An optimized workflow for MS-based quantitative proteomics of challenging clinical bronchoalveolar lavage fluid (BALF) samples.

BACKGROUND: Clinical bronchoalveolar lavage fluid (BALF) samples are rich in biomolecules, including proteins, and useful for molecular studies of lung health and disease. However, mass spectrometry (MS)-based proteomic analysis of BALF is challenged by the dynamic range of protein abundance, and potential for interfering contaminants. A robust, MS-based proteomics compatible sample preparation workflow for BALF samples, including those of small and large volume, would be useful for many researchers. RESULTS: We have developed a workflow that combines high abundance protein depletion, protein trapping, clean-up, and in-situ tryptic digestion, that is compatible with either qualitative or quantitative MS-based proteomic analysis. The workflow includes a value-added collection of endogenous peptides for peptidomic analysis of BALF samples, if desired, as well as amenability to offline semi-preparative or microscale fractionation of complex peptide mixtures prior to LC-MS/MS analysis, for increased depth of analysis. We demonstrate the effectiveness of this workflow on BALF samples collected from COPD patients, including for smaller sample volumes of 1-5 mL that are commonly available from the clinic. We also demonstrate the repeatability of the workflow as an indicator of its utility for quantitative proteomic studies. CONCLUSIONS: Overall, our described workflow consistently provided high quality proteins and tryptic peptides for MS analysis. It should enable researchers to apply MS-based proteomics to a wide-variety of studies focused on BALF clinical specimens.

Metabolic and Metabolomic Effects of Metformin in Murine Model of Pulmonary Adenoma Formation.

Epidemiologic studies of diabetic patients treated with metformin identified significantly lower incidences of cancer. From this, there is growing interest in the use of metformin to treat and prevent cancer. Studies have investigated chemopreventive mechanisms including alterations in calorie intake, cancer metabolism, and cell signaling. Repurposing the drug is challenging due to its metabolic effects and non-uniform effects on different types of cancer. In our previously published studies, we observed that benzo[a]pyrene treated mice receiving metformin significantly reduced lung adenomas; however, mice had reduced weight gain. In this study, we compared chemoprevention diets with and without metformin to evaluate the effects of diet vs. effects of metformin. We also performed tandem mass spectrometry on mouse serum to assess metabolomic alterations associated with metformin treatment. In metformin cohorts, the rate of weight gain was reduced, but weights did not vary between diets. There was no weight difference between diets without metformin. Interestingly, caloric intake was increased in metformin treated mice. Metabolomic analysis revealed metabolite alterations consistent with metformin treatment. Based on these results, we conclude that previous reductions in lung adenomas may have been occurred from anticancer effects of metformin rather than a potentially toxic effect such as calorie restriction.

Global phosphoproteomic profiling of skeletal muscle in ovarian hormone-deficient mice.

Protein phosphorylation is important in skeletal muscle development, growth, regeneration, and contractile function. Alterations in the skeletal muscle phosphoproteome due to aging have been reported in males; however, studies in females are lacking. We have demonstrated that estrogen deficiency decreases muscle force, which correlates with decreased myosin regulatory light chain phosphorylation. Thus, we questioned whether the decline of estrogen in females that occurs with aging might alter the skeletal muscle phosphoproteome. C57BL/6J female mice (6 mo) were randomly assigned to a sham-operated (Sham) or ovariectomy (Ovx) group to investigate the effects of estrogen deficiency on skeletal muscle protein phosphorylation in a resting, noncontracting condition. After 16 wk of estrogen deficiency, the tibialis anterior muscle was dissected and prepped for label-free nano-liquid chromatography-tandem mass spectrometry phosphoproteomic analysis. We identified 4,780 phosphopeptides in tibialis anterior muscles of ovariectomized (Ovx) and Sham-operated (Sham) control mice. Further analysis revealed 647 differentially regulated phosphopeptides (Benjamini-Hochberg adjusted P value < 0.05 and 1.5-fold change ratio) that corresponded to 130 proteins with 22 proteins differentially phosphorylated (3 unique to Ovx, 2 unique to Sham, 6 upregulated, and 11 downregulated). Differentially phosphorylated proteins associated with the sarcomere, cytoplasm, and metabolic and calcium signaling pathways were identified. Our work provides the first global phosphoproteomic analysis in females and how estrogen deficiency impacts the skeletal muscle phosphoproteome.

