Investigation of the pharmacokinetics and metabolic fate of Fasiglifam (TAK-875) in male and female rats following oral and intravenous administration.

The metabolism and pharmacokinetics of fasiglifam (TAK-875, 2-[(3S)-6-[[3-[2,6-dimethyl-4-(3-methylsulfonylpropoxy)phenyl]phenyl]methoxy]-2,3-dihydro-1-benzofuran-3-yl]acetic acid), a selective free fatty acid receptor 1 (FFAR1)/GPR40 agonist, were studied following intravenous (5 mg/kg) and oral administration (10 and 50 mg/kg) to male and female Sprague Dawley rats.Following intravenous dosing at 5 mg/kg, peak observed plasma concentrations of 8.8/9.2 µg/ml were seen in male and female rats respectively.Following oral dosing, peak plasma concentrations at 1 h of ca. 12.4/12.9 µg/ml for 10 mg/kg and 76.2/83.7 µg/ml for 50 mg/kg doses were obtained for male and female rats respectively. Drug concentrations then declined in the plasma of both sexes with t1/2's of 12.4 (male) and 11.2 h (female). Oral bioavailability was estimated to be 85-120% in males and females at both dose levels.Urinary excretion was low, but in a significant sex-related difference, female rats eliminated ca. 10-fold more drug-related material by this route.Fasiglifam was the principal drug-related compound in plasma, with 15 metabolites, including the acyl glucuronide, also detected. In addition to previously identified metabolites, a novel biotransformation, that produced a side-chain shortened metabolite via elimination of CH2 from the acetyl side chain was noted with implications for drug toxicity.

High Throughput UHPLC-MS-Based Lipidomics Using Vacuum Jacketed Columns.

Reversed-phase UHPLC-MS is extensively employed for both the profiling of biological fluids and tissues to characterize lipid dysregulation in disease and toxicological studies. With conventional LC-MS systems the chromatographic performance and throughput are limited due to dispersion from the fluidic connections as well as radial and longitudinal thermal gradients in the LC column. In this study vacuum jacketed columns (VJC), positioned at the source of the mass spectrometer, were applied to the lipidomic analysis of plasma extracts. Compared to conventional UHPLC, the VJC-based methods offered greater resolution, faster analysis, and improved peak intensity. For a 5 min VJC analysis, the peak capacity increased by 66%, peak tailing reduced by up to 34%, and the number of lipids detected increased by 30% compared to conventional UHPLC. The narrower peaks, and thus increased resolution, compared to the conventional system resulted in a 2-fold increase in peak intensity as well a significant improvement in MS and MS/MS spectral quality resulting in a 22% increase in the number of lipids identified. When applied to mouse plasma samples, reproducibility of the lipid intensities in the pooled QC ranged from 1.8-12%, with no related drift in tR observed.

High Throughput LC-MS Platform for Large Scale Screening of Bioactive Polar Lipids in Human Plasma and Serum.

Lipids play a key role in many biological processes, and their accurate measurement is critical to unraveling the biology of diseases and human health. A high throughput HILIC-based (LC-MS) method for the semiquantitative screening of over 2000 lipids, based on over 4000 MRM transitions, was devised to produce an accessible and robust lipidomic screen for phospholipids in human plasma/serum. This methodology integrates many of the advantages of global lipid analysis with those of targeted approaches. Having used the method as an initial "wide class" screen, it can then be easily adapted for a more targeted analysis and quantification of key, dysregulated lipids. Robustness was assessed using 1550 continuous injections of plasma extracts onto a single column and via the evaluation of columns from 5 different batches of stationary phase. Initial screens in positive (239 lipids, 431 MRM transitions) and negative electrospray ionization (ESI) mode (232 lipids, 446 MRM transitions) were assessed for reproducibility, sensitivity, and dynamic range using analysis times of 8 min. The total number of lipids monitored using these screening methods was 433 with an overlap of 38 lipids in both modes. A polarity switching method for accurate quantification, using the same LC conditions, was assessed for intra- and interday reproducibility, accuracy, dynamic range, stability, carryover, dilution integrity, and matrix interferences and found to be acceptable. This polarity switching method was then applied to lipids important in the stratification of human prostate cancer samples.

