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.

Study of thermal spin crossover in [Fe(II)(isoxazole)6](BF4)2 with Mössbauer spectroscopy

57Fe Mössbauer spectroscopy of the mononuclear [Fe(II)(isoxazole)6](BF4) 2compound has been studied to reveal the thermal spin crossover of Fe(II) between low-spin (S = 0) and high-spin (S = 2) states. A temperature-dependent spin transition curve has been constructed with the least-square fitted data obtained from the Mössbauer spectra measured at various temperatures in the 240-60K range during the cooling and heating cycle. The compound exhibits a temperature-dependent two-step spin transition phenomenon with Tsco (step 1) = 92 and Tsco (step2) = 191K. The compound has three high-spin Fe(II) sites at the highest temperature of study; among them, two have slightly different coordination environments. These two Fe(II) sites are found to undergo a spin transition, while the third Fe(II) site retains the high-spin state over the whole temperature range. Possible reasons for the formation of the two steps in the spin transition curve are discussed. The observations made from the present study are in complete agreement with those envisaged from earlier magnetic and structural studies made on [Fe(II)(isoxazole)6](BF4)2, but highlights the nature of the spin crossover mechanism.

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.

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.

Trace elemental analysis of glass by inductively coupled plasma-mass spectrometry (ICP-MS) and laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS)

This work evaluated the capabilities of inductively coupled plasma mass spectrometry (ICP-MS) for elemental analysis of trace evidence. A method was developed and validated for the analysis of glass by ICP-MS. A database of ∼700 glass samples was analyzed for elemental composition by external calibration with internal standardization (EC) ICP-MS and refractive index (RI). Additional methods were developed during the course of this work using two well-known techniques, isotope dilution (ID) and laser ablation (LA). These methods were then applied to analyze subsets of this database. ICP-MS data from 161 containers, 45 headlamps, and 458 float glasses (among them at least 143 vehicle windows) are presented and summarized. Data from the analysis of ∼190 glass samples collected from a single glass manufacturing facility over a period of 53 months at different intervals, including 97 samples collected in a 24 hour period are presented. Data from the analysis of 125 glass samples representing 36 manufacturing plants in the U.S. are also presented. ^ The three methods used, ICP-MS, ID-ICP-MS and LA-ICP-MS, were shown to be excellent methods for distinguishing between different glass samples. The database provided information about the variability of refractive index and elemental composition in glasses from diverse population types. Using the proposed methods, the database supports the hypothesis that different glass samples have different elemental profiles and a comparison between fragments from the same source results in indistinguishable profiles. ^

Improved sampling and detection of ignitable liquid residues and explosives by mass spectrometry and ion mobility spectrometry

Fire debris evidence is submitted to crime laboratories to determine if an ignitable liquid (IL) accelerant was used to commit arson. An ignitable liquid residue (ILR) may be difficult to analyze due to interferences, complex matrices, degradation, and low concentrations of analytes. Debris from an explosion and pre-detonated explosive compounds are not trivial to detect and identify due to sampling difficulties, complex matrices, and extremely low amounts (nanogram) of material present. The focus of this research is improving the sampling and detection of ILR and explosives through enhanced sensitivity, selectivity, and field portable instrumentation. Solid Phase MicroExtraction (SPME) enhanced the extraction of ILR by two orders of magnitude over conventional activated charcoal strip (ACS) extraction. Gas chromatography tandem mass spectrometry (GC/MS/MS) improved sensitivity of ILR by one order of magnitude and explosives by two orders of magnitude compared to gas chromatography mass spectrometry (GC/MS). Improvements in sensitivity were attributed to enhanced selectivity. An interface joining SPME to ion mobility spectrometry (IMS) has been constructed and evaluated to improve field detection of hidden explosives. The SPME-IMS interface improved the detection of volatile and semi-volatile explosive compounds and successfully adapted the IMS from a particle sampler into a vapor sampler. ^

Elemental analysis of glass and ink by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and laser induced breakdown spectroscopy (LIBS)

