Urinary Leukotriene E4 and Prostaglandin D2 Metabolites Increase in Adult and Childhood Severe Asthma Characterized by Type 2 Inflammation. A Clinical Observational Study.

Rationale: New approaches are needed to guide personalized treatment of asthma.Objectives: To test if urinary eicosanoid metabolites can direct asthma phenotyping.Methods: Urinary metabolites of prostaglandins (PGs), cysteinyl leukotrienes (CysLTs), and isoprostanes were quantified in the U-BIOPRED (Unbiased Biomarkers for the Prediction of Respiratory Diseases Outcomes) study including 86 adults with mild-to-moderate asthma (MMA), 411 with severe asthma (SA), and 100 healthy control participants. Validation was performed internally in 302 participants with SA followed up after 12-18 months and externally in 95 adolescents with asthma.Measurement and Main Results: Metabolite concentrations in healthy control participants were unrelated to age, body mass index, and sex, except for the PGE2 pathway. Eicosanoid concentrations were generally greater in participants with MMA relative to healthy control participants, with further elevations in participants with SA. However, PGE2 metabolite concentrations were either the same or lower in male nonsmokers with asthma than in healthy control participants. Metabolite concentrations were unchanged in those with asthma who adhered to oral corticosteroid treatment as documented by urinary prednisolone detection, whereas those with SA treated with omalizumab had lower concentrations of LTE4 and the PGD2 metabolite 2,3-dinor-11ß-PGF2α. High concentrations of LTE4 and PGD2 metabolites were associated with lower lung function and increased amounts of exhaled nitric oxide and eosinophil markers in blood, sputum, and urine in U-BIOPRED participants and in adolescents with asthma. These type 2 (T2) asthma associations were reproduced in the follow-up visit of the U-BIOPRED study and were found to be as sensitive to detect T2 inflammation as the established biomarkers.Conclusions: Monitoring of urinary eicosanoids can identify T2 asthma and introduces a new noninvasive approach for molecular phenotyping of adult and adolescent asthma.Clinical trial registered with www.clinicaltrials.gov (NCT01976767).

Impact of first-line cancer treatment on the follicle quality in cryopreserved ovarian samples from girls and young women.

STUDY QUESTION: Does first-line chemotherapy affect the quality of ovarian pre-antral follicles and stromal tissue in a population of young patients? SUMMARY ANSWER: Exposure to first-line chemotherapy significantly impacts follicle viability, size of residual intact follicles, steroid secretion in culture and quality of the stromal compartment. WHAT IS KNOWN ALREADY: First-line chemotherapy is considered to have a low gonadotoxic potential, and as such, does not represent an indication for fertility preservation. Studies investigating the effects of chemotherapy on the quality of ovarian tissue stored for fertility preservation in young patients are limited and the results sometimes contradictory. STUDY DESIGN, SIZE, DURATION: We conducted a retrospective cohort study including young patients referred to three centers (Helsinki, Oslo and Tampere) to perform ovarian tissue cryopreservation for fertility preservation between 2003 and 2018. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 43 patients (age 1-24 years) were included in the study. A total of 25 were exposed to first-line chemotherapy before cryopreservation, whereas 18 patients were not. Density and size of follicles divided by developmental stages, prevalence of atretic follicles, health of the stromal compartment and functionality of the tissue in culture were evaluated and related to age and chemotherapy exposure. Activation of dormant follicles and DNA damage were also assessed. MAIN RESULTS AND THE ROLE OF CHANCE: Patients exposed to first-line chemotherapy showed a significantly higher density of atretic primordial and intermediary follicles than untreated patients. The intact primordial and intermediary follicles were significantly smaller in size in patients exposed to chemotherapy. Production of steroids in culture was also significantly impaired and a higher content of collagen and DNA damage was observed in the stromal compartment of treated patients. Collectively, these observations may indicate reduced quality and developmental capacity of follicles as a consequence of first-line chemotherapy exposure. Neither increased activation of dormant follicles nor elevated levels of DNA damage in oocyte nuclei were found in patients exposed to chemotherapy. LIMITATIONS, REASONS FOR CAUTION: The two groups were not homogeneous in terms of age and the patients were exposed to different treatments, which did not allow us to distinguish the effect of specific agents. The limited material availability did not allow us to perform all the analyses on the entire set of patients. WIDER IMPLICATION OF THE FINDINGS: This study provides for the first time a comprehensive analysis of the effects of first-line chemotherapy on the health, density and functionality of follicles categorized according to the developmental stage in patients under 24 years of age. When exposed to these treatments, patients were considered at low/medium risk of infertility. Our data suggest a profound impact of these relatively safe therapies on ovarian health and encourages further exploration of this effect in follow-up studies in order to optimize fertility preservation for young cancer patients. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the Swedish Childhood Cancer Foundation, the Finnish Cancer Society, the Finnish Pediatric Research Foundation, the Väre Foundation for Pediatric Cancer Research, The Swedish Research Council, the Stockholm County Council (ALF project) and Karolinska Institutet. The authors have no conflict of interest to declare.

