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Mitochondrial dysfunction is an early event in the pathogenesis of neurologic disorders and aging. Sirtuin 3 (SIRT3) regulates mitochondrial function in response to the cellular environment through the reversible deacetylation of proteins involved in metabolism and reactive oxygen species detoxification. As the primary mitochondrial deacetylase, germline, or peripheral tissue-specific deletion of SIRT3 produces mitochondrial hyperacetylation and the accelerated development of age-related diseases. Given the unique metabolic demands of neurons, the role of SIRT3 in the brain is only beginning to emerge. Using mass spectrometry-based acetylomics, high-resolution respirometry, video-EEG, and cognition testing, we report targeted deletion of SIRT3 from select neurons in the cortex and hippocampus produces altered neuronal excitability and metabolic dysfunction in female mice. Targeted deletion of SIRT3 from neuronal helix-loop-helix 1 (NEX)-expressing neurons resulted in mitochondrial hyperacetylation, female-specific superoxide dismutase-2 (SOD2) modification, increased steady-state superoxide levels, metabolic reprogramming, altered neuronal excitability, and working spatial memory deficits. Inducible neuronal deletion of SIRT3 likewise produced female-specific deficits in spatial working memory. Together, the data demonstrate that deletion of SIRT3 from forebrain neurons selectively predisposes female mice to deficits in mitochondrial and cognitive function.SIGNIFICANCE STATEMENTMitochondrial SIRT3 is an enzyme shown to regulate energy metabolism and antioxidant function, by direct deacetylation of proteins. In this study, we show that neuronal SIRT3 deficiency renders female mice selectively vulnerable to impairment in redox and metabolic function, spatial memory, and neuronal excitability. The observed sex-specific effects on cognition and neuronal excitability in female SIRT3-deficient mice suggest that mitochondrial dysfunction may be one factor underlying comorbid neuronal diseases, such as Alzheimer's disease and epilepsy. Furthermore, the data suggest that SIRT3 dysfunction may predispose females to age-related metabolic and cognitive impairment.

Succinylation is a post-translational modification of protein lysine residues with succinyl groups derived from succinyl CoA. Succinylation is considered a significant post-translational modification with the potential to impact protein function which is highly conserved across numerous species. The role of succinylation in the heart, especially in heart failure and myofibril mechanics, remains largely unexplored. Mechanical parameters were measured in myofibrils isolated from failing hearts of ischemic cardiomyopathy patients and non-failing donor controls. We employed mass spectrometry to quantify differential protein expression in myofibrils from failing ischemic cardiomyopathy hearts compared to non-failing hearts. In addition, we combined peptide enrichment by immunoprecipitation with liquid chromatography tandem mass spectrometry to quantitatively analyze succinylated lysine residues in these myofibrils. Several key parameters of sarcomeric mechanical interactions were altered in myofibrils isolated from failing ischemic cardiomyopathy hearts, including lower resting tension and a faster rate of activation. Of the 100 differentially expressed proteins, 46 showed increased expression in ischemic heart failure, while 54 demonstrated decreased expression in ischemic heart failure. Our quantitative succinylome analysis identified a total of 572 unique succinylated lysine sites located on 181 proteins, with 307 significantly changed succinylation events. We found that 297 succinyl-Lys demonstrated decreased succinylation on 104 proteins, while 10 residues demonstrated increased succinylation on 4 proteins. Investigating succinyl CoA generation, enzyme activity assays demonstrated that α-ketoglutarate dehydrogenase and succinate dehydrogenase activities were significantly decreased in ischemic heart failure. An activity assay for succinyl CoA synthetase demonstrated a significant increase in ischemic heart failure. Taken together, our findings support the hypothesis that succinyl CoA production is decreased and succinyl CoA turnover is increased in ischemic heart failure, potentially resulting in an overall decrease in the mitochondrial succinyl CoA pool, which may contribute to decreased myofibril protein succinylation in heart failure.

Identifying and annotating the molecular composition of individual foods will improve scientific understanding of how foods impact human health and how much variation exists in the molecular composition of foods of the same species. The complexity of this task includes distinct varieties and variations in natural occurring pigments of foods. Lipidomics, a sub-field of metabolomics, has emerged as an effective tool to help decipher the molecular composition of foods. For this proof-of-principle research, we determined the lipidomic profiles of green, yellow and red bell peppers (Capsicum annuum) using liquid chromatography mass spectrometry and a novel tool for automated annotation of compounds following database searches. Among 23 samples analyzed from 6 peppers (2 green, 1 yellow, and 3 red), over 8000 lipid compounds were detected with 315 compounds (106 annotated) found in all three colors. Assessments of relationships between these compounds and pepper color, using linear mixed effects regression and false discovery rate (p-value = 7.4 × 10−5; FDR adjusted p-value = 0.0080). These results support lipidomics as a viable analytical technique to identify molecular compounds that can be used for unique characterization of foods.

