation of organophosphorus chemical warfare agents by electrospray ionisation tandem mass spectrometry

Amiton (O,O-diethyl-S-[2-(diethylamino)ethyl]phosphorothiolate), otherwise known as VG, is listed in schedule 2 of the Chemical Weapons Convention (CWC) and has a structure closely related to VX (O-ethyl-S-(2-diisopropylamino)ethylmethylphosphonothiolate). Fragmentation of protonated VG in the gas phase was performed using electrospray ionisation ion trap mass spectrometry (ESI-ITMS) and revealed several characteristic product ions. Quantum chemical calculations provide the most probable structures for these ions as well as the likely unimolecular mechanisms by which they are formed. The decomposition pathways predicted by computation are consistent with deuterium-labeling studies. The combination of experimental and theoretical data suggests that the fragmentation pathways of VG and analogous organophosphorus nerve agents, such as VX and Russian VX, are predictable and thus ESI tandem mass spectrometry is a powerful tool for the verification of unknown compounds listed in the CWC. Copyright (c) 2006 Commonwealth of Australia. Published by John Wiley & Sons, Ltd.

Production and isolation of ligated metal(IV)-oxo ions by tandem mass spectrometry

High valent metal(IV)-oxo species, \[M(=O)(Melm)(n)(OAc)](+) (M = Mn-Ni, MeIm = 1-methylimidazole, n = 1-2), which are relevant to biology and oxidative catalysis, were produced and isolated in gas-phase reactions of the metal(II) precursor ions \[M(MeIm)(n)(OAc)](+) (M = Mn-Zn, n = 1-3) with ozone. The precursor ions \[M(MeIm)(OAc)](+) and \[M(MeIm)(2)(OAc)](+) were generated via collision-induced dissociation of the corresponding \[M(MeIm)(3)(OAc)](+) ion. The dependence of ozone reactivity on metal and coordination number is discussed. Copyright (C) 2010 John Wiley & Sons, Ltd.

Desorption electrospray ionisation mass spectrometry reveals in situ modification of a hindered amine light stabiliser resulting from direct N–OR bond cleavage

Detection and characterisation of structural modifications of a hindered amine light stabiliser (HALS) directly from a polyester-based coil coating have been achieved by desorption electrospray ionisation mass spectrometry (DESI-MS) for the first time. In situ detection is made possible by exposing the coating to an acetone vapour atmosphere prior to analysis. This is a gentle and non-destructive treatment that allows diffusion of analyte to the surface without promoting lateral migration. Using this approach a major structural modification of the HALS TINUVIN®123 (bis(1-octyloxy-2,2,6,6-tetramethyl-4-piperidyl) sebacate) was discovered where one N-ether piperidine moiety (N-OC8H17) is converted to a secondary piperidine (N–H). With the use of 2-dimensional DESI-MS imaging the modification was observed to arise during high curing temperatures (ca. 260 °C) and under simulated physiological conditions (80 °C, full solar spectrum). It is proposed that the secondary piperidine derivative is a result of a highly reactive aminyl radical intermediate produced by N–O homolytic bond cleavage. The nature of the bond cleavage is also suggested by ESR spin-trapping experiments employing α-phenyl-N-tert-butyl nitrone (PBN) in toluene at 80 °C. The presence of a secondary piperidine derivative in situ and the implication of N–OR competing with NO–R bond cleavage suggest an alternative pathway for generation of the nitroxyl radical—an essential requirement in anti-oxidant activity that has not previously been described for the N-ether sub-class of HALS.

Identification of phospholipids in human meibum by nano-electrospray ionisation tandem mass spectrometry

Meibum is believed to be the major source of tear film lipids, which are vital in the prevention of excess evaporation of the aqueous phase. The complete lipid composition of meibum has yet to be established. While earlier studies reported the presence of phospholipids in human meibum, recent mass spectrometric studies have not detected them. In this study we use electrospray ionisation tandem mass spectrometry to investigate the presence of phospholipids in meibum and provide comparison to the phospholipid profile of tears.Lipids were extracted from human meibum and tear samples using standard biphasic methods and analysed by nano-electrospray ionisation tandem mass spectrometry using targeted ion scans. A total of 35 choline-containing phospholipids were identified in meibum and the profile of these was similar to that observed in tears, suggesting tear lipids are derived from meibum. The results shown here highlight the need for a combination of optimised techniques to enable the identification of the large range of lipid classes in meibum. © 2011 Elsevier Ltd.

Time to face the fats : What can mass spectrometry reveal about the structure of lipids and their interactions with proteins?