Gestational exposure to an epidemiologically defined mixture of phthalates leads to gonadal dysfunction in mouse offspring of both sexes.

The increasing concern for the reproductive toxicity of abundantly used phthalates requires reliable tools for exposure risk assessment to mixtures of chemicals, based on real life human exposure and disorder-associated epidemiological evidence. We herein used a mixture of four phthalate monoesters (33% mono-butyl phthalate, 16% mono-benzyl phthalate, 21% mono-ethyl hexyl phthalate, and 30% mono-isononyl phthalate), detected in 1st trimester urine of 194 pregnant women and identified as bad actors for a shorter anogenital distance (AGD) in their baby boys. Mice were treated with 0, 0.26, 2.6 and 13 mg/kg/d of the mixture, corresponding to 0x, 10x, 100x, 500x levels detected in the pregnant women. Adverse outcomes detected in the reproductive system of the offspring in pre-puberty and adulthood included reduced AGD index and gonadal weight, changes in gonadal histology and altered expression of key regulators of gonadal growth and steroidogenesis. Most aberrations were apparent in both sexes, though more pronounced in males, and exhibited a non-monotonic pattern. The phthalate mixture directly affected expression of steroidogenesis as demonstrated in a relevant in vitro model. The detected adversities at exposures close to the levels detected in pregnant women, raise concern on the existing safety limits for early-life human exposures and emphasizes the need for re-evaluation of the exposure risk.

Toxicokinetics of Perfluorinated Alkyl Acids Influences Their Toxic Potency in the Zebrafish Embryo ( Danio rerio).

Perfluorinated alkyl acids (PFAA) are highly persistent and bioaccumulative and have been associated with several adverse health effects. The chemical structure mainly differs in two ways: the length of the hydrophobic alkyl chain and the type of hydrophilic end group. Little is known how the chemical structure affects the toxicokinetics (TK) in different organisms. We studied the TK of four PFAA (PFOS, PFHxS, PFOA, and PFBA) with different chain lengths (4-8 carbons) and functional groups (sulfonic and carboxylic acid) in zebrafish ( Danio rerio) embryo. The time courses of the external (ambient water) and internal concentrations were determined at three exposure concentrations from 2 up to 120 h postfertilization (hpf). Three of the four PFAA showed a biphasic uptake pattern with slow uptake before hatching (around 48 hpf) and faster uptake thereafter. A two-compartment TK model adequately described the biphasic uptake pattern, suggesting that the chorion functions as an uptake barrier until 48 hpf. The bioconcentration factors (BCF) determined at 120 hpf varied widely between PFAA with averages of approximately 4000 (PFOS), 200 (PFHxS), 50 (PFOA), and 0.8 (PFBA) L kg dry weight-1, suggesting that both the alkyl chain length and the functional group influence the TK. The differences in toxic potency were reduced by 3 orders of magnitude when comparing internal effect concentrations instead of effective external concentrations.

