Protein expression profiles of intestinal epithelial co-cultures: effect of functionalised carbon nanotube exposure.

To assess the biological effects of low level, water dispersible, functionalised carbon nanotube (f-CNT) exposure in an in vitro model simulating the digestive tract, cellular protein expression was quantified and compared using label-free quantitative mass spectrometry (LFQMS). Co-cultured cells were exposed to well-characterised SWCNT-COOH, MWCNT-COOH, and MWCNT-PVP. The relative expression of 2,282 unique proteins was compared across the dose groups. 428 proteins were found to be differentially expressed. At the high dose, the extent of differential protein expression was CNT-specific and directly related to CNT colloidal stability. Cells responded to low level MWCNT-PVP exposure with three-fold greater differential expression. Bioinformatic analysis indicated significant and f-CNT-specific effects on relevant molecular and cellular functions and canonical pathways, with little overlap across f-CNT type and in the absence of overt toxicity.

Ethanol exposure alters protein expression in a mouse model of fetal alcohol spectrum disorders.

Alcohol exposure during development can result in variable growth retardation and facial dysmorphology known as fetal alcohol spectrum disorders. Although the mechanisms underlying the disorder are not fully understood, recent progress has been made that alcohol induces aberrant changes in gene expression and in the epigenome of embryos. To inform the gene and epigenetic changes in alcohol-induced teratology, we used whole-embryo culture to identify the alcohol-signature protein profile of neurulating C6 mice. Alcohol-treated and control cultures were homogenized, isoelectrically focused, and loaded for 2D gel electrophoresis. Stained gels were cross matched with analytical software. We identified 40 differentially expressed protein spots (P < 0.01), and 9 spots were selected for LC/MS-MS identification. Misregulated proteins include serotransferrin, triosephosphate isomerase and ubiquitin-conjugating enzyme E2 N. Misregulation of serotransferrin and triosephosphate isomerase was confirmed with immunologic analysis. Alteration of proteins with roles in cellular function, cell cycle, and the ubiquitin-proteasome pathway was induced by alcohol. Several misregulated proteins interact with effectors of the NF-κB and Myc transcription factor cascades. Using a whole-embryo culture, we have identified misregulated proteins known to be involved in nervous system development and function.

Ethanol-induced changes in the expression of proteins related to neurotransmission and metabolism in different regions of the rat brain.

Despite extensive description of the damaging effects of chronic alcohol exposure on brain structure, mechanistic explanations for the observed changes are just emerging. To investigate regional brain changes in protein expression levels following chronic ethanol treatment, one rat per sibling pair of male Wistar rats was exposed to intermittent (14 h/day) vaporized ethanol, the other to air for 26 weeks. At the end of 24 weeks of vapor exposure, the ethanol group had blood ethanol levels averaging 450 mg%, had not experienced a protracted (> 16 h) withdrawal from ethanol, and revealed only mild evidence of hepatic steatosis. Extracted brains were micro-dissected to isolate the prefrontal cortex (PFC), dorsal striatum (STR), corpus callosum genu (CCg), CC body (CCb), anterior vermis (AV), and anterior dorsal lateral cerebellum (ADLC) for protein analysis with two-dimensional gel electrophoresis. Expression levels for 54 protein spots were significantly different between the ethanol- and air-treated groups. Of these 54 proteins, tandem mass spectroscopy successfully identified 39 unique proteins, the levels of which were modified by ethanol treatment: 13 in the PFC, 7 in the STR, 2 in the CCg, 7 in the CCb, 7 in the AV, and 5 in the ADLC. The functions of the proteins altered by chronic ethanol exposure were predominantly associated with neurotransmitter systems in the PFC and cell metabolism in the STR. Stress response proteins were elevated only in the PFC, AV, and ADLC perhaps supporting a role for frontocerebellar circuitry disruption in alcoholism. Of the remaining proteins, some had functions associated with cytoskeletal physiology (e.g., in the CCb) and others with transcription/translation (e.g., in the ADLC). Considered collectively, all but 4 of the 39 proteins identified in the present study have been previously identified in ethanol gene- and/or protein-expression studies lending support for their role in ethanol-related brain alterations.

Differential expression of renal proteins in a rodent model of Meckel syndrome.