High-Throughput Microbore Ultrahigh-Performance Liquid Chromatography-Ion Mobility-Enabled-Mass Spectrometry-Based Proteomics Methodology for the Exploratory Analysis of Serum Samples from Large Cohort Studies.

The deployment of proteomic analysis in clinical studies represents a significant opportunity to detect and validate biomarkers in translational medicine, improve disease understanding, and provide baseline information on population health. However, comprehensive proteome studies usually employ nanoscale chromatography and often require several hours of analysis/sample. Here, we describe a high-throughput liquid chromatography tandem mass spectrometry (LC/MS/MS) methodology using 1 mm scale chromatography requiring only 15 min/sample, coupled to ion mobility-enabled mass spectrometry. The short run time effected a 6-fold increase in productivity compared with nanoscale LC/MS. The method demonstrated excellent reproducibility with retention time coefficient of variations of less than 0.05% and peak area reproducibility ranging from 5 to 15%. The 1 mm system produced similar chromatographic peak capacity values to the nanoscale miniaturized system, detecting 90% of the Escherichia coli proteins identified by the 75 µm LC/MS system (albeit based on only 75% of the peptides found by the latter). Application to the analysis of serum samples from a human prostate cancer study group resulted in the identification of a total of 533 proteins revealing the differential expression of proteins linked to patients receiving hormone-radiotherapy or undergoing surgery.

High-Throughput UHPLC/MS/MS-Based Metabolic Profiling Using a Vacuum Jacketed Column.

In UHPLC, frictional heating from the eluent flowing through the column at pressures of ca. 10-15 Kpsi causes radial diffusion via temperature differences between the center of the column and its walls. Longitudinal dispersion also occurs due to temperature gradients between the inlet and outlet. These effects cause band broadening but can be mitigated via a combination of vacuum jacketed stainless steel tubing, reduced column end nut mass, and a constant temperature in the column from heating the inlet fitting. Here, vacuum jacketed column (VJC) technology, employing a novel column housing located on the source of the mass spectrometer and minimized tubing from the column outlet to the electrospray probe, was applied to profiling metabolites in urine. For a 75 s reversed-phase gradient separation, the average peak widths for endogenous compounds in urine were 1.2 and 0.6 s for conventional LC/MS and VJC systems, respectively. The peak tailing factor was reduced from 1.25 to 1.13 when using the VJC system compared to conventional UHPLC, and the peak capacity increased from 65 to 120, with a 25% increase in features detected in urine. The increased resolving power of the VJC system reduced co-elution, simplifying MS and MS/MS spectra, providing a more confident metabolite identification. The increased LC performance also gave more intense MS peaks, with a 10-120% increase in response, improving the quality of the MS data and detection limits. Reducing the LC gradient duration to 37 s gave peak widths of ca. 0.4 s and a peak capacity of 84.

Glycerophospholipid and detoxification pathways associated with small for gestation age pathophysiology: discovery metabolomics analysis in the SCOPE cohort.

INTRODUCTION: Small for gestational age (SGA) may be associated with neonatal morbidity and mortality. Our understanding of the molecular pathways implicated is poor. OBJECTIVES: Our aim was to determine the metabolic pathways involved in the pathophysiology of SGA and examine their variation between maternal biofluid samples. METHODS: Plasma (Cork) and urine (Cork, Auckland) samples were collected at 20 weeks' gestation from nulliparous low-risk pregnant women participating in the SCOPE study. Women who delivered an SGA infant (birthweight < 10th percentile) were matched to controls (uncomplicated pregnancies). Metabolomics (urine) and lipidomics (plasma) analyses were performed using ultra performance liquid chromatography-mass spectrometry. Features were ranked based on FDR adjusted p-values from empirical Bayes analysis, and significant features putatively identified. RESULTS: Lipidomics plasma analysis revealed that 22 out of the 33 significantly altered lipids annotated were glycerophospholipids; all were detected in higher levels in SGA. Metabolomic analysis identified reduced expression of metabolites associated with detoxification (D-Glucuronic acid, Estriol-16-glucuronide), nutrient absorption and transport (Sulfolithocholic acid) pathways. CONCLUSIONS: This study suggests higher levels of glycerophospholipids, and lower levels of specific urine metabolites are implicated in the pathophysiology of SGA. Further research is needed to confirm these findings in independent samples.