Soluble epoxide hydrolase derived lipid mediators are elevated in bronchoalveolar lavage fluid from patients with sarcoidosis: a cross-sectional study.

BACKGROUND: Sarcoidosis is a systemic inflammatory multi-organ disease almost always affecting the lungs. The etiology remains unknown, but the hallmark of sarcoidosis is formation of non-caseating epithelioid cells granulomas in involved organs. In Scandinavia, > 30% of sarcoidosis patients have Löfgren's syndrome (LS), an acute disease onset mostly indicating a favorable prognosis. The impact of dysregulation of lipid mediators, which has been investigated in other inflammatory disorders, is still unknown. METHODS: Using three different liquid chromatography coupled to tandem mass spectrometry targeted platforms (LC-MS/MS), we quantified a broad suite of lipid mediators including eicosanoids, sphingolipids and endocannabinoids in bronchoalveolar lavage (BAL) fluid from pulmonary sarcoidosis patients (n = 41) and healthy controls (n = 16). RESULTS: A total of 47 lipid mediators were consistently detected in BAL fluid of patients and controls. After false discovery rate adjustment, two products of the soluble epoxide hydrolase (sEH) enzyme, 11,12-dihydroxyeicosa-5,8,14-trienoic acid (11,12-DiHETrE, p = 4.4E-5, q = 1.2E-3, median fold change = 6.0) and its regioisomer 14,15-dihydroxyeicosa-5,8,11-trienoic acid (14,15-DiHETrE, p = 3.6E-3, q = 3.2E-2, median fold change = 1.8) increased in patients with sarcoidosis. Additional shifts were observed in sphingolipid metabolism, with a significant increase in palmitic acid-derived sphingomyelin (SM16:0, p = 1.3E-3, q = 1.7E-2, median fold change = 1.3). No associations were found between these 3 lipid mediators and LS, whereas levels of SM 16:0 and 11,12-DiHETrE associated with radiological stage (p < 0.05), and levels of 14,15-DiHETrE were associated with the BAL fluid CD4/CD8 ratio. CONCLUSIONS: These observed shifts in lipid mediators provide new insights into the pathobiology of sarcoidosis and in particular highlight the sEH pathway to be dysregulated in disease.

Plasma stearoyl-CoA desaturase activity indices and bile acid concentrations after a low-fat meal: association with a genetic variant in the FTO gene.

PURPOSE: Dietary macronutrient composition, stearoyl-CoA desaturase (SCD) activity indices, and primary bile acid (BA) concentrations are among the factors that have been associated with lipid metabolism and contributed to obesity. We investigated the association between the polymorphic expression of the fat mass and obesity-associated (FTO) gene and its relationship with SCD activity indices and primary BA concentrations after a low-fat meal. SUBJECTS AND METHODS: Blood plasma samples were collected from 56 young (20-36 years) healthy subjects with different rs9939609 FTO genotypes. Fasting and post-meal (2 hours after a low-fat breakfast) blood samples were collected on the subsequent morning for the analysis of DNA methylation, SCD activity indices (product-to-precursor fatty acid ratios; 16:1n-7/16:0 and 18:1n-9/18:0), and chenodeoxycholic acid (CDCA) and cholic acid (CA) concentrations. Expression of lipogenic genes was investigated post-meal to assess the relationship between the CDCA and CA concentrations and mRNA levels of lipogenic genes. RESULTS: The FTO AA (obesity risk) genotype group (n=18) had higher (P<0.05) post-meal SCD-16 activity index than the FTO TT (wild type) genotype group (n=26). In both the FTO TT (n=16) and AA (n=8) genotype groups, the post-meal concentrations of CDCA and CA were lower (P<0.05) compared with the fasted state. No difference in BA concentrations between the FTO TT and AA genotype groups in both meal states was observed. After adjusting for the body mass index, the highest 50% post-meal concentrations of CA were inversely (P=0.010) correlated with the level of mRNA SCD expression. CONCLUSION: FTO AA carriers may be at a higher risk for obesity through higher SCD activity in a low-fat diet environment. This effect may be partly pronounced by very low CA concentrations.

