Matrix-free mass spectrometric imaging using laser desorption ionisation Fourier transform ion cyclotron resonance mass spectrometry

Mass spectrometry imaging (MSI) is a powerful tool in metabolomics and proteomics for the spatial localization and identification of pharmaceuticals, metabolites, lipids, peptides and proteins in biological tissues. However, sample preparation remains a crucial variable in obtaining the most accurate distributions. Common washing steps used to remove salts, and solvent-based matrix application, allow analyte spreading to occur. Solvent-free matrix applications can reduce this risk, but increase the possibility of ionisation bias due to matrix adhesion to tissue sections. We report here the use of matrix-free MSI using laser desorption ionisation performed on a 12 T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. We used unprocessed tissue with no post-processing following thaw-mounting on matrix-assisted laser desorption ionisation (MALDI) indium-tin oxide (ITO) target plates. The identification and distribution of a range of phospholipids in mouse brain and kidney sections are presented and compared with previously published MALDI time-of-flight (TOF) MSI distributions.

Protein oxidation:role in signalling and detection by mass spectrometry

Proteins can undergo a wide variety of oxidative post-translational modifications (oxPTM); while reversible modifications are thought to be relevant in physiological processes, non-reversible oxPTM may contribute to pathological situations and disease. The oxidant is also important in determining the type of oxPTM, such as oxidation, chlorination or nitration. The best characterized oxPTMs involved in signalling modulation are partial oxidations of cysteine to disulfide, glutathionylated or sulfenic acid forms that can be reversed by thiol reductants. Proline hydroxylation in HIF signalling is also quite well characterized, and there is increasing evidence that specific oxidations of methionine and tyrosine may have some biological roles. For some proteins regulated by cysteine oxidation, the residues and molecular mechanism involved have been extensively studied and are well understood, such as the protein tyrosine phosphatase PTP1B and MAP3 kinase ASK1, as well as transcription factor complex Keap1-Nrf2. The advances in understanding of the role oxPTMs in signalling have been facilitated by advances in analytical technology, in particular tandem mass spectrometry techniques. Combinations of peptide sequencing by collisionally induced dissociation and precursor ion scanning or neutral loss to select for specific oxPTMs have proved very useful for identifying oxidatively modified proteins and mapping the sites of oxidation. The development of specific labelling and enrichment procedures for S-nitrosylation or disulfide formation has proved invaluable, and there is ongoing work to establish analogous methods for detection of nitrotyrosine and other modifications.

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.

Redox signalling and detection of protein oxidation by mass spectrometry

It is now recognised that redox control of proteins plays an important role in many signalling pathways both in health and disease. Proteins can undergo a wide variety of oxidative post-translational modifications (oxPTM); while the reversible modifications are thought to be most important in physiological processes, non-reversible oxPTM may contribute to pathological situations and disease. The oxidant is also important in determining the type of oxPTM (chlorination, nitration, etc.), and the susceptibilities of residues vary depending on their structural location. The best characterized oxPTMs involved in signalling modulation are partial oxidations of cysteine to the disulfide, glutathionylated or sulfenic acid forms, but there is increasing evidence that specific oxidations of methionine and tyrosine may have some biological roles. Well understood examples of oxidative regulation include protein tyrosine phosphatases, e.g. PTP1B/C, and members of the MAPK pathways such as MEKK1 and ASK1. Transcription factors such as NFkB and Nrf-2 are also regulated by redox-active cysteines. Improved methods for analysing specific oxPTMs in biological samples are critical for understanding the physiological and pathological roles of these changes, and tandem or MS3 mass spectrometry techniques interfaced with nano-LC separation are being now used. MS3 fragmentation markers for a variety of oxidized residues including tyrosine, tryptophan and proline have been identified, and a precursor ion scanning method that allows the selective identification of these oxPTMs in complex samples has been developed. Such advances in technology offer potential for biomarker development, disease diagnosis and understanding pathology.

