For label-free expression profiling of tissue proteomes, efficient protein extraction, thorough and quantitative sample cleanup and digestion procedures, as well as sufficient and reproducible chromatographic separation, are highly desirable but remain challenging. However, optimal methodology has remained elusive, especially for proteomes that are rich in membrane proteins, such as the mitochondria. Here we describe a straightforward and reproducible sample preparation procedure, coupled with a highly selective and sensitive nano-LC/Orbitrap analysis, which enables reliable and comprehensive expression profiling of tissue mitochondria. The mitochondrial proteome of swine heart was selected as a test system. Efficient protein extraction was accomplished using a strong buffer containing both ionic and non-ionic detergents. Overnight precipitation was used for cleanup of the extract, and the sample was subjected to an optimized 2-step, on-pellet digestion approach. In the first step, the protein pellet was dissolved via a 4 h tryptic digestion under vigorous agitation, which nano-LC/LTQ/ETD showed to produce large and incompletely cleaved tryptic peptides. The mixture was then reduced, alkylated, and digested into its full complement of tryptic peptides with additional trypsin. This solvent precipitation/on-pellet digestion procedure achieved significantly higher and more reproducible peptide recovery of the mitochondrial preparation, than observed using a prevalent alternative procedure for label-free expression profiling, SDS-PAGE/ingel digestion (87% vs. 54%). Furthermore, uneven peptide losses were lower than observed with SDS-PAGE/in-gel digestion. The resulting peptides were sufficiently resolved by a 5 h gradient using a nano-LC configuration that features a low-void-volume, high chromatographic reproducibility, and an LTQ/Orbitrap analyzer for protein identification and quantification. The developed method was employed for label-free comparison of the mitochondrial proteomes of myocardium from healthy animals vs. those with hibernating myocardium. Each experimental group consisted of a relatively large number of animals (n=10), and samples were analyzed in random order to minimize quantitative false-positives. Using this approach, 904 proteins were identified and quantified with high confidence, and those mitochondrial proteins that were altered significantly between groups were

Abstract-Hibernating myocardium is accompanied by a downregulation in energy utilization that prevents the immediate development of ischemia during stress at the expense of an attenuated level of regional contractile function. We used a discovery based proteomic approach to identify novel regional molecular adaptations responsible for this phenomenon in subendocardial samples from swine instrumented with a chronic LAD stenosis. After 3 months (nϭ8), hibernating myocardium was present as reflected by reduced resting LAD flow (0.75Ϯ0.14 versus 1.19Ϯ0.14 mL ⅐ min Ϫ1 ⅐ g Ϫ1 in remote) and wall thickening (1.93Ϯ0.46 mm versus 5.46Ϯ0.41 mm in remote, PϽ0.05). Regionally altered proteins were quantified with 2D Differential-in-Gel Electrophoresis (2D-DIGE) using normal myocardium as a reference with identification of candidates using MALDI-TOF mass spectrometry. Hibernating myocardium developed a significant downregulation of many mitochondrial proteins and an upregulation of stress proteins. Of particular note, the major entry points to oxidative metabolism (eg, pyruvate dehydrogenase complex and Acyl-CoA dehydrogenase) and enzymes involved in electron transport (eg, complexes I, III, and V) were reduced (PϽ0.05). Multiple subunits within an enzyme complex frequently showed a concordant downregulation in abundance leading to an amplification of their cumulative effects on activity (eg, "total" LAD PDC activity was 21.9Ϯ3.1 versus 42.8Ϯ1.9 mU, PϽ0.05). After 5-months (nϭ10), changes in mitochondrial and stress proteins persisted whereas cytoskeletal proteins (eg, desmin and vimentin) normalized. These data indicate that the proteomic phenotype of hibernating myocardium is dynamic and has similarities to global changes in energy substrate metabolism and function in the advanced failing heart. These proteomic changes may limit oxidative injury and apoptosis and impact functional recovery after revascularization. Key Words: metabolism Ⅲ proteomics Ⅲ hibernating myocardium Ⅲ ischemic heart disease H ibernating myocardium is characterized by viable, chronically dysfunctional myocardium that develops in response to repetitive myocardial ischemia. 1,2 We have previously demonstrated that the relation between regional oxygen consumption, coronary flow, and function in response to stress is attenuated in hibernating myocardium and thus dissociated from the usual determinants of myocardial oxygen demand. 3 By reducing regional energy utilization, hibernation prevents the development of ischemia after submaximal stress. This is supported by a lack of biochemical markers of ischemia and preservation of total ATP and creatine phosphate content in swine with hibernating myocardium 3,4 as well as human biopsies from patients without significant fibrosis. 5 Although there has been interest in identifying the role of increased glucose uptake in these responses, maximal insulin stimulated glucose uptake is unchanged in chronic hibernating myocardium, and alterations in other metabolic pathways responsible for the attenuated increase...