Synaptotagmin-like protein 4 (Slp-4), also known as granuphilin, is a Rab effector responsible for docking secretory vesicles to the plasma membrane before exocytosis. Slp-4 binds vesicular Rab proteins via an N-terminal Slp homology (SHD) domain, interacts with plasma membrane SNARE complex proteins via a central linker region, and contains tandem C-terminal C2 domains (C2A and C2B) with affinity for phosphatidylinositol-(4,5)-bisphosphate (PIP2). The Slp-4 C2A domain binds with low nanomolar apparent affinity to PIP2 in lipid vesicles that also contain background anionic lipids such as phosphatidylserine (PS), but much weaker when either the background anionic lipids or PIP2 are removed. Through computational and experimental approaches, we show that this high affinity membrane interaction arises from concerted interaction at multiple sites on the C2A domain. In addition to a conserved PIP2-selective lysine cluster, there exists a larger cationic surface surrounding the cluster which contributes substantially to the affinity for physiologically relevant lipid compositions. While mutations at the PIP2-selective site decrease affinity for PIP2, multiple mutations are needed to decrease binding to physiologically relevant lipid compositions. Docking and molecular dynamics simulations indicate several conformationally flexible loops that contribute to the nonspecific cationic surface. We also identify and characterize a covalently modified variant in the bacterially expressed protein, which arises through reactivity of the PIP2-binding lysine cluster with endogenous bacterial compounds and has a low membrane affinity. Overall, multivalent lipid binding by the Slp-4 C2A domain provides selective recognition and high affinity docking of large dense-core secretory vesicles to the plasma membrane.

OBJECTIVES: Astaxanthin, a predominately marine-source carotenoid, is the subject of a large number of studies for its antioxidant and anti-inflammatory properties. Astaxanthin is not generally a primary carotenoid in human plasma due to relatively low dietary intake. Salmon is the one of the few dietary sources of astaxanthin in typical American diets and the concentration may vary by the source of salmon foods. A study was performed to 1) Compare astaxanthin concentration in various sources of salmon; 2) Compare astaxanthin plasma concentrations before and after salmon consumption. METHODS: An assortment of salmon types and forms was purchased in the greater Denver, CO region: wild Pacific, farmed Atlantic, canned and pouch. Plasma samples were collected from five participants prior to and after a five week Mediterranean diet intervention study, which included two servings of salmon per week. Salmon samples were freeze-dried, then both salmon (in triplicate) and plasma samples were prepared by liquid-liquid extraction for untargeted liquid chromatography-mass spectrometry analysis. An accurate mass and retention time database was used to identify and quantify astaxanthin. ANOVA with Tukey multiple testing corrections was used to assess the relationship between astaxanthin and the different salmon products, and paired t-tests for astaxanthin in plasma. RESULTS: Astaxanthin concentration was significantly higher in fresh salmon compared to pouch packaged (23.0-fold; P = 1.70e-04) and canned (34.9-fold; P = 1.23e-08). Interestingly, astaxanthin levels were similar between fresh wild Pacific and fresh farmed Atlantic salmon (0.91-fold, P = 0.82) and by mode of cooking (i.e., fresh, cooked, frozen; P = 0.81). Astaxanthin concentration in plasma was significantly increased after farmed Atlantic salmon consumption (1.98-fold, P = 6.16e-09). CONCLUSIONS: Our data suggest that astaxanthin concentration varies among different processed salmon products compared to wild and farmed salmon. After salmon consumption, plasma astaxanthin concentration increased and may have potential as a biomarker of salmon consumption. FUNDING SOURCES: National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK).

OBJECTIVES: The carotenoid β-cryptoxanthin is a natural pigment that is both an antioxidant and a precursor to retinol. Research supports that β-cryptoxanthin has greater bioavailability than β-carotene in humans. Red bell peppers have more than double the amount of β-cryptoxanthin than any of the top seven consumed vegetables, as ranked by the USDA. To determine if the amounts of β-cryptoxanthin in bell peppers are dependent upon the organic status, color, cooking, season or location that the fruit was grown within North America, β-cryptoxanthin was measured and compared in green, red and yellow bell peppers. METHODS: An assortment of bell peppers were purchased in the greater Denver, CO region. Green, red and yellow peppers; organic and non-organic; and peppers grown in Canada, the US and Mexico during two different seasons were selected for analysis. The effects of lightly sautéing compared to fresh peppers and season of growth were compared. Samples (100 mg/1 mL) were freeze-dried, then prepared by liquid-liquid extraction for untargeted liquid chromatography mass spectrometry (LC-MS)-based metabolomics analysis. An accurate mass and retention time (AMRT) database was used to identify and quantify β-cryptoxanthin. Linear regression was used to assess the relationship between β-cryptoxanthin and pepper qualities. RESULTS: β-cryptoxanthin concentration was significantly higher in red bell peppers compared to green (11.8-fold) and yellow peppers (7.1-fold) (P = 1.624e-11). β-cryptoxanthin concentration does not appear to be influenced by organic status, season or geographic location. Likewise, the cooked peppers were similar in β-cryptoxanthin content compared to their fresh counterparts. CONCLUSIONS: Our results suggest that the consumption of bell peppers as a source of β-cryptoxanthin is consistent across organic status, fresh, cooked, season and the location in which they were grown. While β-cryptoxanthin concentration in significantly higher in red bell peppers, more research is necessary in order to determine whether these differences result in any altered health outcomes. FUNDING SOURCES: National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK).

Mitochondrial dysfunction is one of many key factors in the etiology of alcoholic liver disease (ALD). Lysine acetylation is known to regulate numerous mitochondrial metabolic pathways, and recent reports demonstrate that alcohol-induced protein acylation negatively impacts these processes. To identify regulatory mechanisms attributed to alcohol-induced protein post-translational modifications, we employed a model of alcohol consumption within the context of wild type (WT), sirtuin 3 knockout (SIRT3 KO), and sirtuin 5 knockout (SIRT5 KO) mice to manipulate hepatic mitochondrial protein acylation. Mitochondrial fractions were examined by label-free quantitative HPLC-MS/MS to reveal competition between lysine acetylation and succinylation. A class of proteins defined as "differential acyl switching proteins" demonstrate select sensitivity to alcohol-induced protein acylation. A number of these proteins reveal saturated lysine-site occupancy, suggesting a significant level of differential stoichiometry in the setting of ethanol consumption. We hypothesize that ethanol downregulates numerous mitochondrial metabolic pathways through differential acyl switching proteins. Data are available via ProteomeXchange with identifier PXD012089.