The necessity of elemental analysis techniques to solve forensic problems continues to expand as the samples collected from crime scenes grow in complexity. Laser ablation ICP-MS (LA-ICP-MS) has been shown to provide a high degree of discrimination between samples that originate from different sources. In the first part of this research, two laser ablation ICP-MS systems were compared, one using a nanosecond laser and another a femtosecond laser source for the forensic analysis of glass. The results showed that femtosecond LA-ICP-MS did not provide significant improvements in terms of accuracy, precision and discrimination, however femtosecond LA-ICP-MS did provide lower detection limits. In addition, it was determined that even for femtosecond LA-ICP-MS an internal standard should be utilized to obtain accurate analytical results for glass analyses. In the second part, a method using laser induced breakdown spectroscopy (LIBS) for the forensic analysis of glass was shown to provide excellent discrimination for a glass set consisting of 41 automotive fragments. The discrimination power was compared to two of the leading elemental analysis techniques, μXRF and LA-ICP-MS, and the results were similar; all methods generated >99% discrimination and the pairs found indistinguishable were similar. An extensive data analysis approach for LIBS glass analyses was developed to minimize Type I and II errors en route to a recommendation of 10 ratios to be used for glass comparisons. Finally, a LA-ICP-MS method for the qualitative analysis and discrimination of gel ink sources was developed and tested for a set of ink samples. In the first discrimination study, qualitative analysis was used to obtain 95.6% discrimination for a blind study consisting of 45 black gel ink samples provided by the United States Secret Service. A 0.4% false exclusion (Type I) error rate and a 3.9% false inclusion (Type II) error rate was obtained for this discrimination study. In the second discrimination study, 99% discrimination power was achieved for a black gel ink pen set consisting of 24 self collected samples. The two pairs found to be indistinguishable came from the same source of origin (the same manufacturer and type of pen purchased in different locations). It was also found that gel ink from the same pen, regardless of the age, was indistinguishable as were gel ink pens (four pens) originating from the same pack.

Improved sampling, pre-concentration, and detection of hidden explosives and illicit drugs by a novel solid phase microextraction geometry coupled to ion mobility spectrometry

The 9/11 Act mandates the inspection of 100% of cargo shipments entering the U.S. by 2012 and 100% inspection of air cargo by March 2010. So far, only 5% of inbound shipping containers are inspected thoroughly while air cargo inspections have fared better at 50%. Government officials have admitted that these milestones cannot be met since the appropriate technology does not exist. This research presents a novel planar solid phase microextraction (PSPME) device with enhanced surface area and capacity for collection of the volatile chemical signatures in air that are emitted from illicit compounds for direct introduction into ion mobility spectrometers (IMS) for detection. These IMS detectors are widely used to detect particles of illicit substances and do not have to be adapted specifically to this technology. For static extractions, PDMS and sol-gel PDMS PSPME devices provide significant increases in sensitivity over conventional fiber SPME. Results show a 50–400 times increase in mass detected of piperonal and a 2–4 times increase for TNT. In a blind study of 6 cases suspected to contain varying amounts of MDMA, PSPME-IMS correctly detected 5 positive cases with no false positives or negatives. One of these cases had minimal amounts of MDMA resulting in a false negative response for fiber SPME-IMS. A La (dihed) phase chemistry has shown an increase in the extraction efficiency of TNT and 2,4-DNT and enhanced retention over time. An alternative PSPME device was also developed for the rapid (seconds) dynamic sampling and preconcentration of large volumes of air for direct thermal desorption into an IMS. This device affords high extraction efficiencies due to strong retention properties under ambient conditions resulting in ppt detection limits when 3.5 L of air are sampled over the course of 10 seconds. Dynamic PSPME was used to sample the headspace over the following: MDMA tablets (12–40 ng detected of piperonal), high explosives (Pentolite) (0.6 ng detected of TNT), and several smokeless powders (26–35 ng of 2,4-DNT and 11–74 ng DPA detected). PSPME-IMS technology is flexible to end-user needs, is low-cost, rapid, sensitive, easy to use, easy to implement, and effective. ^