G protein–coupled receptors (GPCRs) that couple to Gi/o proteins modulate neurotransmission presynaptically by inhibiting exocytosis. Release of Gβγ subunits from activated G proteins decreases the activity of voltage-gated Ca2+ channels (VGCCs), decreasing excitability. A less understood Gβγ-mediated mechanism downstream of Ca2+ entry is the binding of Gβγ to SNARE complexes, which facilitate the fusion of vesicles with the cell plasma membrane in exocytosis. Here, we generated mice expressing a form of the SNARE protein SNAP25 with premature truncation of the C terminus and that were therefore partially deficient in this interaction. SNAP25Δ3 homozygote mice exhibited normal presynaptic inhibition by GABAB receptors, which inhibit VGCCs, but defective presynaptic inhibition by receptors that work directly on the SNARE complex, such as 5-hydroxytryptamine (serotonin) 5-HT1b receptors and adrenergic α2a receptors. Simultaneously stimulating receptors that act through both mechanisms showed synergistic inhibitory effects. SNAP25Δ3 homozygote mice had various behavioral phenotypes, including increased stress-induced hyperthermia, defective spatial learning, impaired gait, and supraspinal nociception. These data suggest that the inhibition of exocytosis by Gi/o-coupled GPCRs through the Gβγ-SNARE interaction is a crucial component of numerous physiological and behavioral processes.

The plasma proteome holds enormous clinical potentials, yet an in-depth analysis of the plasma proteome remains a daunting challenge due to its high complexity and the extremely-wide dynamic range in protein concentrations. Furthermore, existing antibody-based approaches for depleting high-abundance proteins are not adaptable to the analysis of animal plasma proteome, which are often essential for experimental pathology/pharmacology. Here we describe a highly-comprehensive method for the investigation of animal plasma proteomes, which employs an optimized combinatorial peptide ligand libraries (CPLL) treatment to reduce the protein concentration dynamic range and a dual-enzyme, dual-activation strategy to achieve high proteomic coverage. The CPLL-treatment enriched the lower-abundance proteins by >100-fold when loading the samples in moderately-denaturing condition with multiple loading-washing cycles. The native and the CPLL-treated plasma were digested in-parallel respectively by two enzymes (trypsin and GluC) carrying orthogonal specificities. By performing this differential proteolysis, the proteome coverage is improved where peptides produced by only one enzyme are poorly detectable. Digests were fractionated with high-resolution SCX chromatography and then resolved on a long, heated nano-LC column. MS analysis was performed on an LTQ/Orbitrap respectively with two complementary activation methods (CID and ETD). We applied this optimized strategy to investigate the plasma proteome from swine, a prominent animal model for cardiovascular diseases(CVD). This large-scale analysis results in an identification of a total 3421 unique proteins, spanning a concentration range of 9–10 orders of magnitude. The proteins were identified under a set of commonly-accepted criteria including precursor mass error<15 ppm, Xcorr cutoffs, ≥two unique peptides at the peptide probability≥95% and protein probability≥99%, and the peptide FDR of the dataset was 1.8% as estimated by searching reversed database. CPLL treatment resulted in 55% more identified proteins over these from native plasma; moreover, compared with using only trypsin and CID, the dual enzyme/activation approach enabled the identification of 2.6-fold more proteins and substantially higher sequence coverage for most individual proteins. Further analysis revealed 657 proteins as significantly associated with CVD (p<0.05), which constitute five CVD-related pathways. This study represents the first in-depth investigation of non-human plasma proteome and the strategy developed here is adaptable to the comprehensive analysis of other highly complex proteomes.

G-coupled G protein-coupled receptors can inhibit neurotransmitter release at synapses via multiple mechanisms. In addition to Gβγ-mediated modulation of voltage-gated calcium channels (VGCC), inhibition can also be mediated through the direct interaction of Gβγ subunits with the soluble -ethylmaleimide attachment protein receptor (SNARE) complex of the vesicle fusion apparatus. Binding studies with soluble SNARE complexes have shown that Gβγ binds to both ternary SNARE complexes, t-SNARE heterodimers, and monomeric SNAREs, competing with synaptotagmin 1(syt1) for binding sites on t-SNARE. However, in secretory cells, Gβγ, SNAREs, and synaptotagmin interact in the lipid environment of a vesicle at the plasma membrane. To approximate this environment, we show that fluorescently labeled Gβγ interacts specifically with lipid-embedded t-SNAREs consisting of full-length syntaxin 1 and SNAP-25B at the membrane, as measured by fluorescence polarization. Fluorescently labeled syt1 undergoes competition with Gβγ for SNARE-binding sites in lipid environments. Mutant Gβγ subunits that were previously shown to be more efficacious at inhibiting Ca-triggered exocytotic release than wild-type Gβγ were also shown to bind SNAREs at a higher affinity than wild type in a lipid environment. These mutant Gβγ subunits were unable to inhibit VGCC currents. Specific peptides corresponding to regions on Gβ and Gγ shown to be important for the interaction disrupt the interaction in a concentration-dependent manner. In fusion assays using full-length t- and v-SNAREs embedded in liposomes, Gβγ inhibited Ca/synaptotagmin-dependent fusion. Together, these studies demonstrate the importance of these regions for the Gβγ-SNARE interaction and show that the target of Gβγ, downstream of VGCC, is the membrane-embedded SNARE complex.