Rationale The goal of this work was to identify phosphorylation sites within the amino acid sequence of human ADAP1. Using traditional mass spectrometry based techniques we were unable to produce interpretable spectra demonstrating modification by phosphorylation. This prompted us to employ a strategy in which phosphorylated peptides were first predicted using peptide mapping followed by targeted MS/MS acquisition. Methods ADAP1 was immunoprecipitated from extracts of HEK293 cells stably transfected with ADAP1 cDNA. Immunoprecipitated ADAP1 was digested with proteolytic enzymes and analyzed by LC/MS in MS1 mode by high-resolution quadrupole time-of-flight mass spectrometry (QTOF-MS). Peptide molecular features were extracted using an untargeted data-mining algorithm. Extracted peptide neutral masses were matched against the ADAP1 amino acid sequence with phosphorylation included as a predicted modification. Peptides with predicted phosphorylation sites were analyzed by targeted LC/MS2 . Acquired MS2 spectra were then analyzed using database search engines to confirm phosphorylation. Spectra of phosphorylated peptides were validated by manual interpretation. Further confirmation was performed by manipulating phospho-peptide abundance using calf intestinal phosphatase (CIP) and the phorbol ester, phorbol 12-myristate 13-acetate (PMA). Results Of five predicted phosphopeptides, one, comprised of the sequence AVDRPMLPQEYAVEAHFK, was confirmed to be phosphorylated on a tyrosine at position 364. Pre-treatment of cells with PMA prior to immunoprecipitation increased the ratio of phosphorylated to unphosphorylated peptide as determined by area counts of extracted ion chromatograms (EIC). Addition of CIP to immunoprecipitation reactions eliminated the phosphorylated form. Conclusions A novel phosphorylation site was identified at tyrosine 364. Phosphorylation at this site is increased by treatment with PMA. PMA promotes membrane translocation and activation of protein kinase C (PKC), indicating that tyrosine 364 is phosphorylated by a PKC-dependent mechanism.

Although health benefits of the Dietary Approaches to Stop Hypertension (DASH) diet are established, it is not understood which food compounds result in these benefits. We used metabolomics to identify unique compounds from individual foods of a DASH-style diet and determined if these Food-Specific Compounds (FSC) are detectable in urine from participants in a DASH-style dietary study. We also examined relationships between urinary compounds and blood pressure (BP). Nineteen subjects were randomized into 6-week controlled DASH-style diet interventions. Mass spectrometry-based metabolomics was performed on 24-hour urine samples collected before and after each intervention and on 12 representative DASH-style foods. Between 66–969 compounds were catalogued as FSC; for example, 4-hydroxydiphenylamine was found to be unique to apple. Overall, 13–190 of these FSC were detected in urine, demonstrating that these unmetabolized food compounds can be discovered in urine using metabolomics. Although linear mixed effects models showed no FSC from the 12 profiled foods were significantly associated with BP, other endogenous and food-related compounds were associated with BP (N = 16) and changes in BP over time (N = 6). Overall, this proof of principle study demonstrates that metabolomics can be used to catalog FSC, which can be detected in participant urine following a dietary intervention.

Synaptotagmin-like protein 4 (Slp-4), also known as granuphilin, is a Rab effector responsible for docking secretory vesicles to the plasma membrane before exocytosis. Slp-4 binds vesicular Rab proteins via an N-terminal Slp homology domain, interacts with plasma membrane SNARE complex proteins via a central linker region, and contains tandem C-terminal C2 domains (C2A and C2B) with affinity for phosphatidylinositol-(4,5)-bisphosphate (PIP2). The Slp-4 C2A domain binds with low nanomolar apparent affinity to PIP2 in lipid vesicles that also contain background anionic lipids such as phosphatidylserine but much weaker when either the background anionic lipids or PIP2 is removed. Through computational and experimental approaches, we show that this high-affinity membrane binding arises from concerted interaction at multiple sites on the C2A domain. In addition to a conserved PIP2-selective lysine cluster, a larger cationic surface surrounding the cluster contributes substantially to the affinity for physiologically relevant lipid compositions. Although the K398A mutation in the lysine cluster blocks PIP2 binding, this mutated protein domain retains the ability to bind physiological membranes in both a liposome-binding assay and MIN6 cells. Molecular dynamics simulations indicate several conformationally flexible loops that contribute to the nonspecific cationic surface. We also identify and characterize a covalently modified variant that arises through reactivity of the PIP2-binding lysine cluster with endogenous bacterial compounds and binds weakly to membranes. Overall, multivalent lipid binding by the Slp-4 C2A domain provides selective recognition and high-affinity docking of large dense core secretory vesicles to the plasma membrane.

