Identification of oxidized phospholipids by electrospray ionization mass spectrometry and LC-MS using a QQLIT instrument

Phospholipids are complex and varied biomolecules that are susceptible to lipid peroxidation after attack by free radicals or electrophilic oxidants and can yield a large number of different oxidation products. There are many available methods for detecting phospholipid oxidation products, but also various limitations and problems. Electrospray ionization mass spectrometry allows the simultaneous but specific analysis of multiple species with good sensitivity and has a further advantage that it can be coupled to liquid chromatography for separation of oxidation products. Here, we explain the principles of oxidized phospholipid analysis by electrospray mass spectrometry and describe fragmentation routines for surveying the structural properties of the analytes, in particular precursor ion and neutral loss scanning. These allow targeted detection of phospholipid headgroups and identification of phospholipids containing hydroperoxides and chlorine, as well as the detection of some individual oxidation products by their specific fragmentation patterns. We describe instrument protocols for carrying out these survey routines on a QTrap5500 mass spectrometer and also for interfacing with reverse-phase liquid chromatography. The article highlights critical aspects of the analysis as well as some limitations of the methodology.

Protein and peptides in pictures:imaging with MALDI mass spectrometry

Imaging using MS has the potential to deliver highly parallel, multiplexed data on the specific localization of molecular ions in tissue samples directly, and to measure and map the variations of these ions during development and disease progression or treatment. There is an intrinsic potential to be able to identify the biomarkers in the same experiment, or by relatively simple extension of the technique. Unlike many other imaging techniques, no a priori knowledge of the markers being sought is necessary. This review concentrates on the use of MALDI-MS for MS imaging (MSI) of proteins and peptides, with an emphasis on mammalian tissue. We discuss the methodologies used, their potential limitations, overall experimental considerations and progress that has been made towards establishing MALDI-MSI as a routine technique for the spatially resolved measurement of peptides and proteins. As well as determining the local abundance of individual molecular ions, there is the potential to determine their identity within the same experiment using relatively simple extensions of the basic techniques. In this way MSI offers an important opportunity for biomarker discovery and identification.

Mass spectrometry imaging of pharmacological compounds in tissue sections

The use of MS imaging (MSI) to resolve the spatial and pharmacodynamic distributions of compounds in tissues is emerging as a powerful tool for pharmacological research. Unlike established imaging techniques, only limited a priori knowledge is required and no extensive manipulation (e.g., radiolabeling) of drugs is necessary prior to dosing. MS provides highly multiplexed detection, making it possible to identify compounds, their metabolites and other changes in biomolecular abundances directly off tissue sections in a single pass. This can be employed to obtain near cellular, or potentially subcellular, resolution images. Consideration of technical limitations that affect the process is required, from sample preparation through to analyte ionization and detection. The techniques have only recently been adapted for imaging and novel variations to the established MSI methodologies will further enhance the application of MSI for pharmacological research.

Chemical studies of plasma etchants used in integrated circuit manufacture

Plasma or "dry" etching is an essential process for the production of modern microelectronic circuits. However, despite intensive research, many aspects of the etch process are not fully understood. The results of studies of the plasma etching of Si and Si02 in fluorine-containing discharges, and the complementary technique of plasma polymerisation are presented in this thesis. Optical emission spectroscopy with argon actinometry was used as the principle plasma diagnostic. Statistical experimental design was used to model and compare Si and Si02 etch rates in CF4 and SF6 discharges as a function of flow, pressure and power. Etch mechanisms m both systems, including the potential reduction of Si etch rates in CF4 due to fluorocarbon polymer formation, are discussed. Si etch rates in CF4 /SF6 mixtures were successfully accounted for by the models produced. Si etch rates in CF4/C2F6 and CHF3 as a function of the addition of oxygen-containing additives (02, N20 and CO2) are shown to be consistent with a simple competition between F, 0 and CFx species for Si surface sites. For the range of conditions studied, Si02 etch rates were not dependent on F-atom concentration, but the presence of fluorine was essential in order to achieve significant etch rates. The influence of a wide range of electrode materials on the etch rate of Si and Si02 in CF4 and CF4 /02 plasmas was studied. It was found that the Si etch rate in a CF4 plasma was considerably enhanced, relative to an anodised aluminium electrode, in the presence of soda glass or sodium or potassium "doped" quartz. The effect was even more pronounced in a CF4 /02 discharge. In the latter system lead and copper electrodes also enhanced the Si etch rate. These results could not be accounted for by a corresponding rise in atomic fluorine concentration. Three possible etch enhancement mechanisms are discussed. Fluorocarbon polymer deposition was studied, both because of its relevance to etch mechanisms and its intrinsic interest, as a function of fluorocarbon source gas (CF4, C2F6, C3F8 and CHF3), process time, RF power and percentage hydrogen addition. Gas phase concentrations of F, H and CF2 were measured by optical emission spectroscopy, and the resultant polymer structure determined by X-ray photoelectron spectroscopy and infrared spectroscopy. Thermal and electrical properties were measured also. Hydrogen additions are shown to have a dominant role in determining deposition rate and polymer composition. A qualitative description of the polymer growth mechanism is presented which accounts for both changes in growth rate and structure, and leads to an empirical deposition rate model.