Cellular accumulation and lipid binding of perfluorinated alkylated substances (PFASs) - A comparison with lysosomotropic drugs.

Many chemicals accumulate in organisms through a variety of different mechanisms. Cationic amphiphilic drugs (CADs) accumulate in lysosomes and bind to membranes causing phospholipidosis, whereas many lipophilic chemicals target adipose tissue. Perfluoroalkyl substances (PFASs) are widely used as surfactants, but many of them are highly bioaccumulating and persistent in the environment, making them notorious environmental toxicants. Understanding the mechanisms of their bioaccumulation is, therefore, important for their regulation and substitution with new, less harmful chemicals. We compared the highly bioaccumulative perfluorooctanesulfonic acid PFOS to its three less bioaccumulative alternatives perfluorooctanoic acid (PFOA), perfluorohexanoic acid (PFHxA) and perfluorobutane sulfonic acid (PFBS), in their ability to accumulate and remain in lung epithelial cells (NCI-H292) and adipocytes (3T3-L1K) in vitro. As a reference point we tested a set of cationic amphiphilic drugs (CADs), known to highly accumulate in cells and strongly bind to phospholipids, together with their respective non-CAD controls. Finally, all compounds were examined for their ability to bind to neutral lipids and phospholipids in cell-free systems. Cellular accumulation and retention of the test compounds were highly correlated between the lung epithelial cells and adipocytes. Interestingly, although an anion itself, intensities of PFOS accumulation and retention in cells were comparable to those of CAD compounds, but PFOS failed to induce phospholipidosis or alter lysosomal volume. Compared to other lipophilicity measures, phospholipophilicity shows the highest correlation (Rˆ2 = 0.75) to cellular accumulation data in both cell types and best distinguishes between high and low accumulating compounds. This indicates that binding to phospholipids may be the most important component in driving high cellular accumulation in lung epithelial cells, as well as in adipocytes, and for both CADs and bioaccumulating PFASs. Obtained continuous PLS models based on compound's affinity for phospholipids and neutral lipids can be used as good prediction models of cellular accumulation and retention of PFASs and CADs.

Resveratrol supports and alpha-naphthoflavone disrupts growth of human ovarian follicles in an in vitro tissue culture model.

Infertility is a global health problem with an estimated incidence of 15%. Exposure to chemicals is a potential causal factor, and there is a lack of studies examining the effects on female germ cells. Here, we have studied the impact of different aryl hydrocarbon receptor (AHR) modulators on human ovarian follicles using a human ovarian tissue culture model. Expression of AHR was analyzed in tissue samples, and effects of the selected ligands resveratrol (RSVL), 6-formylindolo(3,2-b)carbazole (FICZ), and alpha-naphthoflavone (aNF) on AHR transactivation studied in a granulosa cell tumor line. Cortical human ovarian tissue containing preantral follicles was exposed to the ligands or vehicle (dimethylsulfoxide, DMSO) for seven days in vitro. Follicle growth was assessed by counting and measuring follicles from serial tissue sections, cell death quantified using in situ Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) assay, and steroid hormone production measured using a newly developed ultra-performance liquid chromatography method. AHR was expressed in all donated ovarian tissue samples. FICZ induced AHR transactivation in the granulosa cell line while aNF antagonised it. Compared to DMSO control, FICZ had no effect on follicles in culture, RSVL increased the proportion of growing follicles, and aNF increased cell death, disrupted growth of secondary follicles, increased testosterone, and reduced estradiol levels. We conclude that RSVL supports and aNF disrupts growth of human ovarian follicles in culture. We further conclude that the human ovarian tissue culture model is suitable for studying effects of chemicals on follicular biology.

Fluoxetine modulates sex steroid levels in vitro.