Background: Metabolomics is emerging as a valuable tool in clinical science. However, one major challenge in clinical metabolomics is the limited use of standardized guidelines for sample collection and handling. In this study, we conducted a pilot analysis of serum and plasma to determine the effects of processing time and collection tube on the metabolome. Methods: Blood was collected in 3 tubes: Vacutainer serum separator tube (SST, serum), EDTA (plasma) and P100 (plasma) and stored at 4 degrees for 0, 0.5, 1, 2, 4 and 24 h prior to centrifugation. Compounds were extracted using liquid-liquid extraction to obtain a hydrophilic and a hydrophobic fraction and analyzed using liquid chromatography mass spectrometry. Differences among the blood collection tubes and sample processing time were evaluated (ANOVA, Bonferroni FWER ≤ 0.05 and ANOVA, Benjamini Hochberg FDR ≤ 0.1, respectively). Results: Among the serum and plasma tubes 93.5% of compounds overlapped, 382 compounds were unique to serum and one compound was unique to plasma. There were 46, 50 and 86 compounds affected by processing time in SST, EDTA and P100 tubes, respectively, including many lipids. In contrast, 496 hydrophilic and 242 hydrophobic compounds differed by collection tube. Forty-five different chemical classes including alcohols, sugars, amino acids and prenol lipids were affected by the choice of blood collection tube. Conclusion: Our results suggest that the choice of blood collection tube has a significant effect on detected metabolites and their overall abundances. Perhaps surprisingly, variation in sample processing time has less of an effect compared to collection tube, however, a larger sample size is needed to confirm this.

Advancements in omics technologies have increased our potential to evaluate molecular changes in a rapid and comprehensive manner. This is especially true in mass spectrometry-based metabolomics where improvements, including ease of use, in high-performance liquid chromatography (HPLC), column chemistries, instruments, software, and molecular databases, have advanced the field considerably. Applications of this relatively new omics technology in clinical research include discovering disease biomarkers, finding new drug targets, and elucidating disease mechanisms. Here we describe a typical clinical metabolomics workflow, which includes the following steps: (1) extraction of metabolites from the lung, plasma, bronchoalveolar lavage, or cells; (2) sample analysis via liquid chromatography-mass spectrometry; and (3) data analysis using commercial and freely available software packages. Overall, the methods delineated here can help investigators use metabolomics to discovery novel biomarkers and to understand lung diseases.

Proteomics has enabled researchers to evaluate global protein changes in a relatively rapid and comprehensive manner. Applications of these technologies in lung research include biomarker and drug discovery, elucidating disease mechanisms, and quantitative clinical assays. Two common workflows exist for quantitative proteomics studies that are aimed at determining differences in protein levels: label-free and labeling methods. Here we describe specific techniques involved in both quantitative workflows; these include extensive sample preparation methods for several lung-specific sample types. Methods are also included for mass spectrometry-based sample analysis and data analysis. While the focus is on quantitative, clinical proteomics, these strategies are appropriate for a wide array of sample types and applications.

The analysis of bronchoalveolar lavage fluid (BALF) using mass spectrometry-based metabolomics can provide insight into lung diseases, such as asthma. However, the important step of compound identification is hindered by the lack of a small molecule database that is specific for BALF. Here we describe prototypic, small molecule databases derived from human BALF samples (n=117). Human BALF was extracted into lipid and aqueous fractions and analyzed using liquid chromatography mass spectrometry. Following filtering to reduce contaminants and artifacts, the resulting BALF databases (BALF-DBs) contain 11,736 lipid and 658 aqueous compounds. Over 10% of these were found in 100% of samples. Testing the BALF-DBs using nested test sets produced a 99% match rate for lipids and 47% match rate for aqueous molecules. Searching an independent dataset resulted in 45% matching to the lipid BALF-DB compared to

Proteomics has enabled researchers to evaluate global protein changes in a relatively rapid and comprehensive manner. Applications of these technologies in lung research include biomarker and drug discovery, elucidating disease mechanisms, and quantitative clinical assays. Two common workflows exist for quantitative proteomics studies that are aimed at determining differences in protein levels: label-free and labeling methods. Here we describe specific techniques involved in both quantitative workflows; these include extensive sample preparation methods for several lung-specific sample types. Methods are also included for mass spectrometry-based sample analysis and data analysis. While the focus is on quantitative, clinical proteomics, these strategies are appropriate for a wide array of sample types and applications.

Advancements in omics technologies have increased our potential to evaluate molecular changes in a rapid and comprehensive manner. This is especially true in mass spectrometry-based metabolomics where improvements, including ease of use, in high-performance liquid chromatography (HPLC), column chemistries, instruments, software, and molecular databases, have advanced the field considerably. Applications of this relatively new omics technology in clinical research include discovering disease biomarkers, finding new drug targets, and elucidating disease mechanisms. Here we describe a typical clinical metabolomics workflow, which includes the following steps: (1) extraction of metabolites from the lung, plasma, bronchoalveolar lavage, or cells; (2) sample analysis via liquid chromatography-mass spectrometry; and (3) data analysis using commercial and freely available software packages. Overall, the methods delineated here can help investigators use metabolomics to discovery novel biomarkers and to understand lung diseases.

Mass spectrometry-based metabolomics is being increasingly utilized in various research fields including investigating human diseases, nutrition, industrial applications, and plant/natural products studies. Although new analytical approaches have enhanced the performance of metabolomic analyses, significant challenges associated with throughput, metabolome coverage, and compound identification still exist. Ion mobility mass spectrometry offers great potential for improving throughput and depth of coverage in metabolomics experiments. For example, multi-dimensional, structural resolution offered by ion mobility enables improved identification of metabolites and chemical classes. This mini-review discusses the advantages, recent developments and limitations of using ion mobility mass spectrometry as part of a metabolomics workflow.