BACKGROUND: Meckel syndrome (MKS) is a fatal autosomal recessive condition with prominent renal cystic pathology. Renal protein misexpression was evaluated in the Wpk rat model of human MKS3 gene disease to identify biomarkers for the staging of renal cystic progression. METHODS: Misexpressed proteins were compared between early and late stages of MKS renal cystic disease using proteomic analysis (two-dimensional gel electrophoresis with LC-MS/MS identification) followed by Western blot analysis. RESULTS: A proteomic analysis identified 76 proteins with statistically different, normalized abundance in at least one group. Subsequently, Western blot was used to confirm differential expression in several of these and polycystic kidney disease (PKD)-associated proteins. Galectin-1 and vimentin were identified as overexpressed proteins, which have been previously found in the jck mouse model of nephronophthisis 9. Ciliopathic PKD proteins, polycystins 1 & 2, and fibrocystin were also differentially expressed in Wpk kidney. CONCLUSION: In the Wpk rat, misexpressed proteins were identified that were also implicated in other forms of cystic disease. Numerous proteins were either over- or underexpressed in late-stage disease. Differences in protein expression may serve as biomarkers of cystic disease and its progression.

Protein changes in macrophages induced by plasma from rats exposed to 35 GHz millimeter waves.

A macrophage assay and proteomic screening were used to investigate the biological activity of soluble factors in the plasma of millimeter wave-exposed rats. NR8383 rat macrophages were incubated for 24 h with 10% plasma from male Sprague-Dawley rats that had been exposed to sham conditions, or exposed to 42 °C environmental heat or 35 GHz millimeter waves at 75 mW/cm² until core temperature reached 41.0 °C. Two-dimensional polyacrylamide gel electrophoresis, image analysis, and Western blotting were used to analyze approximately 600 protein spots in the cell lysates for changes in protein abundance and levels of 3-nitrotyrosine, a marker of macrophage stimulation. Proteins of interest were identified using peptide mass fingerprinting. Compared to plasma from sham-exposed rats, plasma from environmental heat- or millimeter wave-exposed rats increased the expression of 11 proteins, and levels of 3-nitrotyrosine in seven proteins, in the NR8383 cells. These altered proteins are associated with inflammation, oxidative stress, and energy metabolism. Findings of this study indicate both environmental heat and 35 GHz millimeter wave exposure elicit the release of macrophage-activating mediators into the plasma of rats.

Serum Proteomic Profiles In Subjects with Heavy Alcohol Abuse.

OBJECTIVES: The abuse of alcohol is a major public health problem, and the diagnosis and care of patients with alcohol abuse and dependence is hindered by the lack of tests that can detect dangerous levels of drinking or relapse during therapy. Gastroenterologists and other healthcare providers find it very challenging to obtain an accurate alcohol drinking history. We hypothesized that the effects of ethanol on numerous systems may well be reflected in changes in quantity or qualities of constituent or novel plasma proteins or protein fragments. Organ/tissue-specific proteins may be released into the blood stream when cells are injured by alcohol, or when systemic changes are induced by alcohol, and such proteins would be detected using a proteomic approach. The objective of this pilot study was to determine if there are plasma proteome profiles that correlate with heavy alcohol use. METHODS: Paired serum samples, before and after intensive alcohol treatment, were obtained from subjects who attended an outpatient alcohol treatment program. Serum proteomic profiles using MALDI -OTOF Mass Spectrometry were compared between pre- and post treatment samples. RESULTS: Of 16 subjects who enrolled in the study, 8 were females. The mean age of the study subjects was 49 yrs. The baseline laboratory data showed elevated AST (54 ± 37 IU/L), ALT (37 ± 19 IU/L), and MCV (99 ± 5 fl). Self-reported pre-treatment drinking levels for these subjects averaged 17 ± 7drinks/day and 103 ± 37 drinks/week. Mass spectrometry analyses showed a novel 5.9 kDa protein, a fragment of alpha fibrinogen, isoform 1, that might be might be a new novel marker for abusive alcohol drinking. CONCLUSIONS: We have shown in this pilot study that several potential protein markers have appeared in mass spectral profiles and that they may be useful clinically to determine the status of alcohol drinking by MALDI -OTOF mass spectrometry, especially a fragment of alpha fibrinogen, isoform 1. However, a large-scale study is needed to confirm and validate our current results.

A proteomic workflow for discovery of serum carrier protein-bound biomarker candidates of alcohol abuse using LC-MS/MS.