BACKGROUND AND AIMS: Selective serotonin reuptake inhibitors (SSRIs) are antidepressants increasingly prescribed against depression during and after pregnancy. However, these compounds cross the placenta and are found in breast milk, thus reaching, and possibly affecting, the fetus and infant during critical developmental stages. Fluoxetine (FLX), a widely used SSRI, can interfere with estrogen signaling, which is important for the development of female sex organs and certain brain areas, among others. Interference with estrogen signaling can take place on different levels, e.g., by affecting receptor activity or hormone levels. FLX has previously been shown to induce estrogen receptor-dependent transcription in vitro at high concentrations. In this study we set out to assess effects of FLX on estradiol levels in vitro. METHODS: FLX was tested using the OECD recommended H295R model, a human adrenocortical carcinoma cell line that is able to produce all steroid hormones found in the gonads and adrenal glands, including estradiol and testosterone. H295R cells were incubated with different doses of FLX for 48h. Subsequently, concentrations of these two steroids were measured in cell culture medium after FLX exposure, using liquid chromatography coupled with tandem mass spectrometry. Aromatase mRNA expression was assessed using qPCR. RESULTS: Fluoxetine significantly increased estradiol secretion in H295R cells after a 48h exposure at low, submicromolar concentrations, but showed no effects on testosterone levels or aromatase mRNA expression. CONCLUSION: Fluoxetine has the potential to interfere with estrogenic signaling by increasing estradiol secretion at low concentrations, which are relevant for fetal and adult human exposure.

De novo design and synthesis of a 30-cistron translation-factor module.

Two of the many goals of synthetic biology are synthesizing large biochemical systems and simplifying their assembly. While several genes have been assembled together by modular idempotent cloning, it is unclear if such simplified strategies scale to very large constructs for expression and purification of whole pathways. Here we synthesize from oligodeoxyribonucleotides a completely de-novo-designed, 58-kb multigene DNA. This BioBrick plasmid insert encodes 30 of the 31 translation factors of the PURE translation system, each His-tagged and in separate transcription cistrons. Dividing the insert between three high-copy expression plasmids enables the bulk purification of the aminoacyl-tRNA synthetases and translation factors necessary for affordable, scalable reconstitution of an in vitro transcription and translation system, PURE 3.0.

Spinal Cord Stimulation Alters Protein Levels in the Cerebrospinal Fluid of Neuropathic Pain Patients: A Proteomic Mass Spectrometric Analysis.

OBJECTIVES: Electrical neuromodulation by spinal cord stimulation (SCS) is a well-established method for treatment of neuropathic pain. However, the mechanism behind the pain relieving effect in patients remains largely unknown. In this study, we target the human cerebrospinal fluid (CSF) proteome, a little investigated aspect of SCS mechanism of action. METHODS: Two different proteomic mass spectrometry protocols were used to analyze the CSF of 14 SCS responsive neuropathic pain patients. Each patient acted as his or her own control and protein content was compared when the stimulator was turned off for 48 hours, and after the stimulator had been used as normal for three weeks. RESULTS: Eighty-six proteins were statistically significantly altered in the CSF of neuropathic pain patients using SCS, when comparing the stimulator off condition to the stimulator on condition. The top 12 of the altered proteins are involved in neuroprotection (clusterin, gelsolin, mimecan, angiotensinogen, secretogranin-1, amyloid beta A4 protein), synaptic plasticity/learning/memory (gelsolin, apolipoprotein C1, apolipoprotein E, contactin-1, neural cell adhesion molecule L1-like protein), nociceptive signaling (neurosecretory protein VGF), and immune regulation (dickkopf-related protein 3). CONCLUSION: Previously unknown effects of SCS on levels of proteins involved in neuroprotection, nociceptive signaling, immune regulation, and synaptic plasticity are demonstrated. These findings, in the CSF of neuropathic pain patients, expand the picture of SCS effects on the neurochemical environment of the human spinal cord. An improved understanding of SCS mechanism may lead to new tracks of investigation and improved treatment strategies for neuropathic pain.

Linoleic acid-derived lipid mediators increase in a female-dominated subphenotype of COPD.