A commercial liquid chromatography/drift tube ion mobility-mass spectrometer (LC/IM-MS) was evaluated for its utility in global metabolomics analysis. Performance was assessed using 12 targeted metabolite standards where the limit of detection (LOD), linear dynamic range, resolving power, and collision cross section (Ω) are reported for each standard. Data were collected in three different instrument operation modes: flow injection analysis with IM-MS (FIA/IM-MS), LC/MS, and LC/IM-MS. Metabolomics analyses of human plasma and HaCaT cells were used to compare the above three operation modes. LC/MS provides linearity in response, data processing automation, improved limits of detection, and ease of use. Advantages of LC/IM-MS and FIA/IM-MS include the ability to develop mobility-mass trend lines for structurally similar biomolecules, increased peak capacity, reduction of chemical/matrix noise, improvement in signal-to-noise, and separations of isobar/isomer compounds that are not resolved by LC. We further tested the feasibility of incorporating IM-MS into conventional LC/MS metabolomics workflows. In general, the addition of ion mobility dimension has increased the separation of compounds in complex biological matrixes and has the potential to largely improve the throughput of metabolomics analysis.

Galectin-9 is a pleiotropic immune modulator affecting numerous cell types of innate and adaptive immunity. Patients with chronic infection with either hepatitis C virus (HCV) or HIV have elevated circulating levels. Limited data exist on the regulation of natural killer (NK) cell function through interaction with galectin-9. We found that galectin-9 ligation downregulates multiple immune-activating genes, including eight involved in the NK cell-mediated cytotoxicity pathway, impairs lymphokine-activated killing, and decreases the proportion of gamma interferon (IFN-γ)-producing NK cells that had been stimulated with interleukin-12 (IL-12)/IL-15. We demonstrate that the transcriptional and functional changes induced by galectin-9 are independent of Tim-3. Consistent with these results for humans, we find that the genetic absence of galectin-9 in mice is associated with greater IFN-γ production by NK cells and enhanced degranulation. We also show that in the setting of a short-term (4-day) murine cytomegalovirus infection, terminally differentiated NKs accumulate in the livers of galectin-9 knockout mice, and that hepatic NKs spontaneously produce significantly more IFN-γ in this setting. Taken together, our results indicate that galectin-9 engagement impairs the function of NK cells, including cytotoxicity and cytokine production.

Background Asthma is a complex lung disease resulting from the interplay of genetic and environmental factors. To understand the molecular changes that occur during the development of allergic asthma without genetic and environmental confounders, an experimental model of allergic asthma in mice was used. Our goals were to (1) identify changes at the small molecule level due to allergen exposure, (2) determine perturbed pathways due to disease, and (3) determine whether small molecule changes correlate with lung function. Methods In this experimental model of allergic asthma, matched bronchoalveolar lavage (BAL) fluid and plasma were collected from three groups of C57BL6 mice (control vs sensitized and/or challenged with ovalbumin, n=3-5/group) 6h, 24h, and 48h after the last challenge. Samples were analyzed using liquid chromatography mass spectrometry-based metabolomics. Airway hyperresponsiveness (AHR) measurements and differential cell counts were performed. Results In total, 398 and 368 dysregulated metabolites in the BAL fluid and plasma of sensitized and challenged mice were identified, respectively. These belonged to four, interconnected pathways relevant to asthma pathogenesis: sphingolipid metabolism (p=6.6x10-5), arginine and proline metabolism (p=1.12x10-7), glycerophospholipid metabolism (p=1.3x10-10), and the neurotrophin signaling pathway (p=7.0x10-6). Furthermore, within the arginine and proline metabolism pathway, a positive correlation between urea-1-carboxyate and AHR was observed in plasma metabolites, while ornithine revealed a reciprocal effect. In addition, agmatine positively correlated with lung eosinophilia. Conclusion These findings point to potential targets and pathways that may be central to asthma pathogenesis and can serve as novel therapeutic targets. This article is protected by copyright. All rights reserved.

The interaction of αβ T-cell antigen receptors (TCRs) with peptides bound to MHC molecules lies at the center of adaptive immunity. Whether TCRs have evolved to react with MHC or, instead, processes in the thymus involving coreceptors and other molecules select MHC-specific TCRs de novo from a random repertoire is a longstanding immunological question. Here, using nuclease-targeted mutagenesis, we address this question in vivo by generating three independent lines of knockin mice with single-amino acid mutations of conserved class II MHC amino acids that often are involved in interactions with the germ-line–encoded portions of TCRs. Although the TCR repertoire generated in these mutants is similar in size and diversity to that in WT mice, the evolutionary bias of TCRs for MHC is suggested by a shift and preferential use of some TCR subfamilies over others in mice expressing the mutant class II MHCs. Furthermore, T cells educated on these mutant MHC molecules are alloreactive to each other and to WT cells, and vice versa, suggesting strong functional differences among these repertoires. Taken together, these results highlight both the flexibility of thymic selection and the evolutionary bias of TCRs for MHC.

OBJECTIVE To investigate autoantigens in β-cells, we have used a panel of pathogenic T-cell clones that were derived from the NOD mouse. Our particular focus in this study was on the identification of the target antigen for the highly diabetogenic T-cell clone BDC-5.2.9. RESEARCH DESIGN AND METHODS To purify β-cell antigens, we applied sequential size exclusion chromatography and reverse-phase high-performance liquid chromatography to membrane preparations of β-cell tumors. The presence of antigen was monitored by measuring the interferon-γ production of BDC-5.2.9 in response to chromatographic fractions in the presence of NOD antigen-presenting cells. Peak antigenic fractions were analyzed by ion-trap mass spectrometry, and candidate proteins were further investigated through peptide analysis and, where possible, testing of islet tissue from gene knockout mice. RESULTS Mass-spectrometric analysis revealed the presence of islet amyloid polypeptide (IAPP) in antigen-containing fractions. Confirmation of IAPP as the antigen target was demonstrated by the inability of islets from IAPP-deficient mice to stimulate BDC-5.2.9 in vitro and in vivo and by the existence of an IAPP-derived peptide that strongly stimulates BCD-5.2.9. CONCLUSIONS IAPP is the target antigen for the diabetogenic CD4 T-cell clone BDC-5.2.9.

