Development of novel mass spectrometric methods for identifying HOCl-induced modifications to proteins

Protein oxidation is thought to contribute to a number of inflammatory diseases, hence the development of sensitive and specific analytical techniques to detect oxidative PTMs (oxPTMs) in biological samples is highly desirable. Precursor ion scanning for fragment ions of oxidized amino acid residues was investigated as a label-free MS approach to mapping specific oxPTMs in a complex mixture of proteins. Using HOCl-oxidized lysozyme as a model system, it was found that the immonium ions of oxidized tyrosine and tryptophan formed in MS(2) analysis could not be used as diagnostic ions, owing to the occurrence of isobaric fragment ions from unmodified peptides. Using a double quadrupole linear ion trap mass spectrometer, precursor ion scanning was combined with detection of MS(3) fragment ions from the immonium ions and collisionally-activated decomposition peptide sequencing to achieve selectivity for the oxPTMs. For chlorotyrosine, the immonium ion at 170.1 m/z fragmented to yield diagnostic ions at 153.1, 134.1, and 125.1 m/z, and the hydroxytyrosine immonium ion at 152.1 m/z gave diagnostic ions at 135.1 and 107.1 m/z. Selective MS(3) fragment ions were also identified for 2-hydroxytryptophan and 5-hydroxytryptophan. The method was used successfully to map these oxPTMs in a mixture of nine proteins that had been treated with HOCl, thereby demonstrating its potential for application to complex biological samples.

Mass spectrometric analysis of HOCl- and free-radical-induced damage to lipids and proteins

In inflammatory diseases, release of oxidants leads to oxidative damage to biomolecules. HOCl (hypochlorous acid), released by the myeloperoxidase/H2O2/Cl- system, can cause formation of phospholipid chlorohydrins, or alpha-chloro-fatty aldehydes from plasmalogens. It can attack several amino acid residues in proteins, causing post-translational oxidative modifications of proteins, but the formation of 3-chlorotyrosine is one of the most stable markers of HOCl-induced damage. Soft-ionization MS has proved invaluable for detecting the occurrence of oxidative modifications to both phospholipids and proteins, and characterizing the products generated by HOCl-induced attack. For both phospholipids and proteins, the application of advanced mass spectrometric methods such as product or precursor ion scanning and neutral loss analysis can yield information both about the specific nature of the oxidative modification and the biomolecule modified. The ideal is to be able to apply these methods to complex biological or clinical samples, to determine the site-specific modifications of particular cellular components. This is important for understanding disease mechanisms and offers potential for development of novel biomarkers of inflammatory diseases. In the present paper, we review some of the progress that has been made towards this goal.

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.

Use of narrow mass-window, high-resolution extracted product ion chromatograms for the sensitive and selective identification of protein modifications

Protein modifications, including oxidative modifications, glycosylations, and oxidized lipid-protein adducts, are becoming increasingly important as biomarkers and in understanding disease etiology. There has been a great deal of interest in mapping these on Apo B100 from low density lipoprotein (LDL). We have used extracted ion chromatograms of product ions generated using a very narrow mass window from high-resolution tandem mass spectrometric data collected on a rapid scanning quadrupole time-of-flight (QTOF) instrument, to selectively and sensitively detect modified peptides and identify the site and nature of a number of protein modifications in parallel. We have demonstrated the utility of this method by characterizing for the first time oxidized phospholipid adducts to LDL and human serum albumin and for the detection of glycosylation and kynurenin formation from the oxidation of tryptophan residues in LDL. © 2013 American Chemical Society.

