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.

Mass transfer and chemical reaction on a distillation plate

A multistage distillation column in which mass transfer and a reversible chemical reaction occurred simultaneously, has been investigated to formulate a technique by which this process can be analysed or predicted. A transesterification reaction between ethyl alcohol and butyl acetate, catalysed by concentrated sulphuric acid, was selected for the investigation and all the components were analysed on a gas liquid chromatograph. The transesterification reaction kinetics have been studied in a batch reactor for catalyst concentrations of 0.1 - 1.0 weight percent and temperatures between 21.4 and 85.0 °C. The reaction was found to be second order and dependent on the catalyst concentration at a given temperature. The vapour liquid equilibrium data for six binary, four ternary and one quaternary systems are measured at atmospheric pressure using a modified Cathala dynamic equilibrium still. The systems with the exception of ethyl alcohol - butyl alcohol mixtures, were found to be non-ideal. Multicomponent vapour liquid equilibrium compositions were predicted by a computer programme which utilised the Van Laar constants obtained from the binary data sets. Good agreement was obtained between the predicted and experimental quaternary equilibrium vapour compositions. Continuous transesterification experiments were carried out in a six stage sieve plate distillation column. The column was 3" in internal diameter and of unit construction in glass. The plates were 8" apart and had a free area of 7.7%. Both the liquid and vapour streams were analysed. The component conversion was dependent on the boilup rate and the reflux ratio. Because of the presence of the reaction, the concentration of one of the lighter components increased below the feed plate. In the same region a highly developed foam was formed due to the presence of the catalyst. The experimental results were analysed by the solution of a series of simultaneous enthalpy and mass equations. Good agreement was obtained between the experimental and calculated results.

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.

Mapping nitro-tyrosine modifications in fibrinogen by mass spectrometry as a biomarker for inflammatory disease

There is a growing awareness that inflammatory diseases have an oxidative pathology, which can result in specific oxidation of amino acids within proteins. It is known that patients with inflammatory disease have higher levels of plasma protein nitro-tyrosine than healthy controls. Fibrinogen is an abundant plasma protein, highly susceptible to such oxidative modifications, and is therefore a potential marker for oxidative protein damage. The aim of this study was to map tyrosine nitration in fibrinogen under oxidative conditions and identify susceptible residues. Fibrinogen was oxidised with 0.25mM and 1mM SIN-1, a peroxynitrite generator, and methionine was used to quench excess oxidant in the samples. The carbonyl assay was used to confirm oxidation in the samples. The carbonyl levels were 2.3, 8.72 and 11.5nmol/mg protein in 0, 0.25mM and 1mM SIN-1 samples respectively. The samples were run on a SDS-PAGE gel and tryptically digested before analysis by HPLC MS-MS. All 3 chains of fibrinogen were observed for all treatment conditions. The overall sequence coverage for fibrinogen determined by Mascot was between 60-75%. The oxidised samples showed increases in oxidative modifications in both alpha and beta chains, commonly methionine sulfoxide and tyrosine nitration, correlating with increasing SIN-1 treatment. Tyrosines that were most susceptible were Tyr135 (tryptic peptide YLQEIYNSNNQK) and Tyr277 (tryptic peptide GGSTSYGTGSETESPR), but several other nitrated tyrosines were also identified with high confidence. Identification of these susceptible peptides will allow design of sequences-specific biomarkers of oxidative and nitrative damage to plasma protein in inflammatory conditions.

Residue-specific investigation of the relationship between oxidation and activity of a dual specificity phosphatase by mass spectrometry