T cells target peptide combos One of the enduring mysteries of autoimmunity is the identity of the specific proteins targeted by autoimmune T cells. Delong et al. used mass spectrometry to elucidate the peptide targets of autoimmune T cells isolated from a mouse model of type 1 diabetes. T cells targeted hybrid peptides formed by the covalent linking of a peptide derived from pro-insulin to other peptides derived from proteins found in pancreatic beta cells. T cells isolated from the pancreatic islets of two individuals with type 1 diabetes also recognized such hybrid peptides, suggesting that they may play an important role in driving disease. Science , this issue p. 711

Large-scale functional genomics approaches are fundamental to the characterization of mammalian transcriptomes annotated by genome sequencing projects. Although current high-throughput strategies systematically survey either transcriptional or biochemical networks, analogous genome-scale investigations that analyze gene function in mammalian cells have yet to be fully realized. Through transient overexpression analysis, we describe the parallel interrogation of approximately 20,000 sequence annotated genes in cancer-related signaling pathways. For experimental validation of these genome data, we apply an integrative strategy to characterize previously unreported effectors of activator protein-1 (AP-1) mediated growth and mitogenic response pathways. These studies identify the ADP-ribosylation factor GTPase-activating protein Centaurin alpha1 and a Tudor domain-containing hypothetical protein as putative AP-1 regulatory oncogenes. These results provide insight into the composition of the AP-1 signaling machinery and validate this approach as a tractable platform for genome-wide functional analysis.

Metabolomics is an emerging field which enables profiling of samples from living organisms in order to obtain insight into biological processes. A vital aspect of metabolomics is sample preparation whereby inconsistent techniques generate unreliable results. This technique encompasses protein precipitation, liquid-liquid extraction, and solid-phase extraction as a means of fractionating metabolites into four distinct classes. Improved enrichment of low abundance molecules with a resulting increase in sensitivity is obtained, and ultimately results in more confident identification of molecules. This technique has been applied to plasma, bronchoalveolar lavage fluid, and cerebrospinal fluid samples with volumes as low as 50 µl. Samples can be used for multiple downstream applications; for example, the pellet resulting from protein precipitation can be stored for later analysis. The supernatant from that step undergoes liquid-liquid extraction using water and strong organic solvent to separate the hydrophilic and hydrophobic compounds. Once fractionated, the hydrophilic layer can be processed for later analysis or discarded if not needed. The hydrophobic fraction is further treated with a series of solvents during three solid-phase extraction steps to separate it into fatty acids, neutral lipids, and phospholipids. This allows the technician the flexibility to choose which class of compounds is preferred for analysis. It also aids in more reliable metabolite identification since some knowledge of chemical class exists.

The Class 3 aldehyde dehydrogenase gene (ALDH3) is expressed differentially in a tissue-specific manner, occurring constitutively in some tissues and in others as a result of xenobiotic induction via the Ah receptor/ARNT pathway. ARNT is also involved in regulating gene expression in response to hypoxia. It dimerizes with hypoxia-inducible factor 1 alpha (HIF-1 alpha) and enhances expression of hypoxia-responsive genes. To determine if ARNT plays a role in regulating ALDH3 in response to low oxygen tension, we studied the effects of 1% oxygen and the hypoxia mimic cobalt chloride on constitutive and inducible ALDH3 expression in rat hepatoma cells and rat corneal epithelial cells. Hypoxia sharply down-regulates constitutive ALDH3 expression in corneal epithelial cells. Likewise, aromatic hydrocarbon-induced ALDH3 expression in H4-II-EC3 cells is significantly reduced by hypoxia. In contrast, hypoxia has no effect on constitutive or aromatic hydrocarbon-inducible ALDH3 expression in HTC cells. Our data indicate that hypoxia exerts cell type-specific effects on both constitutive and induced ALDH3 expression.

Cigarette smoke exposure is linked to the development of a variety of chronic lung and systemic diseases in susceptible individuals. Metabolomics approaches may aid in defining disease phenotypes, may help predict responses to treatment, and could identify biomarkers of risk for developing disease. Using a mouse model of chronic cigarette smoke exposure sufficient to cause mild emphysema, we investigated whether cigarette smoke induces distinct metabolic profiles and determined their persistence following smoking cessation. Metabolites were extracted from plasma and fractionated based on chemical class using liquid-liquid and solid-phase extraction prior to performing liquid chromatography mass spectrometry-based metabolomics. Metabolites were evaluated for statistically significant differences among group means (p-value≤0.05) and fold change ≥1.5). Cigarette smoke exposure was associated with significant differences in amino acid, purine, lipid, fatty acid, and steroid metabolite levels compared to air exposed animals. Whereas 60% of the metabolite changes were reversible, 40% of metabolites remained persistently altered even following 2 months of smoking cessation, including nicotine metabolites. Validation of metabolite species and translation of these findings to human plasma metabolite signatures induced by cigarette smoking may lead to the discovery of biomarkers or pathogenic pathways of smoking-induced disease.

