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.

Insulin sensitivity and secretion in obese Type 2 diabetic women after various bariatric operations

Objective: To compare the effects of biliopancreatic diversion (BPD) and laparoscopic gastric banding (LAGB) on insulin sensitivity and secretion with the effects of laparoscopic gastric plication (P). Methods: A total of 52 obese women (age 30-66 years) suffering from type 2 diabetes mellitus (T2DM) were prospectively recruited into three study groups: 16 BPD; 16 LAGB, and 20 P. Euglycemic clamps and mixed meal tolerance tests were performed before, at 1 month and at 6 months after bariatric surgery. Beta cell function derived from the meal test parameters was evaluated using mathematical modeling. Results: Glucose disposal per kilogram of fat free mass (a marker of peripheral insulin sensitivity) increased significantly in all groups, especially after 1 month. Basal insulin secretion decreased significantly after all three types of operations, with the most marked decrease after BPD compared with P and LAGB. Total insulin secretion decreased significantly only following the BPD. Beta cell glucose sensitivity did not change significantly post-surgery in any of the study groups. Conclusion: We documented similar improvement in insulin sensitivity in obese T2DM women after all three study operations during the 6-month postoperative follow-up. Notably, only BPD led to decreased demand on beta cells (decreased integrated insulin secretion), but without increasing the beta cell glucose sensitivity.

Second messenger pathways in the action of cachectic factors

Cachexia in cancer is characterised by progressive depletion of both adipose tissue stores and skeletal muscle mass. Two catabolic factors produced by cachexia-inducing tumours have the potential for inducing these changes in body composition: (i) proteolysis-inducing factor (PIF) which acts on skeletal muscle to induce both protein degradation and inhibit protein synthesis, (ii) lipid-mobilising factor (LMF), which has been shown to directly induce lipolysis in isolated epididymal murine white adipocytes. Administration of lipid-mobilising factor (LMF) to mice produced a specific reduction in carcass lipid with a tendency to increase non-fat carcass mass. Treatment of murine myoblasts, myotubes and tumour cells with tumour-produced LMF, caused concentration dependent stimulation of protein synthesis, within a 24hr period. It produced an increase in intracellular cyclic AMP levels, which was linearly related to the increase in protein synthesis. The observed effect was attenuated by pretreating cells with the adenylate cyclase inhibitor, MDL12330A and was additive with stimulation produced by forskolin. Both propranolol and a specific 3 adrenergic antagonist SR59230A, significantly reduced the stimulation of protein synthesis induced by LMF. LMF also affected protein degradation in vitro, as demonstrated by a reduction in proteasome activity, a key component of the ubiquitin-dependent proteolytic pathway. These effects were opposite to those produced by PIF which caused both a decrease in the rate of protein synthesis and an elevation on protein breakdown when incubated in vitro.Incubation of LMF with a fat cell line produced alterations in the levels of guanine-nucleotide binding proteins (G proteins). This was also evident in adipocyte plasma membranes isolated from mice bearing the tumour model of cachexia, MAC16 adenocarcinoma and from patients with cancer cachexia. Progression through the cachectic state induced an upregulation of stimulatory G proteins paralleled with a downregulation of inhibitory G proteins. These changes would contribute to the increased lipid mobilisation seen in cancer cachexia.

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.

Identification of oxidized phospholipids by electrospray ionization mass spectrometry and LC-MS using a QQLIT instrument

Phospholipids are complex and varied biomolecules that are susceptible to lipid peroxidation after attack by free radicals or electrophilic oxidants and can yield a large number of different oxidation products. There are many available methods for detecting phospholipid oxidation products, but also various limitations and problems. Electrospray ionization mass spectrometry allows the simultaneous but specific analysis of multiple species with good sensitivity and has a further advantage that it can be coupled to liquid chromatography for separation of oxidation products. Here, we explain the principles of oxidized phospholipid analysis by electrospray mass spectrometry and describe fragmentation routines for surveying the structural properties of the analytes, in particular precursor ion and neutral loss scanning. These allow targeted detection of phospholipid headgroups and identification of phospholipids containing hydroperoxides and chlorine, as well as the detection of some individual oxidation products by their specific fragmentation patterns. We describe instrument protocols for carrying out these survey routines on a QTrap5500 mass spectrometer and also for interfacing with reverse-phase liquid chromatography. The article highlights critical aspects of the analysis as well as some limitations of the methodology.