Multiomic profiling of iron-deficient infant monkeys reveals alterations in neurologically important biochemicals in serum and cerebrospinal fluid before the onset of anemia.

The effects of iron deficiency (ID) during infancy extend beyond the hematologic compartment and include short- and long-term adverse effects on many tissues including the brain. However, sensitive biomarkers of iron-dependent brain health are lacking in humans. To determine whether serum and cerebrospinal fluid (CSF) biomarkers of ID-induced metabolic dysfunction are concordant in the pre/early anemic stage of ID before anemia in a nonhuman primate model of infantile iron deficiency anemia (IDA). ID (n = 7), rhesus infants at 4 mo (pre-anemic period) and 6 mo of age (anemic) were examined. Hematological, metabolomic, and proteomic profiles were generated via HPLC/MS at both time points to discriminate serum biomarkers of ID-induced brain metabolic dysfunction. We identified 227 metabolites and 205 proteins in serum. Abnormalities indicating altered liver function, lipid dysregulation, and increased acute phase reactants were present in ID. In CSF, we measured 210 metabolites and 1,560 proteins with changes in ID infants indicative of metabolomic and proteomic differences indexing disrupted synaptogenesis. Systemic and CSF proteomic and metabolomic changes were present and concurrent in the pre-anemic and anemic periods. Multiomic serum and CSF profiling uncovered pathways disrupted by ID in both the pre-anemic and anemic stages of infantile IDA, including evidence for hepatic dysfunction and activation of acute phase response. Parallel changes observed in serum and CSF potentially provide measurable serum biomarkers of ID that reflect at-risk brain processes prior to progression to clinical anemia.

Cerebral adrenoleukodystrophy is associated with loss of tolerance to profilin.

Childhood cerebral adrenoleukodystrophy (cALD) is a devastating manifestation of ALD accompanied by demyelination, inflammation, and blood brain barrier (BBB) disruption with shared characteristics of an auto-immune disease. We utilized plasma samples pre- and postdevelopment of cALD to determine the presence of specific auto-antibodies. Mass spectrometry of protein specifically bound with post-cALD plasma antibody identified Profilin1 (PFN1) as the target. In a screen of 94 boys with cALD 48 (51%) had anti-PFN1 antibodies, whereas only 2/29 boys with ALD but without cerebral disease, and 0/30 healthy controls showed anti-PFN1 immunoreactivity. Cerebral spinal fluid from those with cALD showed higher levels of PFN1 protein compared with non-cALD samples (324 ± 634 versus 42 ± 23 pg/mL, p = 0.04). Boys that were anti-PFN positive had a significant increase in the amount of gadolinium signal observed on MRI when compared to boys that were anti-PFN1 negative (p = 0.04) possibly indicating increased BBB disruption. Anti-PFN1 positivity was also associated with elevated levels of very long chain fatty acids (C26 of 1.12 ± 0.41 versus 0.97 ± 0.30 mg/dL, p = 0.03) and increased plasma BAFF (973 ± 277 versus 733 ± 269 pg/mL, p = 0.03). In conclusion, anti-PFN may be a novel biomarker associated with the development of cALD in boys with ALD.

Quantitative proteomics of forebrain subcellular fractions in fragile X mental retardation 1 knockout mice following acute treatment with 2-Methyl-6-(phenylethynyl)pyridine: Relevance to developmental study of schizophrenia.