Genomics and proteomics have emerged as key technologies in biomedical research, resulting in a surge of interest in training by investigators keen to incorporate these technologies into their research. At least two types of training can be envisioned in order to produce meaningful results, quality publications and successful grant applications: (1) immediate short-term training workshops and (2) long-term graduate education or visiting scientist programs. We aimed to fill the former need by providing a comprehensive hands-on training course in genomics, proteomics and informatics in a coherent, experimentally-based framework. This was accomplished through a National Heart, Lung, and Blood Institute (NHLBI)-sponsored 10-day Genomics and Proteomics Hands-on Workshop held at National Jewish Health (NJH) and the University of Colorado School of Medicine (UCD). The course content included comprehensive lectures and laboratories in mass spectrometry and genomics technologies, extensive hands-on experience with instrumentation and software, video demonstrations, optional workshops, online sessions, invited keynote speakers, and local and national guest faculty. Here we describe the detailed curriculum and present the results of short- and long-term evaluations from course attendees. Our educational program consistently received positive reviews from participants and had a substantial impact on grant writing and review, manuscript submissions and publications.

Motivation: Although R packages exist for the pre-processing of metabolomic data, they currently do not incorporate additional analysis steps of summarization, filtering and normalization of aligned data. We developed the MSPrep R package to complement other packages by providing these additional steps, implementing a selection of popular normalization algorithms and generating diagnostics to help guide investigators in their analyses. Availability: http://www.sourceforge.net/projects/msprep Contact: grant.hughes@ucdenver.edu Supplementary Information: Supplementary materials are available at Bioinformatics online.

Technological innovations have increased our potential to evaluate global changes in protein and small molecule levels in a rapid and comprehensive manner. This is especially true in mass spectrometry-based research where improvements, including ease-of-use, in high performance liquid chromatography (HPLC), column chemistries, instruments, software, and molecular databases have advanced the fields of proteomics and metabolomics considerably. Applications of these technologies in clinical research include biomarker discovery, drug targeting, and elucidating molecular networks, and a systems-based approach, utilizing multiple "omics," can also be taken. While the exact choice of workflow can dramatically impact the results of a study, the basic steps are similar, both within and between metabolomics and proteomics experiments. Although gel-based methods of quantitation are still widely used, our laboratory focuses on mass spectrometry-based methods, specifically protein and small molecule profiling.

Autoreactive CD4(+) T cells are involved in the pathogenesis of many autoimmune diseases, but the antigens that stimulate their responses have been difficult to identify and in most cases are not well defined. In the nonobese diabetic (NOD) mouse model of type 1 diabetes, we have identified the peptide WE14 from chromogranin A (ChgA) as the antigen for highly diabetogenic CD4(+) T cell clones. Peptide truncation and extension analysis shows that WE14 bound to the NOD mouse major histocompatibility complex class II molecule I-A(g7) in an atypical manner, occupying only the carboxy-terminal half of the I-A(g7) peptide-binding groove. This finding extends the list of T cell antigens in type 1 diabetes and supports the idea that autoreactive T cells respond to unusually presented self peptides.

Objective To describe clinical proteomics from discovery techniques and their limitations, to applications in allergy, asthma, and immunology, and finally to how proteomics can be integrated into clinical practice. Data Sources Despite many inherent challenges, proteomics-based methods have become a powerful and popular means of profiling clinical samples for the purpose of biomarker discovery. Although several strategies exist, clinical proteomics for the purpose of biomarker discovery generally focuses on 1 of 3 basic workflows: (1) 2-dimensional gel electrophoresis to quantitate relative protein levels followed by mass spectrometry (MS) to identify proteins of interest, (2) non-gel-based methods that rely on liquid chromatography MS (LCMS) for both quantitation and identification of proteins, and (3) protein profiling methods that do not directly result in the identification of proteins but rather generate "fingerprints" that are compared among individuals or samples. Study Selection Regardless of the strategy being pursued, a few general experimental steps are followed that will be expounded on in the text. These proteomics techniques have been applied to discover new biomarkers in biofluids and tissues from individuals with a variety of conditions, including allergy, asthma, atopic dermatitis, inflammatory diseases, chronic obstructive pulmonary disease, and other lung diseases. Results After biomarker discovery, LCMS-based proteomics offers several advantages over traditional antibody-based clinical assays, including greater specificity, cost- and time-effectiveness, and the potential to multiplex up to hundreds of peptides in a single assay. Conclusion With many guidelines now in place and model studies on which to design future experiments, there is reason to be optimistic that candidate protein biomarkers will be discovered using proteomics and translated into clinical assays.

Background Recombinant human activated protein C (rhAPC) is associated with improved survival in high-risk patients with severe sepsis; however, the effects of both lipopolysaccharide (LPS) and rhAPC on the bronchoalveolar lavage fluid (BALF) proteome are unknown. Methods Using differential in gel electrophoresis (DIGE) we identified changes in the BALF proteome from 10 healthy volunteers given intrapulmonary LPS in one lobe and saline in another lobe. Subjects were randomized to pretreatment with saline or rhAPC. Results An average of 255 protein spots were detected in each proteome. We found 31 spots corresponding to 8 proteins that displayed abundance increased or decreased at least 2-fold after LPS. Proteins that decreased after LPS included surfactant protein A, immunoglobulin J chain, fibrinogen-γ, α1-antitrypsin, immunoglobulin, and α2-HS-glycoprotein. Haptoglobin increased after LPS-treatment. Treatment with rhAPC was associated with a larger relative decrease in immunoglobulin J chain, fibrinogen-γ, α1-antitrypsin, and α2-HS-glycoprotein. Conclusion Intrapulmonary LPS was associated with specific protein changes suggesting that the lung response to LPS is more than just a loss of integrity in the alveolar epithelial barrier; however, pretreatment with rhAPC resulted in minor changes in relative BALF protein abundance consistent with its lack of affect in ALI and milder forms of sepsis.

