Guard cells (GC)1 are highly specialized cells that form tiny pores called stomata on the leaf surface. When environmental conditions change, guard cells can rapidly change shape so that the pores open or close to control leaf gas exchange and water transpiration. Mesophyll cells (MC) are mainly parenchyma cells between the upper and lower epidermis specialized for photosynthesis. Previous studies that focused on guard cell metabolism and response to environmental signals have revealed important features of functional differentiation of GC (1, 2). Compared with MC, GC contain few chloroplasts with very limited structures and thus possess very low photosynthetic capability. The Calvin cycle in GC only assimilates 2-4% of CO 2 fixed in MC (3). In contrast, GC contain abundant mitochondria and display a high respiratory rate, suggesting that oxidative phosphorylation is an important source of ATP to fuel the guard cell machinery (4). Using microarrays covering just one-third of the Arabidopsis genome, Leonhardt et al. (5) observed a differential abscisic acid (ABA) modulation of many guard cell ABA signaling components as well as key enzymes involved in carbon metabolism in GC and MC. This only available large scale genomics study identified 1309 guard cell expressed genes of which 64 transcripts mainly involved in transcription, signaling, and cytoskeleton were preferentially expressed in GC compared with MC. However, functional grouping of the genes revealed only a 1.9% higher representation of photosynthesis genes in MC than in GC. The percentages of genes in all other categories such as protein turnover, defense, signaling, channels and transporters, and metabolism are similar between the two distinct cell types (5). These proteins are known to play specific roles in guard cell functions (6). This highlights the necessity of studying guard cell functions at the protein level.To date, there have been very few analyses of single celltype proteomes in plants. Proteome analyses of trichomes from Arabidopsis (7) and tobacco (8) and root hairs from soybean (9) exist but have identified fewer than 100 proteins per proteome. The proteomes of pollen from different species have been relatively well studied, but the pollen grains are not single cells because they contain two/three-cell gametophytes (10). A critical factor for large scale proteomics analFrom the ‡Department

Androgenic chemicals are present in the environment at concentrations that impair reproductive processes in fish. The objective of this experiment was to identify proteins and cell processes mediated through androgen receptor signaling using an androgen receptor agonist (17beta-trenbolone) and antagonist (flutamide) in the liver. Female fathead minnows were exposed to nominal concentrations of either 17beta-trenbolone (0.05, 0.5, or 5 microg/L), flutamide (50, 150, or 500 microg/L), or a mixture (500 microg flutamide/L and 0.5 microg 17beta-trenbolone/L) for 48 h. The iTRAQ method was used to label peptides after protein extraction and trypsin-digestion from livers of untreated controls or from fish treated with 17beta-trenbolone (5 microg/L), flutamide (500 microg/L), or a mixture of both compounds. Forty-five proteins were differentially altered by one or more treatments (p<0.05). Many altered proteins were involved in cellular metabolism (e.g., glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate mutase), general and oxidative stress response (e.g., superoxide dismutase and heat shock proteins), and the regulation of translation (e.g., ribosomal proteins). Cellular pathway analysis identified additional signaling cascades activated or inhibited by flutamide that may not be androgen receptor mediated. We also compared changes in select proteins to changes in their mRNA levels and observed, in general, that proteins and mRNA changes did not correlate, suggesting complex regulation at the level of both the transcriptome and proteome. It is concluded that both transcriptomic and proteomic approaches offer unique and complementary insights into mechanisms of regulation. We demonstrate the utility of proteomic profiling for use on a model species with value to ecotoxicology but having limited genomic information.

The extracellular accumulation of β-amyloid peptide is a key trigger in the pathogenesis of Alzheimer's disease (AD). In humans, amyloid deposition precedes the appearance of intracellular inclusion pathology formed by cytosolic proteins such as Tau, α-synuclein and TDP-43. These secondary pathologies have not been observed in mice that model Alzheimer-type amyloidosis by expressing mutant amyloid precursor protein, with or without mutant presenilin 1. The lack of secondary pathology in these models has made it difficult to establish how amyloid deposition initiates the cascade of events that leads to secondary intracellular pathology that characterizes human AD. In transgenic mice that model Alzheimer-type amyloidosis, we sought to determine whether there is evidence of altered cytosolic protein folding by assessing whether amyloid deposition causes normally soluble proteins to misfold. Using a method that involved detergent extraction and sedimentation coupled with proteomic approaches, we identified numerous cytosolic proteins that show specific losses in solubility as amyloid accumulates. The proteins identified included glycolytic enzymes and members of the 14-3-3 chaperone family. A substantial accumulation of lysine 48-linked polyubiquitin was also detected. Overall, the data demonstrate that the accumulation of amyloid by some manner causes the loss of solubility intracellular cytosolic proteins.

Heat-shock is an acute insult to the mammalian proteome. The sudden elevation in temperature has far-reaching effects on protein metabolism, leads to a rapid inhibition of most protein synthesis, and the induction of protein chaperones. Using heat-shock in cells of neuronal (SH-SY5Y) and glial (CCF-STTG1) lineage, in conjunction with detergent extraction and sedimentation followed by LC-MS/MS proteomic approaches, we sought to identify human proteins that lose solubility upon heat-shock. The two cell lines showed largely overlapping profiles of proteins detected by LC-MS/MS. We identified 58 proteins in detergent insoluble fractions as losing solubility in after heat shock; 10 were common between the 2 cell lines. A subset of the proteins identified by LC-MS/MS was validated by immunoblotting of similarly prepared fractions. Ultimately, we were able to definitively identify 3 proteins as putatively metastable neural proteins; FEN1, CDK1, and TDP-43. We also determined that after heat-shock these cells accumulate insoluble polyubiquitin chains largely linked via lysine 48 (K-48) residues. Collectively, this study identifies human neural proteins that lose solubility upon heat-shock. These proteins may represent components of the human proteome that are vulnerable to misfolding in settings of proteostasis stress.

We report the rapid discovery of putative protein biomarkers of traumatic brain injury (TBI) by SDS-PAGE-capillary liquid chromatography-tandem mass spectrometry (SDS-PAGE-Capillary LC-MS(2)). Ipsilateral hippocampus (IH) samples were collected from naive rats and rats subjected to controlled cortical impact (a rodent model of TBI). Protein database searching with 15,558 uninterpreted MS(2) spectra, collected in 3 days via data-dependent capillary LC-MS(2) of pooled cyanine dye-labeled samples separated by SDS-PAGE, identified more than 306 unique proteins. Differential proteomic analysis revealed differences in protein sequence coverage for 170 mammalian proteins (57 in naive only, 74 in injured only, and 39 of 64 in both), suggesting these are putative biomarkers of TBI. Confidence in our results was obtained by the presence of several known biomarkers of TBI (including alphaII-spectrin, brain creatine kinase, and neuron-specific enolase) in our data set. These results show that SDS-PAGE prior to in vitro proteolysis and capillary LC-MS(2) is a promising strategy for the rapid discovery of putative protein biomarkers associated with a specific physiological state (i.e., TBI) without a priori knowledge of the molecules involved.