Protein oxidative modifications:new mass spectrometry approaches to detection in biological samples

In inflammatory diseases, release of oxidants leads to oxidative damage to proteins. The precise nature of oxidative damage to individual proteins depends on the oxidant involved. Chlorination and nitration are markers of modification by the myeloperoxidase-H2O2-Cl- system and nitric oxide-derived oxidants, respectively. Although these modifications can be detected by western blotting, currently no reliable method exists to identify the specific sites damage to individual proteins in complex mixtures such as clinical samples. We are developing novel LCMS2 and precursor ion scanning methods to address this. LC-MS2 allows separation of peptides and detection of mass changes in oxidized residues on fragmentation of the peptides. We have identified indicative fragment ions for chlorotyrosine, nitrotyrosine, hydroxytyrosine and hydroxytryptophan. A nano-LC/MS3 method involving the dissociation of immonium ions to give specific fragments for the oxidized residues has been developed to overcome the problem of false positives from ions isobaric to these immonium ions that exist in unmodified peptides. The approach has proved able to identify precise protein modifications in individual proteins and mixtures of proteins. An alternative methodology involves multiple reaction monitoring for precursors and fragment ions are specific to oxidized and chlorinated proteins, and this has been tested with human serum albumin. Our ultimate aim is to apply this methodology to the detection of oxidative post-translational modifications in clinical samples for disease diagnosis, monitoring the outcomes of therapy, and improved understanding of disease biochemistry.

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.

Post-translational modifications and mass spectrometry detection

In this review, we provide a comprehensive bibliographic overview of the role of mass spectrometry and the recent technical developments in the detection of post-translational modifications (PTMs). We briefly describe the principles of mass spectrometry for detecting PTMs and the protein and peptide enrichment strategies for PTM analysis, including phosphorylation, acetylation and oxidation. This review presents a bibliographic overview of the scientific achievements and the recent technical development in the detection of PTMs is provided. In order to ascertain the state of the art in mass spectrometry and proteomics methodologies for the study of PTMs, we analyzed all the PTM data introduced in the Universal Protein Resource (UniProt) and the literature published in the last three years. The evolution of curated data in UniProt for proteins annotated as being post-translationally modified is also analyzed. Additionally, we have undertaken a careful analysis of the research articles published in the years 2010 to 2012 reporting the detection of PTMs in biological samples by mass spectrometry. © 2013 Elsevier Inc.

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.

Development of novel mass spectrometry methodology to detect post-translational modifications in oxidative stress and disease

This paper presented at the European Meeting of the Society-for-Free-Radical-Research-Europe 2007, discusses the development of novel mass spectrometry methodology to detect post-translational modifications in oxidative stress and disease.

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.

Interfacing low-energy SAW nebulization with liquid chromatography-mass spectrometry for the analysis of biological samples

Soft ionization methods for the introduction of labile biomolecules into a mass spectrometer are of fundamental importance to biomolecular analysis. Previously, electrospray ionization (ESI) and matrix assisted laser desorption-ionization (MALDI) have been the main ionization methods used. Surface acoustic wave nebulization (SAWN) is a new technique that has been demonstrated to deposit less energy into ions upon ion formation and transfer for detection than other methods for sample introduction into a mass spectrometer (MS). Here we report the optimization and use of SAWN as a nebulization technique for the introduction of samples from a low flow of liquid, and the interfacing of SAWN with liquid chromatographic separation (LC) for the analysis of a protein digest. This demonstrates that SAWN can be a viable, low-energy alternative to ESI for the LC-MS analysis of proteomic samples.

Mass spectrometry-based methods for identifying oxidized proteins in disease:advances and challenges

Many inflammatory diseases have an oxidative aetiology, which leads to oxidative damage to biomolecules, including proteins. It is now increasingly recognized that oxidative post-translational modifications (oxPTMs) of proteins affect cell signalling and behaviour, and can contribute to pathology. Moreover, oxidized proteins have potential as biomarkers for inflammatory diseases. Although many assays for generic protein oxidation and breakdown products of protein oxidation are available, only advanced tandem mass spectrometry approaches have the power to localize specific oxPTMs in identified proteins. While much work has been carried out using untargeted or discovery mass spectrometry approaches, identification of oxPTMs in disease has benefitted from the development of sophisticated targeted or semi-targeted scanning routines, combined with chemical labeling and enrichment approaches. Nevertheless, many potential pitfalls exist which can result in incorrect identifications. This review explains the limitations, advantages and challenges of all of these approaches to detecting oxidatively modified proteins, and provides an update on recent literature in which they have been used to detect and quantify protein oxidation in disease.

Sucrose cryo-protection facilitates imaging of whole eye sections by MALDI mass spectrometry

Sucrose is used as a cryo-preservation agent on large mammalian eyes post formalin fixation and is shown to reduce freezing artefacts allowing the collection of 12-μm thick sections from these large aqueous samples. The suitability of this technique for use in MALDI imaging experiments is demonstrated by the acquisition of the first images of lipid distributions within whole sagittal porcine eye sections. © 2012 John Wiley & Sons, Ltd.