The rat class 3 aldehyde dehydrogenase gene (ALDH3A1) is expressed constitutively or by xenobiotic induction depending on the tissue in which it occurs. Although the mechanism that mediates inducible expression has been well characterized, relatively little is known about constitutive regulatory mechanisms. Previous ALDH3A1 promoter analyses have indicated that primary regulatory regions within the ALDH3A1 5' flanking region exert similar effects on both constitutive and inducible ALDH3A1 expression. However, promoter gene analyses that served as the basis of early work were limited by the lack of sufficient 5' flanking region sequence. To gain a more complete picture of how the 5' flanking region regulates both modes of expression, we have subcloned an 8.0-kilobase (kb) fragment from the 5' flanking region of the ALDH3A1 gene and subjected it to reporter gene analyses. We found a region located between 4.8 and 7.8 kb upstream of the noncoding first exon that drives strong ALDH3A1 reporter activity. This region contains xenobiotic response element consensus sequences that mediate constitutive and inducible ALDH3A1 reporter gene expression. Using the new generation of ALDH3A1 reporter constructs, we were unable to confirm the presence of a negative regulatory region that was apparent in previous studies using a shorter fragment of the 5' flanking region. We also demonstrate that 3-methylcholanthrene induces ALDH3A1 expression above high constitutive background in corneal epithelial cells.

Expression of cyclin dependent kinase (Cdk) inhibitor p27(Kip1), which blocks cell cycle progression from G(1) to S phase, can be regulated via multiple mechanisms including transcription, protein degradation, and translation. Recently, it was shown that p27(Kip1) plays an important role in the cellular response to hypoxia. However, the mechanisms involved in the hypoxia-induced regulation of p27(Kip1) expression are still not clear. In this study, we compare the expression of p27(Kip1) in two related murine hepatoma cell lines, Hepa-1 and c4. Hepa-1 produces functional aryl hydrocarbon receptor nuclear translocator (ARNT). c4 cells are derived from Hepa-1, but are ARNT deficient. Interestingly, we observed cell line-dependent effects of hypoxia on the expression of p27(Kip1). The level of p27(Kip1) protein in Hepa-1 cells is enhanced by hypoxia, but is reduced by hypoxia in c4 cells. Further investigation demonstrated that hypoxia-induced, ARNT-mediated, transactivation of the p27(Kip1) gene in Hepa-1 cells is responsible for the increase in p27(Kip1) protein. Once c4 cells were stably transfected with the wild type ARNT gene, a hypoxia-induced increase in p27(Kip1) mRNA was observed and reduction of p27(Kip1) protein caused by hypoxia was blocked. Hence, our data indicate that ARNT is involved in transcriptional upregulation of the p27(Kip1) gene under hypoxic conditions.

We have previously shown that expression of the Class 3 aldehyde dehydrogenase gene (ALDH3) is abrogated by hypoxia. This phenomenon occurs in rat hepatoma systems in which ALDH3 expression is xenobiotic-inducible as well as in rat primary corneal epithelial cells that exhibit high constitutive ALDH3 expression. We have begun to test various segments of the ALDH3 5' flanking region for elements that may mediate this effect using CAT reporter gene constructs. In addition, although the involvement of the Ah receptor nuclear translocator (ARNT) in xenobiotic induction of ALDH3 is well established, the role of ARNT in constitutive ALDH3 expression is not clear. Moreover, ARNT is also a component of the hypoxia inducible factor-1 (HIF-1) bipartite transcription factor complex that mediates hypoxic induction of a variety of genes. Concomitant activation of the xenobiotic and hypoxia pathways results in cross-talk and functional interference. It has been hypothesized that this interference is due to limiting levels of ARNT. To examine if ARNT levels are limiting during hypoxic and xenobiotic induction in the context of ALDH3 expression and to examine possible roles of ARNT in constitutive expression of ALDH3 in corneal epithelial cells we co-transfected rat corneal epithelial cells and H4-II-EC3 rat hepatoma cells with ALDH3 5' UTR-CAT reporter genes and expression vectors containing either wild type or dominant negative forms of ARNT. Our results indicate that during hypoxia and xenobiotic induction of ALDH3 in H4-II-EC3 cells ARNT is not the limiting transcription factor. Further, neither wild type nor dominant negative ARNT had effects on constitutive ALDH3 expression in corneal epithelial cells.

Class ldehyde dehydrogenase (ALDH3) is an enzyme that efficiently converts a varietf endogenous as well as exogenous aldehydes to their corresponding carboxylic acids.r some time now the tissue distribution, protein and gene sequence of ALDH3 have b known. A low Km for medium length aliphatic as well as aromatic aldehydes is the maof ALDH3. The ALDH3 monomer is 453 amino acids long, and unlike other mammal ALDHs, the native protein is a homodimer. Recently, the ALDH3 crystal struct has been determined and, arguably, nearly understood. ALDHS is expressed differentially in mamian tissues, being constitutively expressed in cornea, stomach, lung and urry tract, and only inducible by xenobiotics in liver; it is also found in certain cancer cells.