Incorporation of N-heterocyclic cations into proteins with a highly directed chemical modification

N-Heterocyclic cations are incorporated into proteins using 5-(2-bromoethyl)phenanthridinium bromide, which selectively reacts with either cysteine or lysine residues, resulting in ethylphenanthridinium (Phen) or highly stable cyclised dihydro-imidazo-phenanthridinium (DIP) adducts respectively; these modifications have been found to manipulate the observed structure of lysozyme and bovine serum albumin by AFM.

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.

The fall and rise of tear albumin levels:a multifactorial phenomenon

Albumin in tears is used as a diagnostic marker of ocular insult and inflammation, but whether its presence in tears is responsive or part of an adaptive reaction remains unresolved. A review of the literature on tear albumin concentration emphasizes that variables such as collection method, stimulus, assay technique, and disease state influence the quoted values to different extents. Influence of assay technique is negligible in comparison to variation in sampling conditions. Ocular disease increases albumin concentrations but not in a specific manner. The literature review also highlighted that little systematic research has been carried out on the daily cycle of tear albumin levels. In order to remedy this shortcoming, we investigated variations in tear albumin concentration during the waking day. The concentration of albumin in 400 tear samples collected from 13 subjects was assessed at 2-hourly intervals throughout the waking day. Highest daytime albumin concentrations were obtained within 10 minutes of waking, with a mean concentration of >50 ± 22 µg/ml. Albumin levels were at their lowest, but most consistent, 2-6 hours post-waking. This pattern was followed by a progressive increase in albumin concentration during the latter part of the day. Although individual subject-to-subject concentration differences were observed, this distinctive pattern of diurnal variation was found in all subjects. The results presented suggest a regulated, not random, pattern of variation within the period of study. © 2013 Elsevier Inc. All rights reserved.

Lipoxidation adducts with peptides and proteins:deleterious modifications or signalling mechanisms?

Protein lipoxidation refers to the modification by electrophilic lipid oxidation products to form covalent adducts, which for many years has been considered as a deleterious consequence of oxidative stress. Oxidized lipids or phospholipids containing carbonyl moieties react readily with lysine to form Schiff bases; alternatively, oxidation products containing α,β-unsaturated moieties are susceptible to nucleophilic attack by cysteine, histidine or lysine residues to yield Michael adducts, overall corresponding to a large number of possible protein adducts. The most common detection methods for lipoxidized proteins take advantage of the presence of reactive carbonyl groups to add labels, or use antibodies. These methods have limitations in terms of specificity and identification of the modification site. The latter question is satisfactorily addressed by mass spectrometry, which enables the characterization of the adduct structure. This has allowed the identification of lipoxidized proteins in physiological and pathological situations. While in many cases lipoxidation interferes with protein function, causing inhibition of enzymatic activity and increased immunogenicity, there are a small number of cases where lipoxidation results in gain of function or activity. For certain proteins lipoxidation may represent a form of redox signaling, although more work is required to confirm the physiological relevance and mechanisms of such processes. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine. © 2013 Elsevier B.V.

Albumin in tears

Albumin is not endogenous to the tear film and is present as a product of plasma leakage. It is used as a diagnostic marker of ocular insult and inflammation. Tear albumin is, however, poorly understood, with large variations in reported concentrations between studies. There is also no authoritative information on whether its presence in tears is responsive or part of an adaptive reaction.The presented research aimed to resolve the disparities in published tear albumin concentrations and investigate the role of albumin in the tear film. Collation and evaluation of the available literature identified collection method, stimulus, assay technique, and disease state as factors able to influence quoted tear albumin to different extents. Difference in sampling technique exhibited the largest variations in mean tear albumin concentrations. Review of the literature also highlighted that little systematic investigations of the daily cycle of tear albumin levels, and subject-to-subject-variation, had been carried out. In order to remedy this shortcoming, variations in tear albumin concentration were investigated in 13 subjects throughout the waking day. Results identified a time period where albumin levels are relatively stable (2-6 hours post-waking). This was designated a suitable baseline for the determinations of tear albumin concentrations and subject-to-subject comparisons. Significantly, a previously unrecognised progressive increase in albumin concentration during the latter part of the day was also identified in the population. This increase suggests that albumin may play a more active and dynamic role in the ocular environment than is commonly perceived. To facilitate the collection of additional tear albumin data, tear sampling and point-of-care analysis in contact lens clinics were investigated. Two instruments were evaluated and were found to be suitable for the analysis of tear albumin in commercial institutions. Collectively, the described research has provided new insight into tear albumin and a strong foundation for further studies.