Chronic obstructive pulmonary disease (COPD) is a leading cause of mortality; however, the role of inflammatory mediators in its pathobiology remains unclear. The aim of this study was to investigate the influence of gender in COPD on lipid mediator levels.Bronchoalveolar lavage fluid (BALF) and serum were obtained from healthy never-smokers, smokers and COPD patients (Global Initiative for Chronic Obstructive Lung Disease stage I-II/A-B) (n=114). 94 lipid mediators derived from the cytochrome-P450, lipoxygenase, and cyclooxygenase pathways were analysed by liquid chromatography-mass spectrometry.Multivariate modelling identified a 9-lipid panel in BALF that classified female smokers with COPD from healthy female smokers (p=6×10(-6)). No differences were observed for the corresponding male population (p=1.0). These findings were replicated in an independent cohort with 92% accuracy (p=0.005). The strongest drivers were the cytochrome P450-derived epoxide products of linoleic acid (leukotoxins) and their corresponding soluble epoxide hydrolase (sEH)-derived products (leukotoxin-diols). These species correlated with lung function (r=0.87; p=0.0009) and mRNA levels of enzymes putatively involved in their biosynthesis (r=0.96; p=0.003). Leukotoxin levels correlated with goblet cell abundance (r=0.72; p=0.028).These findings suggest a mechanism by which goblet cell-associated cytochrome-P450 and sEH activity produce elevated leukotoxin-diol levels, which play a putative role in the clinical manifestations of COPD in a female-dominated disease sub-phenotype.

Lipid mediator profile in vernix caseosa reflects skin barrier development.

Vernix caseosa (VC) is a protective layer that covers the skin of most human newborns. This study characterized the VC lipid mediator profile, and examined its relationship to gestational period, gender of the newborn and maternal lifestyle. VC collected at birth from 156 newborns within the ALADDIN birth cohort was analyzed and 3 different groups of lipid mediators (eicosanoids and related oxylipin analogs, endocannabinoids and sphingolipids) were screened using LC-MS/MS. A total of 54 compounds were detected in VC. A number of associations between lipid mediators and the gestational period were observed, including increases in the ceramide to sphingomyelin ratio as well as the endocannabinoids anandamide and 2-arachidonoylglycerol. Gender-specific differences in lipid mediator levels were observed for all 3 lipid classes. In addition, levels of the linoleic acid oxidation products 9(10)-epoxy-12Z-octadecenoic and 12(13)-epoxy-9Z-octadecenoic acid (EpOMEs) as well as 12,13-dihydroxy-9Z-octadecenoic acid (DiHOME) were increased in VC of children from mothers with an anthroposophic lifestyle. Accordingly, VC was found to be rich in multiple classes of bioactive lipid mediators, which evidence lifestyle, gender and gestational week dependencies. Levels of lipid mediators in VC may therefore be useful as early stage non-invasive markers of the development of the skin as a protective barrier.

Urinary excretion of lipid mediators in response to repeated eucapnic voluntary hyperpnea in asthmatic subjects.

Exercise-induced bronchoconstriction displays refractoriness manifested as a decreased response to repeated exercise challenge within hours. The refractoriness may be attenuated by inhibition of the biosynthesis of prostaglandins (PG). The aim of the study was to determine which PGs and other lipid mediators are excreted during the refractory period. First, 16 subjects with mild stable asthma performed two repeated 4-min challenges with eucapnic voluntary hyperpnea (EVH) 1 and 3 h apart. There was a similar degree of refractoriness in both protocols (∼15% protection). The 1-h interval was too short to study mediator excretion because the urinary levels did not return to baseline before the second challenge. With the 3-h protocol, there was increased urinary excretion of cysteinyl-leukotrienes and metabolites of the mast cell product PGD2 after both challenges. Next, another eight subjects performed two 6-min challenges with EVH 3 h apart, which produced a greater bronchoconstrictor response than the 4-min protocol (30.0 ± 5.4 vs. 17.7 ± 1.5%; P = 0.0029) and a greater degree of refractoriness (∼30%). Analysis by ultra-performance liquid chromatography triple quadrupole mass spectrometry confirmed excretion of the bronchoconstrictor cysteinyl-leukotrienes and PGD2 during both challenges. In addition, there was increased excretion of the bronchoprotective PGE2, and also of the main metabolite of PGI2. This is the first report of excretion of PGE2 and PGI2 during the refractory period to EVH challenge, suggesting that they may mediate the refractoriness. Maintained excretion of PGD2 and leukotriene E4 following the repeat challenge argues against mast cell mediator depletion as the mechanism of refractoriness.