Redox regulation of signalling pathways is critical in proliferation and apoptosis; redox imbalance can lead to pathologies such as inflammation and cancer. Vaccinia H1-related protein (VHR; DUSP3) is a dual-specificity phosphatase important in controlling MAP kinase activity during cell cycle. the active-site motif contains a cysteine that acts as a nucleophile during catalysis. We used VHR to investigate the effect of oxidation in vitro on phosphatase activity, with the aim of determining how the profile of site-specific modification related to catalytic activity. Recombinant human VHR was expressed in E. coli and purified using a GST-tag. Protein was subjected to oxidation with various concentrations of SIN-1 or tetranitromethane (TNM) as nitrating agents, or HOCl. the activity was assayed using either 3-O-methylfluorescein phosphate with fluorescence detection or PIP3 by phosphate release with malachite green. the sites of oxidation were mapped using HPLC coupled to tandem mass spectrometry on an ABSciex 5600TripleTOF following in-gel digestion. More than 25 different concentration-dependent oxidative modifications to the protein were detected, including oxidations of methionine, cysteine, histidine, lysine, proline and tyrosine, and the % oxidized peptide (versus unmodified peptide) was determined from the extracted ion chromatograms. Unsurprisingly, methionine residues were very susceptible to oxidation, but there was a significant different in the extent of their oxidation. Similarly, tyrosine residues varied greatly in their modifications: Y85 and Y138 were readily nitrated, whereas Y38, Y78 and Y101 showed little modification. Y138 must be phosphorylated for MAPK phosphatase activity, so this susceptibility impacts on signalling pathways. Di- and tri- oxidations of cysteine residues were observed, but did not correlate directly with loss of activity. Overall, the catalytic activity did not correlate with redox state of any individual residue, but the total oxidative load correlated with treatment concentration and activity. This study provides the first comprehensive analysis of oxidation modifications of VHR, and demonstrates both heterogenous oxidant effects and differential residue susceptibility in a signalling phosphatase.

Detection and quantification of protein oxidation in sarcopenic models:a mass spectrometry study

Oxidised biomolecules in aged tissue could potentially be used as biomarkers for age-related diseases; however, it is still unclear whether they causatively contribute to ageing or are consequences of the ageing process. To assess the potential of using protein oxidation as markers of ageing, mass spectrometry (MS) was employed for the identification and quantification of oxidative modifications in obese (ob/ob) mice. Lean muscle mass and strength is reduced in obesity, representing a sarcopenic model in which the levels of oxidation can be evaluated for different muscular systems including calcium homeostasis, metabolism and contractility. Several oxidised residues were identified by tandem MS (MS/MS) in both muscle homogenate and isolated sarcoplasmic reticulum (SR), an organelle that regulates intracellular calcium levels in muscle. These modifications include oxidation of methionine, cysteine, tyrosine, and tryptophan in several proteins such as sarcoplasmic reticulum calcium ATPase (SERCA), glycogen phosphorylase, and myosin. Once modifications had been identified, multiple reaction monitoring MS (MRM) was used to quantify the percentage modification of oxidised residues within the samples. Preliminary data suggests proteins in ob/ob mice are more oxidised than the controls. For example SERCA, which constitutes 60-70% of the SR, had approximately a 2-fold increase in cysteine trioxidation of Cys561 in the obese model when compared to the control. Other obese muscle proteins have also shown a similar increase in oxidation for various residues. Further analysis with complex protein mixtures will determine the potential diagnostic use of MRM experiments for analysing protein oxidation in small biological samples such as muscle needle biopsies.

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.

The redoxomics of PTEN: walking a fine line between damage and signaling:mass spectrometry-based approaches to study the effect of oxidation on PTEN function, structure, and protein-protein interactions

The research described in this PhD thesis focuses on proteomics approaches to study the effect of oxidation on the modification status and protein-protein interactions of PTEN, a redox-sensitive phosphatase involved in a number of cellular processes including metabolism, apoptosis, cell proliferation, and survival. While direct evidence of a redox regulation of PTEN and its downstream signaling has been reported, the effect of cellular oxidative stress or direct PTEN oxidation on PTEN structure and interactome is still poorly defined. In a first study, GST-tagged PTEN was directly oxidized over a range of hypochlorous acid (HOCl) concentration, assayed for phosphatase activity, and oxidative post-translational modifications (oxPTMs) were quantified using LC-MS/MS-based label-free methods. In a second study, GSTtagged PTEN was prepared in a reduced and reversibly H2O2-oxidized form, immobilized on a resin support and incubated with HCT116 cell lysate to capture PTEN interacting proteins, which were analyzed by LC-MS/MS and comparatively quantified using label-free methods. In parallel experiments, HCT116 cells transfected with a GFP-tagged PTEN were treated with H2O2 and PTENinteracting proteins immunoprecipitated using standard methods. Several high abundance HOCl-induced oxPTMs were mapped, including those taking place at amino acids known to be important for PTEN phosphatase activity and protein-protein interactions, such as Met35, Tyr155, Tyr240 and Tyr315. A PTEN redox interactome was also characterized, which identified a number of PTEN-interacting proteins that vary with the reversible inactivation of PTEN caused by H2O2 oxidation. These included new PTEN interactors as well as the redox proteins peroxiredoxin-1 (Prdx1) and thioredoxin (Trx), which are known to be involved in the recycling of PTEN active site following H2O2-induced reversible inactivation. The results suggest that the oxidative modification of PTEN causes functional alterations in PTEN structure and interactome, with fundamental implications for the PTEN signaling role in many cellular processes, such as those involved in the pathophysiology of disease and ageing.