Rapid determination of the pharmacokinetics and metabolic fate of gefitinib in the mouse using a combination of UPLC/MS/MS, UPLC/QToF/MS, and ion mobility (IM)-enabled UPLC/QToF/MS.

The metabolism and pharmacokinetics of gefitinib (Iressa®, N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholino-propoxy)quinazolin-4-amine), a selective thymidylate kinase inhibitor for the epidermal growth factor receptor (EGFR), was studied after IV and PO administration to male C57BL6 mice at 10 and 50 mg/kg respectively.The pharmacokinetics and metabolism of gefitinib were investigated using a range of rapid UHPLC-MS and UHPLC-IM-HRMS methods, using both reversed-phase (RP) and hydrophilic interaction liquid chromatography (HILIC), to rapidly determine the drugs pharmacokinetics and metabolic fate.Rapid oral absorption resulted in peak plasma concentrations at 1 h of ca. 7 µg/mL, that declined with a half-life of 3.8 h (2.6 h for the IV route), and providing an estimated oral bioavailability of 53%. Gefitinib itself was the major circulating drug-related compound in plasma extracts, with a total of 11 metabolites identified.The urinary profiles determined using both HILIC and RP-UPLC-IM-MS detected gefitinib and 10 metabolites or 15 metabolites respectively including the detection of a number of novel glucuronide conjugates.Despite rapid, sub 5 min, LC profiling methods being employed metabolite coverage was shown to be high and compared well with that of previous studies.

Association between phospholipid metabolism in plasma and spontaneous preterm birth: a discovery lipidomic analysis in the cork pregnancy cohort.

INTRODUCTION: Preterm birth (PTB) is defined as birth occurring before 37 weeks' gestation, affects 5-9% of all pregnancies in developed countries, and is the leading cause of perinatal mortality. Spontaneous preterm birth (sPTB) accounts for 31-50% of all PTB, but the underlying pathophysiology is poorly understood. OBJECTIVE: This study aimed to decipher the lipidomics pathways involved in pathophysiology of sPTB. METHODS: Blood samples were taken from SCreening fOr Pregnancy Endpoints (SCOPE), an international study that recruited 5628 nulliparous women, with a singleton low-risk pregnancy. Our analysis focused on plasma from SCOPE in Cork. Discovery profiling of the samples was undertaken using liquid chromatography-mass spectrometry Lipidomics, and features significantly altered between sPTB (n = 16) and Control (n = 32) groups were identified using empirical Bayes testing, adjusting for multiple comparisons. RESULTS: Twenty-six lipids showed lower levels in plasma of sPTB compared to controls (adjusted p < 0.05), including 20 glycerophospholipids (12 phosphatidylcholines, 7 phosphatidylethanolamines, 1 phosphatidylinositol) and 6 sphingolipids (2 ceramides and 4 sphingomyelines). In addition, a diaglyceride, DG (34:4), was detected in higher levels in sPTB compared to controls. CONCLUSIONS: We report reduced levels of plasma phospholipids in sPTB. Phospholipid integrity is linked to biological membrane stability and inflammation, while storage and breakdown of lipids have previously been implicated in pregnancy complications. The contribution of phospholipids to sPTB as a cause or effect is still unclear; however, our results of differential plasma phospholipid expression represent another step in advancing our understanding of the aetiology of sPTB. Further work is needed to validate these findings in independent pregnancy cohorts.

The influence of hypoxia on the prostate cancer proteome.