Comparative study of label and label-free techniques using shotgun proteomics for relative protein quantification.

The analytical performance of three different strategies, iTRAQ (isobaric tag for relative and absolute quantification), dimethyl labeling (DML) and label free (LF) for relative protein quantification using shotgun proteomics have been evaluated. The methods have been explored using samples containing (i) Bovine proteins in known ratios and (ii) Bovine proteins in known ratios spiked into Escherichia coli. The latter case mimics the actual conditions in a typical biological sample with a few differentially expressed proteins and a bulk of proteins with unchanged ratios. Additionally, the evaluation was performed on both QStar and LTQ-FTICR mass spectrometers. LF LTQ-FTICR was found to have the highest proteome coverage while the highest accuracy based on the artificially regulated proteins was found for DML LTQ-FTICR (54%). A varying linearity (k: 0.55-1.16, r(2): 0.61-0.96) was shown for all methods within selected dynamic ranges. All methods were found to consistently underestimate Bovine protein ratios when matrix proteins were added. However, LF LTQ-FTICR was more tolerant toward a compression effect. A single peptide was demonstrated to be sufficient for a reliable quantification using iTRAQ. A ranking system utilizing several parameters important for quantitative proteomics demonstrated that the overall performance of the five different methods was; DML LTQ-FTICR>iTRAQ QStar>LF LTQ-FTICR>DML QStar>LF QStar.

Multiplexed quantification of proteins adsorbed to surface-modified and non-modified microdialysis membranes.

A simple and straightforward method for discovery and quantification of proteins adsorbed onto delicate and sensitive membrane surfaces is presented. The adsorbed proteins were enzymatically cleaved while still adsorbed onto the membranes using an on-surface enzymatic digestion (oSED). This was followed by isobaric tagging, nanoliquid chromatography, and tandem mass spectrometry. Protein adsorption on tri-block copolymer Poloxamer 407 surface-modified microdialysis (MD) membranes were compared with protein adsorption on unmodified MD membranes. Ventricular cerebrospinal fluid (vCSF) kept at 37 °C was used as sample matrix. In total, 19 proteins were quantified in two biological replicates. The surface-modified membranes adsorbed 33% less proteins than control membranes and the most abundant proteins were subunits of hemoglobin and clusterin. The adsorption of clusterin on the modified membranes was on average 36% compared to control membranes. The most common protein in vCSF, Albumin, was not identified adsorbed to the surface at all. It was also experimentally verified that oSED, in conjunction with tandem mass spectrometry can be used to quantify femtomole amounts of proteins adsorbed on limited and delicate surfaces, such as MD membranes. The method has great potential and can be used to study much more complex protein adsorption systems than previously reported.

Quantitative subproteomic analysis of age-related changes in mouse liver peroxisomes by iTRAQ LC-MS/MS.

Aging is a complex multifactorial phenomenon, which is believed to result from the accumulation of cellular damage to biological macromolecules. Peroxisomes recently emerged as another important source of reactive oxygen species (ROS) production in addition to mitochondria. However, the role of these organelles in the process of aging is still not clear. The aim of this study was to characterize the changes in protein expression profiles of young (10 weeks old) versus old (18 months old) mouse liver peroxisome-enriched fractions. We have applied shotgun proteomic approach based on liquid chromatography and tandem mass spectrometry (LC-MS/MS) combined with iTRAQ (isobaric tags for relative and absolute quantitation) labeling that allows comparative quantitative multiplex analysis. Our analysis led to identification and quantification of 150 proteins, 8 out of which were differentially expressed between two age groups at a statistically significant level (p<0.05), with folds ranging from 1.2 to 4.1. These proteins involved in peroxisomal ß-oxidation, detoxification of xenobiotics and production of ROS. Noteworthy, differences in liver proteome have been observed between as well as within different age groups. In conclusion, our subproteomic quantitative study suggests that mouse liver proteome is sufficiently maintained until certain age.