Immunocytes of the Atlantic bottlenose dolphin (Tursiops truncatus) and West Indian manatee (Trichechus manatus latirostris): Morphologic characterizations and correlations between healthy and disease states under free-ranging and captive conditions

Interest in the health of marine mammals has increased due, in part, to the attention given to human impact on the marine environment. Recent mass strandings of the Atlantic bottlenose dolphin (Tursiops truncatus) and rising mortalities of the endangered Florida manatee (Trichechus manatus latirostris) have raised questions on the extent to which pollution, infectious disease, "stress," and captivity influence the immune system of these animals. This study has provided the first in-depth characterization of immunocytes in the peripheral blood of dolphins (n = 190) and manatees (n = 56). Immunocyte morphology and baseline values were determined in clinically normal animals under free-ranging, stranded and captive living conditions as well as by age and sex. Additionally, immunocyte population dynamics were characterized in sick animals. This was accomplished with traditional cytochemical techniques and new lymphocyte phenotyping methodology which was validated in this study. Traditional cytochemical techniques demonstrated that blood immunocyte morphology and cell numbers are similar to terrestrial mammals with some notable exceptions. The manatee heterophilic granulocyte is a morphologically unique cell and probably functions similarly to the typical mammalian neutrophil. Eosinophils were rarely found in manatees but were uncommonly high in healthy and sick dolphins. Basophils were not identified. Manatees had higher total lymphocyte numbers compared to dolphins and most terrestrial mammals. Lymphocyte subsets identified in healthy animals included T$\rm\sb{h}$, T$\rm\sb{c/s}$, B and NK cells. Dolphin and manatee T and B cell values were higher than those reported in man and most terrestrial mammals. The manatee has extraordinarily high absolute numbers of circulating T$\rm\sb{h}$ cells which suggests an enhanced immunological response capability. With few exceptions, immunocyte types and absolute numbers were not significantly different between free-ranging, stranded and captive categories or between sex and age categories. The evaluation of immunocyte dynamics in various disease states demonstrated a wide variation in cellular responses which provided new insights into innate, humoral and cell-mediated immunity in these species. Additionally, this study demonstrated that lymphocyte phenotyping has diagnostic significance and could be developed into a potential indicator of immunocompetence in both free-ranging and captive dolphin and manatee populations.

Low-fat food consumption by people with diabetes decreases fat saturated fat, and cholesterol intake

This study investigated the effect of providing free-access to several fat-modified foods on dietary energy and fat intake in free-living individuals with and without diabetes mellitus. Five low/no-fat products or their regular-fat versions were provided to volunteers to take home and use for 3 days. Energy and nutrient intakes of all foods consumed were determined through a weighed food diary and by weighing the food provided before and after consumption. Fifteen individuals with diabetes and 15 case-matched controls without diabetes participated in the study. Individuals with diabetes and controls responded similarly to the fat-modified foods. In both groups there was a significant reduction in the percent of kcals and grams of fat consumed during the low-fat condition compared to the regular-fat condition (p

Immunocytes of the Atlantic bottlenose dolphin (Tursiops truncatus) and West Indian manatee (Trichechus manatus latirostris) : morphologic characterizations and correlations between healthy and disease states under free-ranging and captive conditions