Tear film lipids:dynamic composition and clinical performance

Purpose: Meibomian-derived lipid secretions are well characterised but their subsequent fate in the ocular environment is less well understood. Phospholipids are thought to facilitate the interface between aqueous and lipid layers of the tear film and to be involved in ocular lubrication processes. We have extended our previous studies on phospholipid levels in the tear film to encompass the fate of polar and non-polar lipids in progressive accumulation and aging processes on both conventional and silicone-modified hydrogel lenses. This is an important aspect of the developing understanding of the role of lipids in the clinical performance of silicone hydrogels. Method: Several techniques were used to identify lipids in the tear film. Mass-spectrometric methods included Agilent 1100-based liquid chromatography coupled to mass spectrometry (LCMS) and Perkin Elmer gas chromatography mass spectrometry (GCMS). Thin layer chromatography (TLC) was used for separation of lipids on the basis of increasing solvent polarity. Routine assay of lipid extractions from patient-worn lenses was carried out using a Hewlett Packard 1090 liquid chromatograph coupled to both uv and Agilent 1100 fluorescence detection. A range of histological together with optical, and electron microscope techniques was used in deposit analysis. Results: Progressive lipid uptake was assessed in various ways, including: composition changes with wear time, differential lipid penetrate into the lens matrix and, particularly, the extent to which lipids become unextractable as a function of wear time. Solvent-based separation and HPLC gave consistent results indicating that the polarity of lipid classes decreased as follows: phospholipids/fatty acids > triglycerides > cholesterol/cholesteryl esters. Tear lipids were found to show autofluorescence—which underpinned the value of fluorescence microscopy and fluorescence detection coupled with HPLC separation. The most fluorescent lipids were found to be cholesteryl esters; histological techniques coupled with fluorescence microscopy indicated that white spots (’’jelly bumps’’) formed on silicone hydrogel lenses contain a high proportion of cholesteryl esters. Lipid profiles averaged for 30 symptomatic and 30 asymptomatic contact lens wearers were compiled. Peak classes were split into: cholesterol (C), cholesteryl esters (CE), glycerides (G), polar fatty acids/phospholipids (PL). The lipid ratio for ymptomatic/symptomatic was 0.6 ± 0.1 for all classes except one—the cholesterol ratio was 0.2 ± 0.05. Significantly the PL ratio was no different from that of any other class except cholesterol. Chromatography indicated that: lipid polarity decreased with depth of penetration and that lipid extractability decreased with wear time. Conclusions: Meibomian lipid composition differs from that in the tear film and on worn lenses. Although the same broad lipid classes were obtained by extraction from all lenses and all patients studied, quantities vary with wear and material. Lipid extractability diminishes with wear time regardless of the use of cleaning regimes. Dry eye symptoms in contact lens wear are frequently linked to lipid layer behaviour but seem to relate more to total lipid than to specific composition. Understanding the detail of lipid related processes is an important element of improving the clinical performance of materials and care solutions.

Targeted mass spectrometry methods for detecting oxidative post-translational modifications

Oxidative post-translational modifications (oxPTMs) can alter the function of proteins, and are important in the redox regulation of cell behaviour. The most informative technique to detect and locate oxPTMs within proteins is mass spectrometry (MS). However, proteomic MS data are usually searched against theoretical databases using statistical search engines, and the occurrence of unspecified or multiple modifications, or other unexpected features, can lead to failure to detect the modifications and erroneous identifications of oxPTMs. We have developed a new approach for mining data from accurate mass instruments that allows multiple modifications to be examined. Accurate mass extracted ion chromatograms (XIC) for specific reporter ions from peptides containing oxPTMs were generated from standard LC-MSMS data acquired on a rapid-scanning high-resolution mass spectrometer (ABSciex 5600 Triple TOF). The method was tested using proteins from human plasma or isolated LDL. A variety of modifications including chlorotyrosine, nitrotyrosine, kynurenine, oxidation of lysine, and oxidized phospholipid adducts were detected. For example, the use of a reporter ion at 184.074 Da/e, corresponding to phosphocholine, was used to identify for the first time intact oxidized phosphatidylcholine adducts on LDL. In all cases the modifications were confirmed by manual sequencing. ApoB-100 containing oxidized lipid adducts was detected even in healthy human samples, as well as LDL from patients with chronic kidney disease. The accurate mass XIC method gave a lower false positive rate than normal database searching using statistical search engines, and identified more oxidatively modified peptides. A major advantage was that additional modifications could be searched after data collection, and multiple modifications on a single peptide identified. The oxPTMs present on albumin and ApoB-100 have potential as indicators of oxidative damage in ageing or inflammatory diseases.

Chemical kinetic investigation of a commercial batch reactor process

The aim of this investigation was to study the chemical reactions occurring during the batchwise production of a butylated melamine-formaldehyde resin, in order to optimise the efficiency and economics of the batch processes. The batch process models are largely empirical in nature as the reaction mechanism is unknown. The process chemistry and the commercial manufacturing method are described. A small scale system was established in glass and the ability to produce laboratory resins with the required quality was demonstrated, simulating the full scale plant. During further experiments the chemical reactions of methylolation, condensation and butylation were studied. The important process stages were identified and studied separately. The effects of variation of certain process parameters on the chemical reactions were also studied. A published model of methylolation was modified and used to simulate the methylolation stage. A major result of this project was the development of an indirect method for studying the condensation and butylation reactions occurring during the dehydration and acid reaction stages, as direct quantitative methods were not available. A mass balance method was devised for this purpose and used to collect experimental data. The reaction scheme was verified using this data. The reactions stages were simulated using an empirical model. This has revealed new information regarding the mechanism and kinetics of the reactions. Laboratory results were shown to be comparable with plant scale results. This work has improved the understanding of the batch process, which can be used to improve product consistency. Future work has been identified and recommended to produce an optimum process and plant design to reduce the batch time.