Prostate cancer accounts for around 15% of male deaths in Western Europe and is the second leading cause of cancer death in men after lung cancer. Mounting evidence suggests that prostate cancer deposits exist within a hypoxic environment and this contributes to radio-resistance thus hampering one of the major therapies for this cancer. Recent reports have shown that nitric oxide (NO) donating non-steroidal anti-inflammatory drugs (NSAIDs) reduced tumour hypoxia as well as maintaining a radio-sensitising/therapeutic effect on prostate cancer cells. The aim of this study was to evaluate the impact of hypoxia on the proteome of the prostate and to establish whether NO-NSAID treatment reverted the protein profiles back to their normoxic status. To this end an established hormone insensitive prostate cancer cell line, PC-3, was cultured under hypoxic and normoxic conditions before and following exposure to NO-NSAID in combination with selected other common prostate cancer treatment types. The extracted proteins were analysed by ion mobility-assisted data independent acquisition mass spectrometry (MS), combined with multivariate statistical analyses, to measure hypoxia-induced alterations in the proteome of these cells. The analyses demonstrated that under hypoxic conditions there were well-defined, significantly regulated/differentially expressed proteins primarily involved with structural and binding processes including, for example, TUBB4A, CIRP and PLOD1. Additionally, the exposure of hypoxic cells to NSAID and NO-NSAID agents, resulted in some of these proteins being differentially expressed; for example, both PCNA and HNRNPA1L were down-regulated, corresponding with disruption in the nucleocytoplasmic shuttling process.

A multiple reaction monitoring method for determining peanut (Arachis hypogea) allergens in serum using quadrupole and time-of-flight mass spectrometry.

Peanut is a major cause of severe IgE-mediated food allergic reactions, which can be exacerbated by factors, such as exercise, that may increase allergen uptake into the circulation. Enzyme-linked immunosorbent assays (ELISAs) have been used to determine allergen uptake into serum, but there are concerns over their specificity and a confirmatory method is required. Mass spectrometry (MS) methods have the potential to provide rigorous alternatives for allergen determination. A suite of peptide targets representing the major clinically relevant peanut allergens previously applied in food analysis were used to develop a targeted multiple reaction monitoring (MRM) method for determination of peanut in serum. Depletion of serum using affinity chromatography was found to be essential to allow detection of the peptide targets. A comparison of triple quadrupole and Q-TOF methods showed that one Ara h 2 peptide was only detected by the Q-TOF, the other peptide targets giving similar assay sensitivities with both MS platforms, although transitions for all the peptides were detected more consistently with the Q-TOF. The Q-TOF MRM assay detected peanut from spiked serum more effectively than the triple quadrupole assay, with Ara h 3 being detected down to 3 mg total peanut protein/L of serum, comparable with an Ara h 3-specific ELISA. The poor recoveries observed for both methods are likely due to loss of peanut immune complexes during the serum depletion process. Nevertheless, the Q-TOF MRM method has much promise to confirm the uptake of peanut proteins in serum samples providing immune complexes can be disrupted effectively prior to depletion. Graphical abstract.

Application of a Novel Mass Spectral Data Acquisition Approach to Lipidomic Analysis of Liver Extracts from Sitaxentan-Treated Liver-Humanized PXB Mice.

The application of a data-independent acquisition (DIA) method ("SONAR") that employs a rapidly scanning quadrupole is described for the lipidomic analysis of complex biological extracts. Using this approach, the MS acquisition window can be varied between 1 and 25 Da, enabling the isolation of ions prior to their entering the collision cell. By rapidly scanning the resolving quadrupole window over a specified mass range, co-eluting precursor ions are transmitted sequentially into the collision cell, where collision energies are cycled between low and elevated levels to induce fragmentation. This method of data generation provides both precursor and fragment ion information at high specificity, allowing for greater accuracy of compound identification, whether using a database, spectral libraries, or comparison to authentic standards. The value of the approach in simplifying and "de-cluttering" the spectra of co-eluting lipids is shown with examples from lipidomic profiles obtained in investigations of the composition of organic extracts of livers obtained from SCID and chimeric liver-humanized mice administered under various experimental conditions.

Development of a rapid profiling method for the analysis of polar analytes in urine using HILIC-MS and ion mobility enabled HILIC-MS.

