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 traffickingdocking (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.

The fast, efficient, and accurate release of proteins from cells and tissues is a critically important initial step in most analytical processes and is essential to reliable proteomic analyses. Two-dimensional gel electrophoresis (2DGE) 1 can be an accurate representation of a proteome only if the entire protein constituency of cells is recovered during the sample preparation process. Pressure cycling technology (PCT) uses alternating cycles of high and low hydrostatic pressure to effectively induce the lysis of cells and tissues in preparation for 2DGE and other analytical or preparative methods. Rapid cycling between high and low pressure is more disruptive than high pressure alone, as evidenced by the increased protein yields from Saccharomyces cerevisae correlating to the number of pressure cycles rather than the total elapsed time at high pressure [1]. Similarly, Herrero and coworkers [2] reported a 20% increase in phycobiliproteins yielded from Spirulina platensis when multiple iterations of a pressurized liquid extraction method were performed. Previously, Geiser and coworkers [3] reported the release of 37% more protein from the nematode Caenorhabditis elegans by PCT than by sonication. From gram-negative bacteria, PCT reportedly yielded 14.2% more protein from Escherichia coli than did bead beating [4,5] and yielded 17.1% more protein from Rhodopseudomonas palustris than did enzymatic lysis with lysozyme [6]. For mammalian tissues, PCT also isolated more protein from liver, including several unique proteins that were not isolated by conventional homogenization techniques [7]. From adipose tissue, PCT extracted more protein than did pulverization under liquid nitrogen and detergent extraction of the triturate. The Barocycler NEP-3229 instrument, disposable polypropylene PULSE Tubes FT-500, and ProteoSOLVE IEF Reagent were obtained from Pressure BioSciences (West Bridgewater, MA, USA). Linear immobilized pH gradients (IPGs, pH 3-10) were obtained from Proteome Systems (Woburn, MA, USA). Nonlinear IPGs and PDQuest version 7.1 image analysis software were obtained from Bio-Rad (Hercules, CA, USA). Ultrafree-CL

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.

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.

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.