The fragile X mental retardation 1 knockout (Fmr1 KO) mouse replicates behavioral deficits associated with autism, fragile X syndrome, and schizophrenia. Less is known whether protein expression changes are consistent with findings in subjects with schizophrenia. In the current study, we used liquid chromatography tandem mass spectrometry (LC-MS/MS) proteomics to determine the protein expression of four subcellular fractions in the forebrains of Fmr1 KO mice vs. C57BL/6 J mice and the effect of a negative allosteric modulator of mGluR5-2-Methyl-6-(phenylethynyl)pyridine (MPEP)-on protein expression. Strain- and treatment-specific differential expression of proteins was observed, many of which have previously been observed in the brains of subjects with schizophrenia. Western blotting verified the direction and magnitude of change for several proteins in different subcellular fractions as follows: neurofilament light protein (NEFL) and 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNP) in the total homogenate; heterogeneous nuclear ribonucleoproteins C1/C2 (HNRNPC) and heterogeneous nuclear ribonucleoprotein D0 (HNRNPD) in the nuclear fraction; excitatory amino acid transporter 2 (EAAT2) and ras-related protein rab 3a (RAB3A) in the synaptic fraction; and ras-related protein rab 35 (RAB35) and neuromodulin (GAP43) in the rough endoplasmic reticulum fraction. Individuals with FXS do not display symptoms of schizophrenia. However, the biomarkers that have been identified suggest that the Fmr1 KO model could potentially be useful in the study of schizophrenia.

An O-Methyltransferase Is Required for Infection of Tick Cells by Anaplasma phagocytophilum.

Anaplasma phagocytophilum, the causative agent of Human Granulocytic Anaplasmosis (HGA), is an obligately intracellular α-proteobacterium that is transmitted by Ixodes spp ticks. However, the pathogen is not transovarially transmitted between tick generations and therefore needs to survive in both a mammalian host and the arthropod vector to complete its life cycle. To adapt to different environments, pathogens rely on differential gene expression as well as the modification of proteins and other molecules. Random transposon mutagenesis of A. phagocytophilum resulted in an insertion within the coding region of an o-methyltransferase (omt) family 3 gene. In wild-type bacteria, expression of omt was up-regulated during binding to tick cells (ISE6) at 2 hr post-inoculation, but nearly absent by 4 hr p.i. Gene disruption reduced bacterial binding to ISE6 cells, and the mutant bacteria that were able to enter the cells were arrested in their replication and development. Analyses of the proteomes of wild-type versus mutant bacteria during binding to ISE6 cells identified Major Surface Protein 4 (Msp4), but also hypothetical protein APH_0406, as the most differentially methylated. Importantly, two glutamic acid residues (the targets of the OMT) were methyl-modified in wild-type Msp4, whereas a single asparagine (not a target of the OMT) was methylated in APH_0406. In vitro methylation assays demonstrated that recombinant OMT specifically methylated Msp4. Towards a greater understanding of the overall structure and catalytic activity of the OMT, we solved the apo (PDB_ID:4OA8), the S-adenosine homocystein-bound (PDB_ID:4OA5), the SAH-Mn2+ bound (PDB_ID:4PCA), and SAM- Mn2+ bound (PDB_ID:4PCL) X-ray crystal structures of the enzyme. Here, we characterized a mutation in A. phagocytophilum that affected the ability of the bacteria to productively infect cells from its natural vector. Nevertheless, due to the lack of complementation, we cannot rule out secondary mutations.

Mosaic composition of ribA and wspB genes flanking the virB8-D4 operon in the Wolbachia supergroup B-strain, wStr.

The obligate intracellular bacterium, Wolbachia pipientis (Rickettsiales), is a widespread, vertically transmitted endosymbiont of filarial nematodes and arthropods. In insects, Wolbachia modifies reproduction, and in mosquitoes, infection interferes with replication of arboviruses, bacteria and plasmodia. Development of Wolbachia as a tool to control pest insects will be facilitated by an understanding of molecular events that underlie genetic exchange between Wolbachia strains. Here, we used nucleotide sequence, transcriptional and proteomic analyses to evaluate expression levels and establish the mosaic nature of genes flanking the T4SS virB8-D4 operon from wStr, a supergroup B-strain from a planthopper (Hemiptera) that maintains a robust, persistent infection in an Aedes albopictus mosquito cell line. Based on protein abundance, ribA, which contains promoter elements at the 5'-end of the operon, is weakly expressed. The 3'-end of the operon encodes an intact wspB, which encodes an outer membrane protein and is co-transcribed with the vir genes. WspB and vir proteins are expressed at similar, above average abundance levels. In wStr, both ribA and wspB are mosaics of conserved sequence motifs from Wolbachia supergroup A- and B-strains, and wspB is nearly identical to its homolog from wCobU4-2, an A-strain from weevils (Coleoptera). We describe conserved repeated sequence elements that map within or near pseudogene lesions and transitions between A- and B-strain motifs. These studies contribute to ongoing efforts to explore interactions between Wolbachia and its host cell in an in vitro system.