Use of a solvent-free dry matrix coating for quantitative matrix-assisted laser desorption ionization imaging of 4-bromophenyl-1,4-diazabicyclo(3.2.2)nonane-4-carboxylate in rat brain and quantitative analysis of the drug from laser microdissected tissue regions

A dry matrix application for matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) was used to profile the distribution of 4-bromophenyl-1,4-diazabicyclo(3.2.2)nonane-4-carboxylate, monohydrochloride (BDNC, SSR180711) in rat brain tissue sections. Matrix application involved applying layers of finely ground dry alpha-cyano-4-hydroxycinnamic acid (CHCA) to the surface of tissue sections thaw mounted onto MALDI targets. It was not possible to detect the drug when applying matrix in a standard aqueous-organic solvent solution. The drug was detected at higher concentrations in specific regions of the brain, particularly the white matter of the cerebellum. Pseudomultiple reaction monitoring imaging was used to validate that the observed distribution was the target compound. The semiquantitative data obtained from signal intensities in the imaging was confirmed by laser microdissection of specific regions of the brain directed by the imaging, followed by hydrophilic interaction chromatography in combination with a quantitative high-resolution mass spectrometry method. This study illustrates that a dry matrix coating is a valuable and complementary matrix application method for analysis of small polar drugs and metabolites that can be used for semiquantitative analysis.

Stabilising cysteinyl thiol oxidation and nitrosation for proteomic analysis

Oxidation and S-nitrosylation of cysteinyl thiols (Cys-SH) to sulfenic (Cys-SOH), sulfinic (Cys-SO2H), sulfonic acids (Cys-SO3H), disulphides and S-nitrosothiols are suggested as important post-translational modifications that can activate or deactivate the function of many proteins. Non-enzymatic post-translational modifications to cysteinyl thiols have been implicated in a wide variety of physiological and pathophysiological states but have been difficult to monitor in a physiological setting because of a lack of experimental tools. The purpose of this review is to bring together the approaches that have been developed for stably trapping cysteine either in its reduced or oxidised forms for enrichment and or subsequent mass spectrometric analysis. These tools are providing insight into potential targets for post-translational modifications to cysteine modification in vivo. This article is part of a Special Issue entitled: Special Issue: Posttranslational Protein modifications in biology and Medicine. © 2013.

Small Ubiquitin-related Modifier (SUMO)-1 promotes glycolysis in hypoxia

Under conditions of hypoxia, most eukaryotic cells undergo a shift in metabolic strategy, which involves increased flux through the glycolytic pathway. Although this is critical for bioenergetic homeostasis, the underlying mechanisms have remained incompletely understood. Here, we report that the induction of hypoxia-induced glycolysis is retained in cells when gene transcription or protein synthesis are inhibited suggesting the involvement of additional post-translational mechanisms. Post-translational protein modification by the small ubiquitin related modifier-1 (SUMO-1) is induced in hypoxia and mass spectrometric analysis using yeast cells expressing tap-tagged Smt3 (the yeast homolog of mammalian SUMO) revealed hypoxia-dependent modification of a number of key glycolytic enzymes. Overexpression of SUMO-1 in mammalian cancer cells resulted in increased hypoxia-induced glycolysis and resistance to hypoxia-dependent ATP depletion. Supporting this, non-transformed cells also demonstrated increased glucose uptake upon SUMO-1 overexpression. Conversely, cells overexpressing the de-SUMOylating enzyme SENP-2 failed to demonstrate hypoxia-induced glycolysis. SUMO-1 overexpressing cells demonstrated focal clustering of glycolytic enzymes in response to hypoxia leading us to hypothesize a role for SUMOylation in promoting spatial re-organization of the glycolytic pathway. In summary, we hypothesize that SUMO modification of key metabolic enzymes plays an important role in shifting cellular metabolic strategies toward increased flux through the glycolytic pathway during periods of hypoxic stress. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

The enantioselective population pharmacokinetics of intravenous ketorolac in children using a stereoselective assay suitable for microanalysis

Objective: To describe the effect of age and body size on enantiomer selective pharmacokinetic (PK) of intravenous ketorolac in children using a microanalytical assay. Methods: Blood samples were obtained at 0, 15 and 30 min and at 1, 2, 4, 6, 8 and 12 h after a weight-dependent dose of ketorolac. Enantiomer concentration was measured using a liquid chromatography tandem mass spectrometry method. Non-linear mixed-effect modelling was used to assess PK parameters. Key findings: Data from 11 children (1.7–15.6 years, weight 10.7–67.4 kg) were best described by a two-compartment model for R(+), S(−) and racemic ketorolac. Only weight (WT) significantly improved the goodness of fit. The final population models were CL = 1.5 × (WT/46)0.75, V1 = 8.2 × (WT/46), Q = 3.4 × (WT/46)0.75, V2 = 7.9 × (WT/46), CL = 2.98 × (WT/46), V1 = 13.2 × (WT/46), Q = 2.8 × (WT/46)0.75, V2 = 51.5 × (WT/46), and CL = 1.1 × (WT/46)0.75, V1 = 4.9 × (WT/46), Q = 1.7 × (WT/46)0.75 and V2 = 6.3 × (WT/46)for R(+), S(−) and racemic ketorolac. Conclusions: Only body weight influenced the PK parameters for R(+) and S(−) ketorolac. Using allometric size scaling significantly affected the clearances (CL, Q) and volumes of distribution (V1, V2).