INTRODUCTION: As large scale metabolic phenotyping is increasingly employed in preclinical studies and in the investigation of human health and disease the current LC-MS/MS profiling methodologies adopted for large sample sets can result in lengthy analysis times, putting strain on available resources. As a result of these pressures rapid methods of untargeted analysis may have value where large numbers of samples require screening. OBJECTIVES: To develop, characterise and evaluate a rapid UHP-HILIC-MS-based method for the analysis of polar metabolites in rat urine and then extend the capabilities of this approach by the addition of IMS to the system. METHODS: A rapid untargeted HILIC LC-MS/MS profiling method for the analysis of small polar molecules has been developed. The 3.3 min separation used a Waters BEH amide (1 mm ID) analytical column on a Waters Synapt G2-Si Q-Tof enabled with ion mobility spectrometry (IMS). The methodology, was applied to the metabolic profiling of a series of rodent urine samples from vehicle-treated control rats and animals administered tienilic acid. The same separation was subsequently linked to IMS and MS to evaluate the benefits that IMS might provide for metabolome characterisation. RESULTS: The rapid HILIC-MS method was successfully applied to rapid analysis of rat urine and found, based on the data generated from the data acquired for the pooled quality control samples analysed at regular intervals throughout the analysis, to be robust. Peak area and retention times for the compounds detected in these samples showed good reproducibility across the batch. When used to profile the urine samples obtained from vehicle-dosed control and those administered tienilic acid the HILIC-MS method detected 3007 mass/retention time features. Analysis of the same samples using HILIC-IMS-MS enabled the detection of 6711 features. Provisional metabolite identification for a number of compounds was performed using the high collision energy MS/MS information compared against the Metlin MS/MS database and, in addition, both calculated and measured CCS values from an experimentally derived CCS database. CONCLUSION: A rapid metabolic profiling method for the analysis of polar metabolites has been developed. The method has the advantages of speed and both reducing sample and solvent consumption compared to conventional profiling methods. The addition of IMS added an additional dimension for feature detection and the identification of metabolites.

Excessive dietary lipid intake provokes an acquired form of lysosomal lipid storage disease in the kidney.

Obesity and dyslipidaemia are features of the metabolic syndrome and risk factors for chronic kidney disease. The cellular mechanisms connecting metabolic syndrome with chronic kidney disease onset and progression remain largely unclear. We show that proximal tubular epithelium is a target site for lipid deposition upon overnutrition with a cholesterol-rich Western-type diet. Affected proximal tubule epithelial cells displayed giant vacuoles of lysosomal or autophagosomal origin, harbouring oxidised lipoproteins and concentric membrane layer structures (multilamellar bodies), reminiscent of lysosomal storage diseases. Additionally, lipidomic analysis revealed renal deposition of cholesterol and phospholipids, including lysosomal phospholipids. Proteomic profiles of renal multilamellar bodies were distinct from those of epidermis or lung multilamellar bodies and of cytoplasmic lipid droplets. Tubular multilamellar bodies were observed in kidney biopsies of obese hypercholesterolaemic patients, and the concentration of the phospholipidosis marker di-docosahexaenoyl (22:6)-bis(monoacylglycerol) phosphate was doubled in urine from individuals with metabolic syndrome and chronic kidney disease. The enrichment of proximal tubule epithelial cells with phospholipids and multilamellar bodies was accompanied by enhanced inflammation, fibrosis, tubular damage markers, and higher urinary electrolyte content. Concomitantly to the intralysosomal lipid storage, a renal transcriptional response was initiated to enhance lysosomal degradation and lipid synthesis. In cultured proximal tubule epithelial cells, inhibition of cholesterol efflux transport or oxysterol treatment induced effects very similar to the in vivo situation, such as multilamellar body and phospholipid amassing, and induction of damage, inflammatory, fibrotic, and lipogenic molecules. The onset of phospholipidosis in proximal tubule epithelial cells is a novel pathological trait in metabolic syndrome-related chronic kidney disease, and emphasises the importance of healthy lysosomes and nutrition for kidney well-being. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Simplifying the Proteome: Analytical Strategies for Improving Peak Capacity.

