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.

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.

The lipid peroxidation product 4-hydroxy-2-nonenal:advances in chemistry and analysis

4-Hydroxy-2-nonenal (HNE) is one of the most studied products of phospholipid peroxidation, owing to its reactivity and cytotoxicity. It can be formed by several radical-dependent oxidative routes involving the formation of hydroperoxides, alkoxyl radicals, epoxides, and fatty acyl cross-linking reactions. Cleavage of the oxidized fatty acyl chain results in formation of HNE from the methyl end, and 9-oxo-nonanoic acid from the carboxylate or esterified end of the chain, although many other products are also possible. HNE can be metabolized in tissues by a variety of pathways, leading to detoxification and excretion. HNE-adducts to proteins have been detected in inflammatory situations such as atherosclerotic lesions using polyclonal and monoclonal antibodies, which have also been applied in ELISAs and western blotting. However, in order to identify the proteins modified and the exact sites and nature of the modifications, mass spectrometry approaches are required. Combinations of enrichment strategies with targetted mass spectrometry routines such as neutral loss scanning are now facilitating detection of HNE-modified proteins in complex biological samples. This is important for characterizing the interactions of HNE with redox sensitive cell signalling proteins and understanding how it may modulate their activities either physiologically or in disease. © 2013 The Author.

Redox regulation of protein damage in plasma

The presence and concentrations of modified proteins circulating in plasma depend on rates of protein synthesis, modification and clearance. In early studies, the proteins most frequently analysed for damage were those which were more abundant in plasma (e.g. albumin and immunoglobulins) which exist at up to 10 orders of magnitude higher concentrations than other plasma proteins e.g. cytokines. However, advances in analytical techniques using mass spectrometry and immuno-affinity purification methods, have facilitated analysis of less abundant, modified proteins and the nature of modifications at specific sites is now being characterised. The damaging reactive species that cause protein modifications in plasma principally arise from reactive oxygen species (ROS) produced by NADPH oxidases (NOX), nitric oxide synthases (NOS) and oxygenase activities; reactive nitrogen species (RNS) from myeloperoxidase (MPO) and NOS activities; and hypochlorous acid from MPO. Secondary damage to proteins may be caused by oxidized lipids and glucose autooxidation.In this review, we focus on redox regulatory control of those enzymes and processes which control protein maturation during synthesis, produce reactive species, repair and remove damaged plasma proteins. We have highlighted the potential for alterations in the extracellular redox compartment to regulate intracellular redox state and, conversely, for intracellular oxidative stress to alter the cellular secretome and composition of extracellular vesicles. Through secreted, redox-active regulatory molecules, changes in redox state may be transmitted to distant sites. © 2014 The Authors.

AllerTOP v.2 - a server for in silico prediction of allergens

Allergy is an overreaction by the immune system to a previously encountered, ordinarily harmless substance - typically proteins - resulting in skin rash, swelling of mucous membranes, sneezing or wheezing, or other abnormal conditions. The use of modified proteins is increasingly widespread: their presence in food, commercial products, such as washing powder, and medical therapeutics and diagnostics, makes predicting and identifying potential allergens a crucial societal issue. The prediction of allergens has been explored widely using bioinformatics, with many tools being developed in the last decade; many of these are freely available online. Here, we report a set of novel models for allergen prediction utilizing amino acid E-descriptors, auto- and cross-covariance transformation, and several machine learning methods for classification, including logistic regression (LR), decision tree (DT), naïve Bayes (NB), random forest (RF), multilayer perceptron (MLP) and k nearest neighbours (kNN). The best performing method was kNN with 85.3% accuracy at 5-fold cross-validation. The resulting model has been implemented in a revised version of the AllerTOP server (http://www.ddg-pharmfac.net/AllerTOP). © Springer-Verlag 2014.

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.

Transglutaminase mediated grafting of silk proteins onto wool fabrics leading to improved physical and mechanical properties

Transglutaminases have the ability to incorporate primary amines and to graft peptides (containing glutamine or lysine residues) into proteins. These properties enable transglutaminases to be used in the grafting of a range of compounds including peptides and/or proteins onto wool fibres, altering their functionality. In this paper we investigated the transglutaminase mediated grafting of silk proteins into wool and its effect on wool properties. A commercial hydrolysed silk preparation was compared with silk sericin. The silk sericin protein was labelled with a fluorescent probe which was used to demonstrate the efficiency of the TGase grafting of such proteins into wool fibres. The TGase mediated grafting of these proteins led to a significant effect on the properties of wool yarn and fabric, resulting in increased bursting strength, as well as reduced levels of felting shrinkage and improved fabric softness. Also observed was an accumulation of deposits on the surface of the treated wool fibres when monitored by SEM and alterations in the thermal behaviour of the modified fibres, in particular for mTGase/sericin treated fibres which, with the confocal studies, corroborate the physical changes observed on the treated wool fabric. © 2006 Elsevier Inc. All rights reserved.

Novel pMDI formulations for pulmonary delivery of proteins

In this paper, we demonstrate that co-spray-drying a model protein with sodium carboxymethylcellulose (NaCMC) protects protein integrity during spray-drying, and that the resultant spray-dried powders can be successfully dispersed in hydrofluoroalkane (HFA) propellant to prepare pressurised metered dose (pMDI) formulations that exhibit high respirable fractions. The spray-dried powders were formulated as HFA-134a pMDI suspensions in the absence of any other excipients (e.g. surfactants) or co-solvents (e.g. ethanol). The in vitro aerosolisation profile of these systems was assessed using the twin stage impinger; fine particle fractions (FPF) ≥50% of the recovered dose were obtained. Following storage for five months, the aerosolisation performance was reassessed; the NaCMC-free formulation demonstrated a significant decrease in FPF, whereas the performance of the NaCMC-modified formulations was statistically equivalent to their initial performance. Thus, formulation of pMDI suspensions using NaCMC-based spray-dried powders is a promising approach for the pulmonary delivery of proteins and peptides. © 2009 Elsevier B.V. All rights reserved.

The use of sodium carboxymethylcellulose in the preparation of spray-dried proteins for pulmonary drug delivery

The use of sodium carboxymethylcellulose (NaCMC) as a spray-drying excipient in the preparation of inhalable formulations of proteins was investigated, using alkaline phosphatase as a model functional protein. Two spray-dried powders were investigated: a control powder comprising 100% (w/w) alkaline phosphatase and a test powder comprising 67% (w/w) NaCMC and 33% (w/w) alkaline phosphatase. Following physicochemical characterisation, the powders were prepared as both dry powder inhaler (DPI) and pressurised metered dose inhaler (pMDI) formulations. The aerosolisation performance of the formulations was assessed using a Multi-Stage Liquid Impinger, both immediately after preparation and over a 16-week storage period. Formulating the control powder as a DPI resulted in a poor fine particle fraction (FPF: 10%), whereas the FPF of the NaCMC-modified DPI formulation was significantly greater (47%). When the powders were formulated as pMDI systems, the control and NaCMC-modified powders demonstrated FPFs of 52% and 55%, respectively. Following storage, reduced FPF was observed for all formulations except the NaCMC-modified pMDI system; the performance of this formulation following storage was statistically equivalent to that immediately following preparation. Co-spray-drying proteins and peptides with NaCMC may therefore offer an alternative method for the preparation of stable and respirable pMDI formulations for pulmonary delivery. © 2010 Elsevier B.V.