Measurement of HNE-protein adducts in human plasma and serum by ELISA-Comparison of two primary antibodies

There is increasing evidence that non-enzymatic post-translational protein modifications might play key roles in various diseases. These protein modifications can be caused by free radicals generated during oxidative stress or by their products generated during lipid peroxidation. 4-Hydroxynonenal (HNE), a major biomarker of oxidative stress and lipid peroxidation, has been recognized as important molecule in pathology as well as in physiology of living organisms. Therefore, its detection and quantification can be considered as valuable tool for evaluating various pathophysiological conditions.The HNE-protein adduct ELISA is a method to detect HNE bound to proteins, which is considered as the most likely form of HNE occurrence in living systems. Since the earlier described ELISA has been validated for cell lysates and the antibody used for detection of HNE-protein adducts is non-commercial, the aim of this work was to adapt the ELISA to a commercial antibody and to apply it in the analysis of human plasma samples.After modification and validation of the protocol for both antibodies, samples of two groups were analyzed: apparently healthy obese (n=62) and non-obese controls (n=15). Although the detected absolute values of HNE-protein adducts were different, depending on the antibody used, both ELISA methods showed significantly higher values of HNE-protein adducts in the obese group. © 2013 The Authors.

Light scattering study of human serum albumin in pre-denaturation:relation to dynamic transition in water at 42°C

Protein functional motions are ultimately connected to water dynamics. The goal of this study is to link the conformational dynamics of albumin to a dynamic transition taking place at ∼ 42°C in water. We report the results of dynamic light scattering measurements of albumin aqueous solution in the temperature interval 20-65°C. The processing of the experimental data produced the temperature dependence of the macromolecular hydrodynamic radius. We demonstrate that the growth of the macromolecular size in this temperature range can be divided into two stages that are connected to the dynamical properties of water. © 2012 Elsevier B.V. All rights reserved.

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.

Glutathione adducts induced by ischemia and deletion of glutaredoxin-1 stabilize HIF-1α and improve limb revascularization

Reactive oxygen species (ROS) are increased in ischemic tissues and necessary for revascularization; however, the mechanism remains unclear. Exposure of cysteine residues to ROS in the presence of glutathione (GSH) generates GSH-protein adducts that are specifically reversed by the cytosolic thioltransferase, glutaredoxin-1 (Glrx). Here, we show that a key angiogenic transcriptional factor hypoxia-inducible factor (HIF)-1α is stabilized by GSH adducts, and the genetic deletion of Glrx improves ischemic revascularization. In mouse muscle C2C12 cells, HIF-1α protein levels are increased by increasing GSH adducts with cell-permeable oxidized GSH (GSSG-ethyl ester) or 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanyl thiocarbonylamino) phenylthiocarbamoylsulfanyl] propionic acid (2-AAPA), an inhibitor of glutathione reductase. A biotin switch assay shows that GSSG-ester-induced HIF-1α contains reversibly modified thiols, and MS confirms GSH adducts on Cys520 (mouse Cys533). In addition, an HIF-1α Cys520 serine mutant is resistant to 2-AAPA–induced HIF-1α stabilization. Furthermore, Glrx overexpression prevents HIF-1α stabilization, whereas Glrx ablation by siRNA increases HIF-1α protein and expression of downstream angiogenic genes. Blood flow recovery after femoral artery ligation is significantly improved in Glrx KO mice, associated with increased levels of GSH-protein adducts, capillary density, vascular endothelial growth factor (VEGF)-A, and HIF-1α in the ischemic muscles. Therefore, Glrx ablation stabilizes HIF-1α by increasing GSH adducts on Cys520 promoting in vivo HIF-1α stabilization, VEGF-A production, and revascularization in the ischemic muscles