The diversity of biological samples and dynamic range of analytes being analyzed can prove to be an analytical challenge and is particularly prevalent to proteomic studies. Maximizing the peak capacity of the workflow employed can extend the dynamic range and increase identification rates. The focus of this chapter is to present means of achieving this for various analytical techniques such as liquid chromatography, mass spectrometry and ion mobility. A combination of these methods can be used as part of a data independent acquisition strategy, thereby limiting issues such as chimericy when analyzing regions of extreme analyte density.

Microfluidic Separation Coupled to Mass Spectrometry for Quantification of Peanut Allergens in a Complex Food Matrix.

Peanut is an important food allergen, but it cannot currently be reliably detected and quantified in processed foods at low levels. A level of 3 mg protein/kg is increasingly being used as a reference dose above which precautionary allergen labeling is applied to food products. Two exemplar matrices (chocolate dessert and chocolate bar) were prepared and incurred with 0, 3, 10, or 50 mg/kg peanut protein using a commercially available lightly roasted peanut flour ingredient. After simple buffer extraction employing an acid-labile detergent, multiple reaction monitoring (MRM) experiments were used to assess matrix effects on the detection of a set of seven peptide targets derived from peanut allergens using either conventional or microfluidic chromatographic separation prior to mass spectrometry. Microfluidic separation provided greater sensitivity and increased ionization efficiency at low levels. Individual monitored transitions were detected in consistent ratios across the dilution series, independent of matrix. The peanut protein content of each sample was then determined using ELISA and the optimized MRM method. Although other peptide targets were detected with three transitions at the 50 mg/kg peanut protein level in both matrices, only Arah2(Q6PSU2)147-155 could be quantified reliably and only in the chocolate dessert at 10 mg/kg peanut protein. Recoveries were consistent with ELISA analysis returning around 30-50% of the incurred dose. MS coupled with microfluidic separation shows great promise as a complementary analytical tool for allergen detection and quantification in complex foods using a simple extraction methodology.

Eukaryotic elongation factor 2 kinase upregulates the expression of proteins implicated in cell migration and cancer cell metastasis.

Eukaryotic elongation factor 2 kinase (eEF2K) negatively regulates the elongation phase of mRNA translation and hence protein synthesis. Increasing evidence indicates that eEF2K plays an important role in the survival and migration of cancer cells and in tumor progression. As demonstrated by two-dimensional wound-healing and three-dimensional transwell invasion assays, knocking down or inhibiting eEF2K in cancer cells impairs migration and invasion of cancer cells. Conversely, exogenous expression of eEF2K or knocking down eEF2 (the substrate of eEF2K) accelerates wound healing and invasion. Importantly, using LC-HDMSE analysis, we identify 150 proteins whose expression is decreased and 73 proteins which are increased upon knocking down eEF2K in human lung carcinoma cells. Of interest, 34 downregulated proteins are integrins and other proteins implicated in cell migration, suggesting that inhibiting eEF2K may help prevent cancer cell mobility and metastasis. Interestingly, eEF2K promotes the association of integrin mRNAs with polysomes, providing a mechanism by which eEF2K may enhance their cellular levels. Consistent with this, genetic knock down or pharmacological inhibition of eEF2K reduces the protein expression levels of integrins. Notably, pharmacological or genetic inhibition of eEF2K almost completely blocked tumor growth and effectively prevented the spread of tumor cells in vivo. High levels of eEF2K expression were associated with invasive carcinoma and metastatic tumors. These data provide the evidence that eEF2K is a new potential therapeutic target for preventing tumor metastasis.

Lipid profiling of complex biological mixtures by liquid chromatography/mass spectrometry using a novel scanning quadrupole data-independent acquisition strategy.

RATIONALE: A novel data-independent acquisition method is detailed that incorporates a scanning quadrupole in front of an orthogonal acceleration time-of-flight (TOF) mass analyser. This approach is described and the attributes are compared and contrasted to other DIA approaches. METHODS: Specific application of the method to both targeted and untargeted lipidomic identification strategies is discussed, with data from both shotgun and LC separated lipidomics experiments presented. RESULTS: The benefits of the fast quadrupole scanning technique are highlighted, and include improvements in speed and specificity for complex mixtures providing high quality qualitative and quantitative data. CONCLUSIONS: In particular the high specificity afforded by the scanning quadrupole improves qualitative information for lipid identification.