Quantitative analysis of the murine lipid droplet-associated proteome during diet-induced hepatic steatosis.

Hepatic steatosis is characterized by the accumulation of lipid droplets (LDs), which are composed of a neutral lipid core surrounded by a phospholipid monolayer embedded with many proteins. Although the LD-associated proteome has been investigated in multiple tissues and organisms, the dynamic changes in the murine LD-associated proteome in response to obesity and hepatic steatosis have not been studied. We characterized the hepatic LD-associated proteome of C57BL/6J male mouse livers following high-fat feeding using isobaric tagging for relative and absolute quantification. Of the 1,520 proteins identified with a 5% local false discovery rate, we report a total of 48 proteins that were increased and 52 proteins that were decreased on LDs in response to high-fat feeding. Most notably, ribosomal and endoplasmic reticulum proteins were increased and extracellular and cytosolic proteins were decreased in response to high-fat feeding. Additionally, many proteins involved in fatty acid catabolism or xenobiotic metabolism were enriched in the LD fraction following high-fat feeding. In contrast, proteins involved in glucose metabolism and liver X receptor or retinoid X receptor activation were decreased on LDs of high-fat-fed mice. This study provides insights into unique biological functions of hepatic LDs under normal and steatotic conditions.

Proteomic profiling of a robust Wolbachia infection in an Aedes albopictus mosquito cell line.

Wolbachia pipientis, a widespread vertically transmitted intracellular bacterium, provides a tool for insect control through manipulation of host-microbe interactions. We report proteomic characterization of wStr, a Wolbachia strain associated with a strong cytoplasmic incompatibility phenotype in its native host, Laodelphax striatellus. In the Aedes albopictus C/wStr1 mosquito cell line, wStr maintains a robust, persistent infection. MS/MS analyses of gel bands revealed a protein 'footprint' dominated by Wolbachia-encoded chaperones, stress response and cell membrane proteins, including the surface antigen WspA, a peptidoglycan-associated lipoprotein and a 73 kDa outer membrane protein. Functional classifications and estimated abundance levels of 790 identified proteins suggested that expression, stabilization and secretion of proteins predominate over bacterial genome replication and cell division. High relative abundances of cysteine desulphurase, serine/glycine hydroxymethyl transferase, and components of the α-ketoglutarate dehydrogenase complex in conjunction with above average abundances of glutamate dehydrogenase and proline utilization protein A support Wolbachia genome-based predictions for amino acid metabolism as a primary energy source. wStr expresses 15 Vir proteins of a Type IV secretion system and its transcriptional regulator. Proteomic characterization of a robust insect-associated Wolbachia strain provides baseline information that will inform further development of in vitro protocols for Wolbachia manipulation.

sdf1 Expression reveals a source of perivascular-derived mesenchymal stem cells in zebrafish.

There is accumulating evidence that mesenchymal stem cells (MSCs) have their origin as perivascular cells (PVCs) in vivo, but precisely identifying them has been a challenge, as they have no single definitive marker and are rare. We have developed a fluorescent transgenic vertebrate model in which PVC can be visualized in vivo based upon sdf1 expression in the zebrafish. Prospective isolation and culture of sdf1(DsRed) PVC demonstrated properties consistent with MSC including prototypical cell surface marker expression; mesodermal differentiation into adipogenic, osteogenic, and chondrogenic lineages; and the ability to support hematopoietic cells. Global proteomic studies performed by two-dimensional liquid chromatography and tandem mass spectrometry revealed a high degree of similarity to human MSC (hMSC) and discovery of novel markers (CD99, CD151, and MYOF) that were previously unknown to be expressed by hMSC. Dynamic in vivo imaging during fin regeneration showed that PVC may arise from undifferentiated mesenchyme providing evidence of a PVC-MSC relationship. This is the first model, established in zebrafish, in which MSC can be visualized in vivo and will allow us to better understand their function in a native environment.

Identification and origin of N-linked ß-D-N-acetylglucosamine monosaccharide modifications on Arabidopsis proteins.