Since the 1950s, X-ray crystallography has been the mainstay of structural biology, providing detailed atomic-level structures that continue to revolutionize our understanding of protein function. From recent advances in this discipline, a picture has emerged of intimate and specific interactions between lipids and proteins that has driven renewed interest in the structure of lipids themselves and raised intriguing questions as to the specificity and stoichiometry in lipid-protein complexes. Herein we demonstrate some of the limitations of crystallography in resolving critical structural features of ligated lipids and thus determining how these motifs impact protein binding. As a consequence, mass spectrometry must play an important and complementary role in unraveling the complexities of lipid-protein interactions. We evaluate recent advances and highlight ongoing challenges towards the twin goals of (1) complete structure elucidation of low, abundant, and structurally diverse lipids by mass spectrometry alone, and (2) assignment of stoichiometry and specificity of lipid interactions within protein complexes.

Imaging of human lens lipids by desorption electrospray ionization mass spectrometry

The lipid composition of the human lens is distinct from most other tissues in that it is high in dihydrosphingomyelin and the most abundant glycerophospholipids in the lens are unusual 1-O-alkyl-ether linked phosphatidylethanolamines and phosphatidylserines. In this study, desorption electrospray ionization (DESI) mass spectrometry-imaging was used to determine the distribution of these lipids in the human lens along with other lipids including, ceramides, ceramide-1-phosphates, and lyso 1-O-alkyl ethers. To achieve this, 25 μm lens slices were mounted onto glass slides and analyzed using a linear ion-trap mass spectrometer equipped with a custom-built, 2-D automated DESI source. In contrast to other tissues that have been previously analyzed by DESI, the presence of a strong acid in the spray solvent was required to desorb lipids directly from lens tissue. Distinctive distributions were observed for [M + H]+ ions arising from each lipid class. Of particular interest were ionized 1-O-alkyl phosphatidylethanolamines and phosphatidylserines, PE (18:1e/18:1), and PS (18:1e/18:1), which were found in a thin ring in the outermost region of the lens. This distribution was confirmed by quantitative analysis of lenses that were sectioned into four distinct regions (outer, barrier, inner, and core), extracted and analyzed by electrospray ionization tandem mass spectrometry. DESI-imaging also revealed a complementary distribution for the structurally-related lyso 1-O-alkyl phosphatidylethanolamine, LPE (18:1e), which was localized closer to the centre of the lens. The data obtained in this study indicate that DESI-imaging is a powerful tool for determining the spatial distribution of human lens lipids. © 2010 American Society for Mass Spectrometry.

A mass spectrometry study of XCO+, X = Si, Ge : Is SiCO+ a main group carbonyl? Comments on the bonding in ground state SiCO and the Si,C,O (+) potential energy surface

The cation\[Si,C,O](+) has been generated by 1) the electron ionisation (EI) of tetramethoxysilane and 2) chemical ionisation (CI) of a mixture of silane and carbon monoxide. Collisional activation (CA) experiments performed for mass-selected \[Si,C,O](+), generated by using both methods, indicate that the structure is not inserted OSiC+; however, a definitive structural assignment as Si+-CO, Si+-OC or some cyclic variant is impossible based on these results alone. Neutralisation-reionisation (+NR+) experiments for EI-generated \[Si,C,O](+) reveal a small peak corresponding to SiC+, but no detectable SiO+ signal, and thus establishes the existence of the Si+-CO isomer. CCSD(T)//B3LYP calculations employing a triple-zeta basis set have been used to explore the doublet and quartet potential-energy surfaces of the cation, as well as some important neutral states The results suggest that both Si+-CO and Si+ - OC isomers are feasible; however, the global minimum is (2)Pi SiCO+. Isomeric (2)Pi SiOC+ is 12.1 kcal mol(-1) less stable than (2)Pi SiCO+, and all quartet isomers are much higher in energy. The corresponding neutrals Si-CO and Si-OC are also feasible, but the lowest energy Si - OC isomer ((3)A") is bound by only 1.5 kcal mol(-1). We attribute most, if nor all, of the recovery signal in the +NR' experiment to SiCO+ survivor ions. The nature of the bonding in the lowest energy isomers of Si+ -(CO,OC) is interpreted with the aid of natural bond order analyses, and the ground stale bonding of SiCO+ is discussed in relation to classical analogues such as metal carbonyls and ketenes.

Advanced mass spectrometry-based multi-omics technologies for exploring the pathogenesis of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the primary hepatic malignancies and is the third most common cause of cancer related death worldwide. Although a wealth of knowledge has been gained concerning the initiation and progression of HCC over the last half century, efforts to improve our understanding of its pathogenesis at a molecular level are still greatly needed, to enable clinicians to enhance the standards of the current diagnosis and treatment of HCC. In the post-genome era, advanced mass spectrometry driven multi-omics technologies (e.g., profiling of DNA damage adducts, RNA modification profiling, proteomics, and metabolomics) stand at the interface between chemistry and biology, and have yielded valuable outcomes from the study of a diversity of complicated diseases. Particularly, these technologies are being broadly used to dissect various biological aspects of HCC with the purpose of biomarker discovery, interrogating pathogenesis as well as for therapeutic discovery. This proof of knowledge-based critical review aims at exploring the selected applications of those defined omics technologies in the HCC niche with an emphasis on translational applications driven by advanced mass spectrometry, toward the specific clinical use for HCC patients. This approach will enable the biomedical community, through both basic research and the clinical sciences, to enhance the applicability of mass spectrometry-based omics technologies in dissecting the pathogenesis of HCC and could lead to novel therapeutic discoveries for HCC.