The diagnosis and care of patients with alcohol abuse and dependence is hampered by a lack of sensitive and specific screening and monitoring tests. Proteomics is a good approach to search for biomarkers of alcohol abuse. Serum carrier protein-bound proteins have attracted significant interest because they remain a relatively un-mined region of the proteome. In the present study, a proteomic workflow including LC-MS/MS with enrichment of serum carrier protein-bound biomarkers technique was applied to profile the changes in quality and quantity of serum carrier protein-bound proteins for the discovery of novel biomarker candidates of alcohol abuse. In total, 311 proteins identified with high confidence were discovered to be bound to serum carrier proteins. Complement isoforms, Ig fragments, and apolipoprotein family proteins are the main serum carrier-bound proteins. Protein quantification analysis with and without concern as to gender revealed that gender is a critical consideration for biomarker development in alcohol abuse. Identified proteins not previously associated with alcohol abuse include gelsolin, selenoprotein P, serotransferrin, tetranectin, hemopexin, histidine-rich glycoprotein, plasma kallikrein, and vitronectin. Altered abundance of these proteins suggests that they may be potential novel biomarkers for alcohol abuse.

Sample complexity reduction for two-dimensional electrophoresis using solution isoelectric focusing prefractionation.

Despite its excellent resolving power, 2-DE is of limited use when analyzing cellular proteomes, especially in differential expression studies. Frequently, fewer than 2000 protein spots are detected on a single 2-D gel (a fraction of the total proteome) regardless of the gel platform, sample, or detection method used. This is due to the vast number of proteins expressed and their equally vast dynamic range. To exploit 2-DE unique ability as both an analytical and a preparative tool, the significant sample prefractionation is necessary. We have used solution isoelectric focusing (sIEF) via the ZOOM IEF Fractionator (Invitrogen) to generate sample fractions from complex bacterial lysates, followed by parallel 2-DE, using narrow-range IPG strips that bracket the sIEF fractions. The net result of this process is a significant enrichment of the bacterial proteome resolved on multiple 2-D gels. After prefractionation, we detected 5525 spots, an approximate 3.5-fold increase over the 1577 spots detected in an unfractionated gel. We concluded that sIEF is an effective means of prefractionation to increase depth of field and improve the analysis of low-abundance proteins.

A proteomic survey of rat cerebral cortical synaptosomes.

Previous findings from our laboratory and others indicate that two-dimensional gel electrophoresis (2-DE) can be used to study protein expression in defined brain regions, but mainly the proteins which are present in high abundance in glia are readily detected. The current study was undertaken to determine the protein profile in a synaptosomal subcellular fraction isolated from the cerebral cortex of the rat. Both 2-DE and liquid chromatography - tandem mass spectrometry (LC-MS/MS) procedures were used to isolate and identify proteins in the synaptosomal fraction and accordingly >900 proteins were detected using 2-DE; the 167 most intense gel spots were isolated and identified with matrix-assisted laser desorption/ionization - time of flight peptide mass fingerprinting or LC-MS/MS. In addition, over 200 proteins were separated and identified with the LC-MS/MS "shotgun proteomics" technique, some in post-translationally modified form. The following classes of proteins associated with synaptic function were detected: (a) proteins involved in synaptic vesicle trafficking-docking (e.g., SNAP-25, synapsin I and II, synaptotagmin I, II, and V, VAMP-2, syntaxin 1A and 1B, etc.); (b) proteins that function as transporters or receptors (e.g., excitatory amino acid transporters 1 and 2, GABA transporter 1); (c) proteins that are associated with the synaptic plasma membrane (e.g., post-synaptic density-95/synapse-associated protein-90 complex, neuromodulin (GAP-43), voltage-dependent anion-selective channel protein (VDACs), sodium-potassium ATPase subunits, alpha 2 spectrin, septin 7, etc.); and (d) proteins that mediate intracellular signaling cascades that modulate synaptic function (e.g., calmodulin, calcium-calmodulin-dependent protein kinase subunits, etc.). Other identified proteins are associated with mitochondrial or general cytosolic function. Of the two proteins identified as endoplasmic reticular, both interact with the synaptic SNARE complex to regulate vesicle trafficking. Taken together, these results suggest that the integrity of the synaptosomes was maintained during the isolation procedure and that this subcellular fractionation technique enables the enrichment of proteins associated with synaptic function. The results also suggest that this experimental approach can be used to study the differential expression of multiple proteins involved in alterations of synaptic function.