A solvent-free matrix application method for matrix-assisted laser desorption/ionization imaging of small molecules

Matrix application continues to be a critical step in sample preparation for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI). Imaging of small molecules such as drugs and metabolites is particularly problematic because the commonly used washing steps to remove salts are usually omitted as they may also remove the analyte, and analyte spreading is more likely with conventional wet matrix application methods. We have developed a method which uses the application of matrix as a dry, finely divided powder, here referred to as dry matrix application, for the imaging of drug compounds. This appears to offer a complementary method to wet matrix application for the MALDI-MSI of small molecules, with the alternative matrix application techniques producing different ion profiles, and allows the visualization of compounds not observed using wet matrix application methods. We demonstrate its value in imaging clozapine from rat kidney and 4-bromophenyl-1,4-diazabicyclo(3.2.2)nonane-4-carboxylic acid from rat brain. In addition, exposure of the dry matrix coated sample to a saturated moist atmosphere appears to enhance the visualization of a different set of molecules.

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.

Octane requirement increase arising from the use of lead free fuel

Lead in petrol has been identified as a health hazard and attempts are being made to create a lead-free atmosphere. Through an intensive study a review is made of the various options available to the automobile and petroleum industry. The economic and atmospheric penalties coupled with automobile fuel consumption trends are calculated and presented in both graphical and tabulated form. Experimental measurements of carbon monoxide and hydrocarbon emissions are also presented for certain selected fuels. Reduction in CO and HC's with the employment of a three-way catalyst is also discussed. All tests were carried out on a Fiat 127A engine at wide open throttle and standard timing setting. A Froude dynamometer was used to vary engine speed. With the introduction of lead-free petrol, interest in combustion chamber deposits in spark ignition engines has ben renewed. These deposits cause octane requirement increase or rise in engine knock and decreased volumetric efficiency. The detrimental effect of the deposits has been attributed to the physical volume of the deposit and to changes in heat transfer. This study attempts to assess why leaded deposits, though often greater in mass and volume, yield relatively lower ORI when compared to lead-free deposits under identical operating conditions. This has been carried out by identifying the differences in the physical nature of the deposit and then through measurement of the thermal conductivity and permeability of the deposits. The measured thermal conductivity results are later used in a mathematical model to determine heat transfer rates and temperature variation across the engine wall and deposit. For the model, the walls of the combustion cylinder and top are assumed to be free of engine deposit, the major deposit being on the piston head. Seven different heat transfer equations are formulated describing heat flow at each part of the four stroke cycle, and the variation of cylinder wall area exposed to gas mixture is accounted for. The heat transfer equations are solved using numerical methods and temperature variations across the wall identified. Though the calculations have been carried out for one particular moment in the cycle, similar calculations are possible for every degree of the crank angle, and thus further information regarding location of maximum temperatures at every degree of the crank angle may also be determined. In conclusion, thermal conductivity values of leaded and lead-free deposits have been found. The fundamental concepts of a mathematical model with great potential have been formulated and it is hoped that with future work it may be used in a simulation for different engine construction materials and motor fuels, leading to better design of future prototype engines.

A study of the drying of single droplets in free-flight

The literature relating to evaporation from single droplets of pure liquids and the drying of solution and slurry droplets, and of droplet sprays has been reviewed. The heat and mass transfer rates for individual droplets suspended in free-flight, were investigated using a specially-designed vertical wind tunnel, to simulate conditions in a spray drier. The technique represented a unique alternative method for investigating evaporation from unrestrained single droplets with variable residence times. The experiments covered droplets of pure liquid allowbreak (water, isopropanol) allowbreak and of significantly different solutions (sucrose, potassium sulphate) over a range of temperatures of 37oC to 97oC, initial concentrations of 5 to 40wt/wt% , and initial drop sizes of 2.8 to 4.6mm. Drop behaviour was recorded photographically and dried particles were examined by Scanning Electron Microscopy. Correlations were developed for mass transfer coefficients for pure water droplets in free-flight; (i) experiencing oscillations, rotation and deformation, Sh = -105 + 3.9 [Ta - Td/Tamb]0.18Re0.5Sc033 for Re approx. > 1380 (ii) when these movements had ceased or diminished, Sh = 2.0 + 0.71 [Ta - Td/Tamb]0.18Re0.5Sc033 for Re approx. < 1060. Data for isopropanol drops were correlated resonably well by these equations. The heat transfer data showed a similar transition range. The drying rate curves for drops of sucrose and potassium sulphate solution exhibited three distinct stages; an initial increase in the drying rate as drop temperature reduced to the wet-bulb temperature, a short constant-rate period and a falling-rate period characterised by formation of a crust which controlled the mass transfer rate. Due to drop perturbation the rates in the high Re number region were up to 5 times greater than predicted from theory for spherical droplets. In the case of sucrose solution a `skin' formed over the drop surface prior to crust formation. This provided an additional resistance to mass transfer and resulted in extended drying times and a smooth crust of low porosity. The relevance of the results to practical spray drying operations is discussed.

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.