Many plant proteins are modified with N-linked oligosaccharides at asparagine-X-serine/threonine sites during transit through the endoplasmic reticulum and the Golgi. We have identified a number of Arabidopsis (Arabidopsis thaliana) proteins with modifications consisting of an N-linked N-acetyl-D-glucosamine monosaccharide (N-GlcNAc). Electron transfer dissociation mass spectrometry analysis of peptides bearing this modification mapped the modification to asparagine-X-serine/threonine sites on proteins that are predicted to transit through the endoplasmic reticulum and Golgi. A mass labeling method was developed and used to study N-GlcNAc modification of two thioglucoside glucohydrolases (myrosinases), TGG1 and TGG2 (for thioglucoside glucohydrolase). These myrosinases are also modified with high-mannose (Man)-type glycans. We found that N-GlcNAc and high-Man-type glycans can occur at the same site. It has been hypothesized that N-GlcNAc modifications are generated when endo-ß-N-acetylglucosaminidase (ENGase) cleaves N-linked glycans. We examined the effects of mutations affecting the two known Arabidopsis ENGases on N-GlcNAc modification of myrosinase and found that modification of TGG2 was greatly reduced in one of the single mutants and absent in the double mutant. Surprisingly, N-GlcNAc modification of TGG1 was not affected in any of the mutants. These data support the hypothesis that ENGases hydrolyze high-Man glycans to produce some of the N-GlcNAc modifications but also suggest that some N-GlcNAc modifications are generated by another mechanism. Since N-GlcNAc modification was detected at only one site on each myrosinase, the production of the N-GlcNAc modification may be regulated.

Non-ATG-initiated translation directed by microsatellite expansions.

Trinucleotide expansions cause disease by both protein- and RNA-mediated mechanisms. Unexpectedly, we discovered that CAG expansion constructs express homopolymeric polyglutamine, polyalanine, and polyserine proteins in the absence of an ATG start codon. This repeat-associated non-ATG translation (RAN translation) occurs across long, hairpin-forming repeats in transfected cells or when expansion constructs are integrated into the genome in lentiviral-transduced cells and brains. Additionally, we show that RAN translation across human spinocerebellar ataxia type 8 (SCA8) and myotonic dystrophy type 1 (DM1) CAG expansion transcripts results in the accumulation of SCA8 polyalanine and DM1 polyglutamine expansion proteins in previously established SCA8 and DM1 mouse models and human tissue. These results have implications for understanding fundamental mechanisms of gene expression. Moreover, these toxic, unexpected, homopolymeric proteins now should be considered in pathogenic models of microsatellite disorders.

Natural aging and ovariectomy induces parallel phosphoproteomic alterations in skeletal muscle of female mice.

The loss of skeletal muscle strength mid-life in females is associated with the decline of estrogen. Here, we questioned how estrogen deficiency might impact the overall skeletal muscle phosphoproteome after contraction, as force production induces phosphorylation of several muscle proteins. Phosphoproteomic analyses of the tibialis anterior muscle after contraction in two mouse models of estrogen deficiency, ovariectomy (Ovariectomized (Ovx) vs. Sham) and natural aging-induced ovarian senescence (Older Adult (OA) vs. Young Adult (YA)), identified a total of 2,593 and 3,507 phosphopeptides in Ovx/Sham and OA/YA datasets, respectively. Further analysis of estrogen deficiency-associated proteins and phosphosites identified 66 proteins and 21 phosphosites from both datasets. Of these, 4 estrogen deficiency-associated proteins and 4 estrogen deficiency-associated phosphosites were significant and differentially phosphorylated or regulated, respectively. Comparative analyses between Ovx/Sham and OA/YA using Ingenuity Pathway Analysis (IPA) found parallel patterns of inhibition and activation across IPA-defined canonical signaling pathways and physiological functional analysis, which were similarly observed in downstream GO, KEGG, and Reactome pathway overrepresentation analysis pertaining to muscle structural integrity and contraction, including AMPK and calcium signaling. IPA Upstream regulator analysis identified MAPK1 and PRKACA as candidate kinases and calcineurin as a candidate phosphatase sensitive to estrogen. Our findings highlight key molecular signatures and pathways in contracted muscle suggesting that the similarities identified across both datasets could elucidate molecular mechanisms that may contribute to skeletal muscle strength loss due to estrogen deficiency.