Quantitative Proteomic Profiling of Peanut Allergens in Food Ingredients Used for Oral Food Challenges.

Profiling allergens in complex food ingredients used in oral food challenges and immunotherapy is crucial for regulatory acceptance. Mass spectrometry based analysis employing data-independent acquisition coupled with ion mobility mass spectrometry-mass spectrometry (DIA-IM-MS) was used to investigate the allergen composition of raw peanuts and roasted peanut flour ingredients used in challenge meals. This comprehensive qualitative and quantitative analysis using label-free approaches identified and quantified 123 unique protein accessions. Semiquantitative analysis indicated that allergens Ara h 1 and Ara h 3 were the most abundant proteins and present in approximately equal amounts and were extracted in reduced amounts from roasted peanut flours. The clinically significant allergens Ara h 2 and 6 were less abundant, but relative quantification was unaffected by roasting. Ara h 5 was undetectable in any peanut sample, while the Bet v 1 homologue Ara h 8 and the lipid transfer protein allergen, Ara h 9, were detected in low abundance. The oleosin allergens, Ara h 10 and 11, were moderately abundant in the raw peanuts but were 100-fold less abundant in the defatted roasted peanut flour than the major allergens Ara h 1, 3, 2, and 6. Certain isoforms of the major allergens dominated the profile. The relative quantitation of the major peanut allergens showed little variation between different batches of roasted peanut flour. These data will support future development of targeted approaches for absolute quantification of peanut allergens which can be applied to both food ingredients used in clinical studies and extracts used for skin testing and to identify trace levels of allergens in foods.

Verification of a Parkinson's disease protein signature in T-lymphocytes by multiple reaction monitoring.

Diagnosis of Parkinson's disease, the second most common neurodegenerative disease, is based on the appearance of motor symptoms. A panel of protein biomarkers in the T-lymphocyte proteome was previously proposed as a Parkinson's disease signature. Here, we designed an LC-MS based method to quantitatively evaluate this protein signature by multiple reaction monitoring (MRM) in T-lymphocytes and peripheral blood mononuclear cells from a new cohort of nine patients with Parkinson's disease and nine unaffected subjects. Patients were classified using the discriminant function obtained from two-dimensional electrophoresis and protein amounts measured by MRM, thus assigning seven controls out of nine as true negatives and nine patients out of nine as true positives. A good discriminant power was obtained by selecting a subset of peptides from the protein signature, with an area under the receiver operating characteristic curve of 0.877. A similar result is achieved by evaluating all peptides of a selected panel of proteins (gelsolin, moesin, septin-6, twinfilin-2, lymphocyte-specific protein 1, vimentin, transaldolase), with an area under the curve of 0.840. Conversely, the signature was not able to classify the enrolled subjects when evaluated in whole mononuclear cells. Overall, this report shows the portability of the proposed method to a large-scale clinical validation study.

Label-free mass spectrometric profiling of urinary proteins and metabolites from paediatric idiopathic nephrotic syndrome.

Idiopathic nephrotic syndrome (INS) is caused by renal diseases that increase the permeability of the glomerular filtration barrier without evidence of a specific systemic cause. The aim of the present work was to reveal inherent molecular features of INS in children using combined urinary proteomics and metabolomics profiling. In this study, label-free mass spectrometric analysis of urinary proteins and small molecule metabolites was carried out in 12 patients with INS versus 12 sex- and age-matched control subjects with normal renal function. Integration and biological interpretation of obtained results were carried out by Ingenuity IPA software. Validation of obtained proteomics data was carried out by Western blot method. Proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD000765. This study indicates for the first time that paediatric INS is associated with up-regulation of afamin, hydroxyphenylacetate and uridine, and concomitant down-regulation in glutamine and phenylalanine levels, and many of these molecular species were previously shown to be involved in oxidative stress. Further studies in larger patient population are underway to investigate the role of oxidative stress in renal injury in paediatric INS.