Analysis of oxidized and chlorinated lipids by mass spectrometry and relevance to signalling

Oxidized and chlorinated phospholipids are generated under inflammatory conditions and are increasingly understood to play important roles in diseases involving oxidative stress. MS is a sensitive and informative technique for monitoring phospholipid oxidation that can provide structural information and simultaneously detect a wide variety of oxidation products, including chain-shortened and -chlorinated phospholipids. MSn technologies involve fragmentation of the compounds to yield diagnostic fragment ions and thus assist in identification. Advanced methods such as neutral loss and precursor ion scanning can facilitate the analysis of specific oxidation products in complex biological samples. This is essential for determining the contributions of different phospholipid oxidation products in disease. While many pro-inflammatory signalling effects of oxPLs (oxidized phospholipids) have been reported, it has more recently become clear that they can also have anti-inflammatory effects in conditions such as infection and endotoxaemia. In contrast with free radical-generated oxPLs, the signalling effects of chlorinated lipids are much less well understood, but they appear to demonstrate mainly pro-inflammatory effects. Specific analysis of oxidized and chlorinated lipids and the determination of their molecular effects are crucial to understanding their role in disease pathology.

An FTO variant is associated with Type 2 diabetes in South Asian populations after accounting for body mass index and waist circumference

Aims - A common variant, rs9939609, in the FTO (fat mass and obesity) gene is associated with adiposity in Europeans, explaining its relationship with diabetes. However, data are inconsistent in South Asians. Our aim was to investigate the association of the FTO rs9939609 variant with obesity, obesity-related traits and Type 2 diabetes in South Asian individuals, and to use meta-analyses to attempt to clarify to what extent BMI influences the association of FTO variants with diabetes in South Asians. Methods - We analysed rs9939609 in two studies of Pakistani individuals: 1666 adults aged = 40 years from the Karachi population-based Control of Blood Pressure and Risk Attenuation (COBRA) study and 2745 individuals of Punjabi ancestry who were part of a Type 2 diabetes case–control study (UK Asian Diabetes Study/Diabetes Genetics in Pakistan; UKADS/DGP). The main outcomes were BMI, waist circumference and diabetes. Regression analyses were performed to determine associations between FTO alleles and outcomes. Summary estimates were combined in a meta-analysis of 8091 South Asian individuals (3919 patients with Type 2 diabetes and 4172 control subjects), including those from two previous studies. Results - In the 4411 Pakistani individuals from this study, the age-, sex- and diabetes-adjusted association of FTO variant rs9939609 with BMI was 0.45 (95% CI 0.24–0.67) kg/m2 per A-allele (P = 3.0 × 10-5) and with waist circumference was 0.88 (95% CI 0.36–1.41) cm per A-allele (P = 0.001). The A-allele (30% frequency) was also significantly associated with Type 2 diabetes [per A-allele odds ratio (95% CI) 1.18 (1.07–1.30); P = 0.0009]. A meta-analysis of four South Asian studies with 8091 subjects showed that the FTO A-allele predisposes to Type 2 diabetes [1.22 (95% CI 1.14–1.31); P = 1.07 × 10-8] even after adjusting for BMI [1.18 (95% CI 1.10–1.27); P = 1.02 × 10-5] or waist circumference [1.18 (95% CI 1.10–1.27); P = 3.97 × 10-5]. Conclusions - The strong association between FTO genotype and BMI and waist circumference in South Asians is similar to that observed in Europeans. In contrast, the strong association of FTO genotype with diabetes is only partly accounted for by BMI.

Protein oxidative modifications:new mass spectrometry approaches to detection in biological samples

In inflammatory diseases, release of oxidants leads to oxidative damage to proteins. The precise nature of oxidative damage to individual proteins depends on the oxidant involved. Chlorination and nitration are markers of modification by the myeloperoxidase-H2O2-Cl- system and nitric oxide-derived oxidants, respectively. Although these modifications can be detected by western blotting, currently no reliable method exists to identify the specific sites damage to individual proteins in complex mixtures such as clinical samples. We are developing novel LCMS2 and precursor ion scanning methods to address this. LC-MS2 allows separation of peptides and detection of mass changes in oxidized residues on fragmentation of the peptides. We have identified indicative fragment ions for chlorotyrosine, nitrotyrosine, hydroxytyrosine and hydroxytryptophan. A nano-LC/MS3 method involving the dissociation of immonium ions to give specific fragments for the oxidized residues has been developed to overcome the problem of false positives from ions isobaric to these immonium ions that exist in unmodified peptides. The approach has proved able to identify precise protein modifications in individual proteins and mixtures of proteins. An alternative methodology involves multiple reaction monitoring for precursors and fragment ions are specific to oxidized and chlorinated proteins, and this has been tested with human serum albumin. Our ultimate aim is to apply this methodology to the detection of oxidative post-translational modifications in clinical samples for disease diagnosis, monitoring the outcomes of therapy, and improved understanding of disease biochemistry.

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.