Desmoglein 2 Functions as a Receptor for Fatty Acid Binding Protein 4 in Breast Cancer Epithelial Cells.

Fatty acid binding protein 4 (FABP4) is a secreted adipokine linked to obesity and progression of a variety of cancers. Obesity increases extracellular FABP4 (eFABP4) levels in animal models and in obese breast cancer patients compared with lean healthy controls. Using MCF-7 and T47D breast cancer epithelial cells, we show herein that eFABP4 stimulates cellular proliferation in a time and concentration dependent manner while the non-fatty acid-binding mutant, R126Q, failed to potentiate growth. When E0771 murine breast cancer cells were injected into mice, FABP4 null animals exhibited delayed tumor growth and enhanced survival compared with injections into control C57Bl/6J animals. eFABP4 treatment of MCF-7 cells resulted in a significant increase in phosphorylation of extracellular signal-regulated kinase 1/2 (pERK), transcriptional activation of nuclear factor E2-related factor 2 (NRF2) and corresponding gene targets ALDH1A1, CYP1A1, HMOX1, SOD1 and decreased oxidative stress, while R126Q treatment did not show any effects. Proximity-labeling employing an APEX2-FABP4 fusion protein revealed several proteins functioning in desmosomes as eFABP4 receptor candidates including desmoglein (DSG), desmocollin, junction plankoglobin, desomoplankin, and cytokeratins. AlphaFold modeling predicted an interaction between eFABP4, and the extracellular cadherin repeats of DSG2 and pull-down and immunoprecipitation assays confirmed complex formation that was potentiated by oleic acid. Silencing of DSG2 in MCF-7 cells attenuated eFABP4 effects on cellular proliferation, pERK levels, and ALDH1A1 expression compared with controls. IMPLICATIONS: These results suggest desmosomal proteins, and in particular desmoglein 2, may function as receptors of eFABP4 and provide new insight into the development and progression of obesity-associated cancers.

Organelle interactions compartmentalize hepatic fatty acid trafficking and metabolism.

Organelle interactions play a significant role in compartmentalizing metabolism and signaling. Lipid droplets (LDs) interact with numerous organelles, including mitochondria, which is largely assumed to facilitate lipid transfer and catabolism. However, quantitative proteomics of hepatic peridroplet mitochondria (PDM) and cytosolic mitochondria (CM) reveals that CM are enriched in proteins comprising various oxidative metabolism pathways, whereas PDM are enriched in proteins involved in lipid anabolism. Isotope tracing and super-resolution imaging confirms that fatty acids (FAs) are selectively trafficked to and oxidized in CM during fasting. In contrast, PDM facilitate FA esterification and LD expansion in nutrient-replete medium. Additionally, mitochondrion-associated membranes (MAM) around PDM and CM differ in their proteomes and ability to support distinct lipid metabolic pathways. We conclude that CM and CM-MAM support lipid catabolic pathways, whereas PDM and PDM-MAM allow hepatocytes to efficiently store excess lipids in LDs to prevent lipotoxicity.

Glycoprotein nonmetastatic melanoma protein B (GNMPB) as a novel biomarker for cerebral adrenoleukodystrophy.

Adrenoleukodystrophy (ALD) is an X-linked peroxisomal disease caused by a mutation in the ABCD1 gene, producing mutations in the very long chain fatty acid transporter, ALD protein. Cerebral ALD (cALD) is a severe phenotype of ALD with neuroinflammation and neurodegeneration. Elevated levels of Glycoprotein Nonmetastatic Melanoma Protein B (GNMPB) have been recently documented in neurodegenerative diseases such as Alzheimer's disease, Multiple Sclerosis and Amyotrophic Lateral Sclerosis. Our objective was to measure the levels cerebral spinal fluid (CSF) GNMPB in cALD patients to determine if GNMPB could be a potential biomarker in tracking cALD disease progression. CSF GNMPB levels were significantly higher in cALD patients versus controls (2407 ± 1672 pg/mL vs. 639.5 ± 404 pg/mL, p = 0.0009). We found a positive correlation between CSF GNMPB and MRI disease severity score levels (R2 = 0.3225, p < 0.0001) as well as the gadolinium intensity score (p = 0.0204). Boys with more severe neurologic deficits also had higher levels of CSF GNMPB (p < 0.0001). A positive correlation was shown between CSF GNMPB and another biomarker, chitotriosidase (R2 = 0.2512, p = 0.0244). These data show that GNMPB could be a potential biomarker of cALD disease state and further studies should evaluate it as a predictor of the disease progression.