Analysis of the extreme diversity of salivary alpha-amylase isoforms generated by physiological proteolysis using liquid chromatography-tandem mass spectrometry

Saliva is a crucial biofluid for oral health and is also of increasing importance as a non-invasive source of disease biomarkers. Salivary alpha-amylase is an abundant protein in saliva, and changes in amylase expression have been previously associated with a variety of diseases and conditions. Salivary alpha-amylase is subject to a high diversity of post-translational modifications, including physiological proteolysis in the oral cavity. Here we developed methodology for rapid sample preparation and non-targeted LC-ESI-MS/MS analysis of saliva from healthy subjects and observed an extreme diversity of alpha-amylase proteolytic isoforms. Our results emphasize the importance of consideration of post-translational events such as proteolysis in proteomic studies, biomarker discovery and validation, particularly in saliva. (C) 2012 Elsevier B.V. All rights reserved.

Collection and determination of nucleotide metabolites in neonatal and adult saliva by high performance liquid chromatography with tandem mass spectrometry

Saliva contains a number of biochemical components which may be useful for diagnosis/monitoring of metabolic disorders, and as markers of cancer or heart disease. Saliva collection is attractive as a non-invasive sampling method for infants and elderly patients. We present a method suitable for saliva collection from neonates. We have applied this technique for the determination of salivary nucleotide metabolites. Saliva was collected from 10 healthy neonates using washed cotton swabs, and directly from 10 adults. Two methods for saliva extraction from oral swabs were evaluated. The analytes were then separated using high performance liquid chromatography (HPLC) with tandem mass spectrometry (MS/MS). The limits of detection for 14 purine/pyrimidine metabolites were variable, ranging from 0.01 to 1.0 mu M. Recovery of hydrophobic purine/pyrimidine metabolites from cotton tips was consistently high using water/acetonitrile extraction (92.7-111%) compared with water extraction alone. The concentrations of these metabolites were significantly higher in neonatal saliva than in adults. Preliminary ranges for nucleotide metabolites in neonatal and adult saliva are reported. Hypoxanthine and xanthine were grossly raised in neonates (49.3 +/- 25.4; 30.9 +/- 19.5 mu M respectively) compared to adults (4.3 +/- 3.3; 4.6 +/- 4.5 mu M); nucleosides were also markedly raised in neonates. This study focuses on three essential details: contamination of oral swabs during manufacturing and how to overcome this; weighing swabs to accurately measure small saliva volumes; and methods for extracting saliva metabolites of interest from cotton swabs. A method is described for determining nucleotide metabolites using HPLC with photo-diode array or MS/MS. The advantages of utilising saliva are highlighted. Nucleotide metabolites were not simply in equilibrium with plasma, but may be actively secreted into saliva, and this process is more active in neonates than adults. (C) 2013 Elsevier B.V. All rights reserved.

Characterization of acyl chain position in unsaturated phosphatidylcholines using differential mobility-mass spectrometry

Glycerophospholipids (GPs) that differ in the relative position of the two fatty acyl chains on the glycerol backbone (i.e., sn-positional isomers) can have distinct physicochemical properties. The unambiguous assignment of acyl chain position to an individual GP represents a significant analytical challenge. Here we describe a workflow where phosphatidylcholines (PCs) are subjected to ESI for characterization by a combination of differential mobility spectrometry and MS (DMS-MS). When infused as a mixture, ions formed from silver adduction of each phospholipid isomer {e.g., [PC (16:0/18:1) + Ag]+ and [PC (18:1/16:0) + Ag]+} are transmitted through the DMS device at discrete compensation voltages. Varying their relative amounts allows facile and unambiguous assignment of the sn-positions of the fatty acyl chains for each isomer. Integration of the well-resolved ion populations provides a rapid method (< 3 min) for relative quantification of these lipid isomers. The DMS-MS results show excellent agreement with established, but time-consuming, enzymatic approaches and also provide superior accuracy to methods that rely on MS alone. The advantages of this DMS-MS method in identification and quantification of GP isomer populations is demonstrated by direct analysis of complex biological extracts without any prior fractionation.

Studies of Brisbane municipal water quality using Inductively Coupled Plasma - Mass Spectrometry and Chemometrics

This research established innovative methods and a predictive model to evaluate water quality using the trace element and heavy metal concentrations of drinking water from the greater Brisbane area. Significantly, the combined use of Inductively Coupled Plasma - Mass Spectrometry and Chemometrics can be used worldwide to provide comprehensive, rapid and affordable analyses of elements in drinking water that can have a considerable impact on human health.