Interest in the health of marine mammals has increased due, in part, to the attention given to human impact on the marine environment. Recent mass strandings of the Atlantic bottlenose dolphin (Tursiops truncatus) and rising mortalities of the endangered Florida manatee (Trichechus manatus latirostris) have raised questions on the extent to which pollution, infectious disease, "stress," and captivity influence the immune system of these animals. This study has provided the first in-depth characterization of immunocytes in the peripheral blood of dolphins (n=180) and manatees (n=56). Immunocyte morphology and baseline values were determined in clinically normal animals under free-ranging, stranded and captive living conditions as well as by age and sex. Additionally, immuocyte population dynamics were characterized in sick animals. This was accomplished with traditional cytochemical techniques and new lymphocyte phenotyping methodology which was validated in this study. Traditional cytochemical techniques demonstrated that blood immunocyte morphology and cell numbers are similar to terrestrial mammals with some notable exceptions. The manatee heterophilic granulocyte is a morphologically unique cell and probably functions similarly to the typical mammalian neutrophil. Eosinophils were rarely found in manatees but were uncommonly high in healthy and sick dolphins. Basophils were not identified. Manatees had higher total lymphocyte numbers compared to dolphins and most terrestrial mammals. Lymphocyte subsets identified in healthy animals included Th, Tes, B and NK cells. Dolphin and manatee T and B cell values were higher than those reported in man and most terrestrial mammals. The manatee has extraordinarily high absolute numbers of circulating Th cells which suggests an enhanced immunological response capability. With few exceptions, immunocyte types and absolute numbers were not significantly different between free-ranging, stranded and captive categories or between sex and age categories. The evaluation of immunocyte dynamics in various disease states demonstrated a wide variation in cellular responses which provided new insights into innate, humoral and cell-mediated immunity in these species. Additionally, this study demonstrated that lymphocyte phenotyping has diagnostic significance and could be developed into a potential indicator of immunocompetence in both free-ranging and captive dolphin and manatee populations.

Supercapattery based on binder-free Co3 (PO4)2·8H2O multilayer nano/microflakes on nickel foam

A binder-free cobalt phosphate hydrate (Co3(PO4)2·8H2O) multilayer nano/microflake structure is synthesized on nickel foam (NF) via a facile hydrothermal process. Four different concentrations (2.5, 5, 10, and 20 mM) of Co2+ and PO4–3 were used to obtain different mass loading of cobalt phosphate on the nickel foam. The Co3(PO4)2·8H2O modified NF electrode (2.5 mM) shows a maximum specific capacity of 868.3 C g–1 (capacitance of 1578.7 F g–1) at a current density of 5 mA cm–2 and remains as high as 566.3 C g–1 (1029.5 F g–1) at 50 mA cm–2 in 1 M NaOH. A supercapattery assembled using Co3(PO4)2·8H2O/NF as the positive electrode and activated carbon/NF as the negative electrode delivers a gravimetric capacitance of 111.2 F g–1 (volumetric capacitance of 4.44 F cm–3). Furthermore, the device offers a high specific energy of 29.29 Wh kg–1 (energy density of 1.17 mWh cm–3) and a specific power of 4687 W kg–1 (power density of 187.5 mW cm–3).

Age, body mass, mass change and dispersal distance of suckling and weaned grey seal (Halichoerus grypus) pups

Grey seal, Halichoerus grypus, pups in the breeding colony at Froan, Norway, have a bimodal pattern of early aquatic behaviour. About 40% of the pups spend their time ashore to save energy, which can be allocated to growth or deposition of energy-rich adipose tissue. The other 60% of the pups enter the sea during suckling and the early postweaning period, and disperse to other locations within the breeding colony. Pups may swim distances up to 12 km. Neonatal aquatic dispersal behaviour may lead to increased energy expenditure for thermoregulation and swimming, and thus lead to a low rate of body mass gain during suckling and a high rate of body mass loss after weaning. Thus, we examined relationships between natal aquatic dispersal behaviour and change in body mass (DeltaBM) in suckling and weaned pups. Suckling pups that had dispersed >2000 m had a significantly lower DBM than suckling pups that dispersed <2000 m or that did not disperse. In weaned pups, there were no effects of aquatic dispersal behaviour on DBM. We suggest that the bimodal natal aquatic dispersal behaviour in grey seals at the study site reflects two different strategies for postweaning survival: to stay ashore and get fat, or to take a swim and acquire diving and feeding skills.