Tandem mass tag proteomic and untargeted metabolomic profiling reveals altered serum and CSF biochemical datasets in iron deficient monkeys.

The effects of early-life iron deficiency anemia (IDA) extend past the blood and include both short- and long-term adverse effects on many tissues including the brain. Prior to IDA, iron deficiency (ID) can cause similar tissue effects, but a sensitive biomarker of iron-dependent brain health is lacking. To determine serum and CSF biomarkers of ID-induced metabolic dysfunction we performed proteomic and metabolomic analysis of serum and CSF at 4- and 6- months from a nonhuman primate model of infantile IDA. LC/MS/MS analyses identified a total of 227 metabolites and 205 proteins in serum. In CSF, we measured 210 metabolites and 1,560 proteins. Data were either processed from a Q-Exactive (Thermo Scientific, Waltham, MA) through Progenesis QI with accurate mass and retention time comparisons to a proprietary small molecule database and Metlin or with raw files imported directly from a Fusion Orbitrap (Thermo Scientific, Waltham, MA) through Sequest in Proteome Discoverer 2.4.0.305 (Thermo Scientific, Waltham, MA) with peptide matches through the latest Rhesus Macaque HMDB database. Metabolite and protein identifiers, p-values, and q-values were utilized for molecular pathway analysis with Ingenuity Pathways Analysis (IPA). We applied multiway distance weighted discrimination (DWD) to identify a weighted sum of the features (proteins or metabolites) that distinguish ID from IS at 4-months (pre-anemic period) and 6-months of age (anemic).

Oxidative stress pathogenically remodels the cardiac myocyte cytoskeleton via structural alterations to the microtubule lattice.

In the failing heart, the cardiac myocyte microtubule network is remodeled, which contributes to cellular contractile failure and patient death. However, the origins of this deleterious cytoskeletal reorganization are unknown. We now find that oxidative stress, a condition characteristic of heart failure, leads to cysteine oxidation of microtubules. Our electron and fluorescence microscopy experiments revealed regions of structural damage within the microtubule lattice that occurred at locations of oxidized tubulin. The incorporation of GTP-tubulin into these damaged, oxidized regions led to stabilized "hot spots" within the microtubule lattice, which suppressed the shortening of dynamic microtubules. Thus, oxidative stress may act inside of cardiac myocytes to facilitate a pathogenic shift from a sparse microtubule network into a dense, aligned network. Our results demonstrate how a disease condition characterized by oxidative stress can trigger a molecular oxidation event, which likely contributes to a toxic cellular-scale transformation of the cardiac myocyte microtubule network.

Identification of candidate biomarkers in ovarian cancer serum by depletion of highly abundant proteins and differential in-gel electrophoresis.

Ovarian cancer is the fifth leading cause of cancer death for women in the US, yet survival rates are over 90% when it is diagnosed at an early stage, highlighting the need for biomarkers for early detection. To enhance the discovery of tumor-specific proteins that could represent novel serum biomarkers for ovarian cancer, we depleted serum of highly abundant proteins which can mask the detection of proteins present in serum at low concentrations. Three commercial immunoaffinity columns were used in parallel to deplete the highly abundant proteins in serum from 60 patients with serous ovarian carcinoma and 60 non-cancer controls. Medium and low abundance serum proteins from each serum pool were then evaluated by the quantitative proteomic technique of differential in-gel electrophoresis. The number of protein spots that were elevated in ovarian cancer sera by at least twofold ranged from 36 to 248, depending upon the depletion and separation methods. From the 33 spots picked for MS analysis, nine different proteins were identified, including the novel candidate ovarian cancer biomarkers leucine-rich alpha2 glycoprotein-1 and ficolin 3. Western blotting validated the relative increases in serum protein levels for three of the proteins identified, demonstrating the utility of this approach for the identification of novel serum biomarkers for ovarian cancer.