Analysis of membrane and hydrophilic proteins simultaneously derived from the mouse brain using cloud-point extraction.

In this study, a temperature-induced phase fractionation known as cloud-point extraction (CPE) with the non-ionic surfactant Triton X-114 was used to simultaneously extract, concentrate, and fractionate hydrophobic and hydrophilic proteins from mouse brain tissue. Two bottom-up proteomic techniques were used to comprehensively identify the extracted proteins. The first "shotgun"-based approach included tryptic digestion of the proteins followed by reversed-phase nanoliquid chromatography (RP-nanoLC) in combination with electrospray ionization (ESI) tandem mass spectrometry (MS/MS). In the second approach, the extracted intact proteins were first separated by one-dimensional (1D) gel electrophoresis and then in-gel digested with trypsin and analyzed with nanoLC-MS/MS. In total, 1,825 proteins were unambiguously identified and the percentage of membrane proteins was 26% which is at the reported genome expression levels of 20-30%. The protein overlap between the two approaches was high. The majority (77%) of the identifications in the first approach was also found by the second method. The protein overlap between the CPE-extracted hydrophilic and hydrophobic fractions was rather small (16-23%) for both methods, which indicates a good phase separation. A quantitative evaluation of the CPE with iTRAQ labeling and nanoLC-ESI-MS/MS analysis gave iTRAQ ratios at the expected levels and an overall variation of the entire method at 17-31%. The results indicate very reproducible sample preparation and analysis methods that readily can be applied on large-scale sample sets.

Cloud-point extraction and delipidation of porcine brain proteins in combination with bottom-up mass spectrometry approaches for proteome analysis.

In this study, temperature-induced phase fractionation also known as cloud-point extraction (CPE) with the nonionic surfactant Triton X-114 was used to simultaneously extract hydrophobic and hydrophilic proteins from porcine brain tissue. Various protein precipitation/delipidation procedures were investigated to efficiently remove lipids and detergents while retaining maximum protein recoveries. The best performing delipidation method was then used in combination with CPE to compare three different mass spectrometry (MS) based "bottom-up" proteomic approaches for protein analysis of the porcine brain. In the first approach, the intact proteins were initially separated by one-dimensional (1D) gel electrophoresis. The excised protein bands were digested with trypsin, and the peptides were separated by reversed phase nanoliquid chromatography (RP-nanoLC) followed by electrospray ionization (ESI) tandem mass spectrometry (MS/MS) analysis. The other bottom-up proteomic approaches were based on first enzymatical digestion of the proteins followed by RP-nanoLC separation in combination with matrix assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS) or on the combination of in-solution isoelectric focusing (IEF) with ESI-nanoLC-MS/MS of the IEF separated peptides. In total, we found and unambiguously identified 331 unique proteins. The overlap between different techniques was about 10%, showing that the use of multiple proteomic approaches is beneficial to yield a better coverage of the proteome. Furthermore, the overlap between the CPE extracted hydrophilic and hydrophobic proteins was rather small (9-16%), indicating an efficient sample preparation technique to extract and separate hydrophilic and hydrophobic proteins from brain tissue. The percentage of identified membrane proteins was 27%, which is in accordance to the fact that about one-third of all genes in various organisms encode for this class of proteins. The results indicate that cloud point extraction is a promising sample preparation tool, which allows simultaneous in depth studies of brain derived membrane proteins as well as hydrophilic proteins. This technique can be very useful when studying human central nervous system (CNS) tissue or animal models of neurological diseases.