Analysis of oxidized and chlorinated lipids by mass spectrometry and relevance to signalling

Oxidized and chlorinated phospholipids are generated under inflammatory conditions and are increasingly understood to play important roles in diseases involving oxidative stress. MS is a sensitive and informative technique for monitoring phospholipid oxidation that can provide structural information and simultaneously detect a wide variety of oxidation products, including chain-shortened and -chlorinated phospholipids. MSn technologies involve fragmentation of the compounds to yield diagnostic fragment ions and thus assist in identification. Advanced methods such as neutral loss and precursor ion scanning can facilitate the analysis of specific oxidation products in complex biological samples. This is essential for determining the contributions of different phospholipid oxidation products in disease. While many pro-inflammatory signalling effects of oxPLs (oxidized phospholipids) have been reported, it has more recently become clear that they can also have anti-inflammatory effects in conditions such as infection and endotoxaemia. In contrast with free radical-generated oxPLs, the signalling effects of chlorinated lipids are much less well understood, but they appear to demonstrate mainly pro-inflammatory effects. Specific analysis of oxidized and chlorinated lipids and the determination of their molecular effects are crucial to understanding their role in disease pathology.

Reporter ion-based mass spectrometry approaches for the detection of non-enzymatic protein modifications in biological samples

Development of mass spectrometry techniques to detect protein oxidation, which contributes to signalling and inflammation, is important. Label-free approaches have the advantage of reduced sample manipulation, but are challenging in complex samples owing to undirected analysis of large data sets using statistical search engines. To identify oxidised proteins in biological samples, we previously developed a targeted approach involving precursor ion scanning for diagnostic MS3 ions from oxidised residues. Here, we tested this approach for other oxidations, and compared it with an alternative approach involving the use of extracted ion chromatograms (XICs) generated from high-resolution MSMS data using very narrow mass windows. This accurate mass XIC data methodology was effective at identifying nitrotyrosine, chlorotyrosine, and oxidative deamination of lysine, and for tyrosine oxidations highlighted more modified peptide species than precursor ion scanning or statistical database searches. Although some false positive peaks still occurred in the XICs, these could be identified by comparative assessment of the peak intensities. The method has the advantage that a number of different modifications can be analysed simultaneously in a single LC-MSMS run. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine. Biological significance: The use of accurate mass extracted product ion chromatograms to detect oxidised peptides could improve the identification of oxidatively damaged proteins in inflammatory conditions. © 2013 Elsevier B.V.

Evidence of lipid degradation during overnight contact lens wear:gas chromatography mass spectrometry as the diagnostic tool

Purpose. We investigated structural differences in the fatty acid profiles of lipids extracted from ex vivo contact lenses by using gas chromatography mass spectrometry (GCMS). Two lens materials (balafilcon A or lotrafilcon A) were worn on a daily or continuous wear schedule for 30 and 7 days. Methods. Lipids from subject-worn lenses were extracted using 1:1 chloroform: methanol and transmethylated using 5% sulfuric acid in methanol. Fatty acid methyl esters (FAMEs) were collected using hexane and water, and analyzed by GCMS (Varian 3800 GC, Saturn 2000 MS). Results. The gas chromatograms of lens extracts that were worn on a continuous wear schedule showed two predominant peaks, C16:0 and C18:0, both of which are saturated fatty acids. This was the case for balafilcon A and lotrafilcon A lenses. However, the gas chromatograms of lens extracts that were worn on a daily wear schedule showed saturated (C16:0, C18:0) and unsaturated (C16:1 and C18:1) fatty acids. Conclusions. Unsaturated fatty acids are degraded during sleep in contact lenses. Degradation occurred independently of lens material or subject-to-subject variability in lipid deposition. The consequences of lipid degradation are the production of oxidative products, which may be linked to contact lens discomfort. © 2014 The Association for Research in Vision and Ophthalmology, Inc.

Studies of phospholipid oxidation by electrospray mass spectrometry: from analysis in cells to biological effects

The oxidation of lipids is important in many pathological conditions and lipid peroxidation products such as 4-hydroxynonenal (HNE) and other aldehydes are commonly measured as biomarkers of oxidative stress. However, it is often useful to complement this with analysis of the original oxidized phospholipid. Electrospray mass spectrometry (ESMS) provides an informative method for detecting oxidative alterations to phospholipids, and has been used to investigate oxidative damage to cells, and low-density lipoprotein, as well as for the analysis of oxidized phosphatidylcholines present in atherosclerotic plaque material. There is increasing evidence that intact oxidized phospholipids have biological effects; in particular, oxidation products of 1-palmitoyl-2-arachidonoyl-sn-glycerophosphocholine (PAPC) have been found to cause inflammatory responses, which could be potentially important in the progression of atherosclerosis. The effects of chlorohydrin derivatives of lipids have been much less studied, but it is clear that free fatty acid chlorohydrins and phosphatidylcholine chlorohydrins are toxic to cells at concentrations above 10 micromolar, a range comparable to that of HNE and oxidized PAPC. There is some evidence that chlorohydrins have biological effects that may be relevant to atherosclerosis, but further work is needed to elucidate their pro-inflammatory properties, and to understand the mechanisms and balance of biological effects that could result from oxidation of complex mixtures of lipids in a pathophysiological situation.