Proteomic Analysis of Integral Plasma Membrane Proteins

Efficient methods for profiling proteins integral to the plasma membrane are highly desirable for the identification of overexpressed proteins in disease cells. Such methods will aid in both understanding basic biological processes and discovering protein targets for the design of therapeutic monoclonal antibodies. Avoiding contamination by subcellular organelles and cytosolic proteins is crucial to the successful proteomic analysis of integral plasma membrane proteins. Here we report a biotin-directed affinity purification (BDAP) method for the preparation of integral plasma membrane proteins, which involves (1) biotinylation of cell surface membrane proteins in viable cells, (2) affinity enrichment using streptavidin beads, and (3) depletion of plasma membrane-associated cytosolic proteins by harsh washes with high-salt and high-pH buffers. The integral plasma membrane proteins are then extracted and subjected to SDS−PAGE separation and HPLC/MS/MS for protein identification. We used the BDAP method to prepare integral plasma membrane proteins from a human lung cancer cell line. Western blotting analysis showed that the preparation was almost completely devoid of actin, a major cytosolic protein. Nano-HPLC/MS/MS analysis of only 30 μg of protein extracted from the affinity-enriched integral plasma membrane preparation led to the identification of 898 unique proteins, of which 781 were annotated with regard to their plasma membrane localization. Among the annotated proteins, at least 526 (67.3%) were integral plasma membrane proteins. Notable among them were 62 prenylated proteins and 45 Ras family proteins. To our knowledge, this is the most comprehensive proteomic analysis of integral plasma membrane proteins in mammalian cells to date. Given the importance of integral membrane proteins for drug design, the described approach will expedite the characterization of plasma membrane subproteomes and the discovery of plasma membrane protein drug targets

Capillary High-Performance Liquid Chromatography/Mass Spectrometric Analysis of Proteins from Affinity-Purified Plasma Membrane

Proteomics analysis of plasma membranes is a potentially powerful strategy for the discovery of proteins involved in membrane remodeling under diverse cellular environments and identification of disease-specific membrane markers. A key factor for successful analysis is the preparation of plasma membrane fractions with low contamination from subcellular organelles. Here we report the characterization of plasma membrane prepared by an affinity-purification method, which involves biotinylation of cell-surface proteins and subsequent affinity enrichment with strepavidin beads. Western blotting analysis showed this method was able to achieve a 1600-fold relative enrichment of plasma membrane versus mitochondria and a 400-fold relative enrichment versus endoplasmic reticulum, two major contaminants in plasma membrane fractions prepared by conventional ultracentrifugation methods. Capillary-HPLC/MS analysis of 30 μg of affinity-purified plasma membrane proteins led to the identification of 918 unique proteins, which include 16.4% integral plasma membrane proteins and 45.5% cytosol proteins (including 8.6% membrane-associated proteins). Notable among the identified membrane proteins include 30 members of ras superfamily, receptors (e.g., EGF receptor, integrins), and signaling molecules. The low number of endoplasmic reticulum and mitochondria proteins (∼3.3% of the total) suggests the plasma membrane preparation has minimum contamination from these organelles. Given the importance of integral membrane proteins for drug design and membrane-associated proteins in the regulation cellular behaviors, the described approach will help expedite the characterization of plasma membrane subproteomes, identify signaling molecules, and discover therapeutic membrane-protein targets in diseases

APN derived from PVAT protects mice from atherosclerosis.

<p>A, HE staining of arteria carotis sections from mice after perivascular collar placement for 12 weeks. B, Quantitative analysis of intimal surface area in arteria carotis of each groups (n = 6 per group). *P<0.05 versus sham. C, Analysis of intima/media ratio in arteriacarotis with each groups (n = 6 per group). D, Bar graph shows quantification of lumen stenosis in arteria carotis with each groups (n = 6 per group). *P<0.05 versus sham. E, HE staining of arteria carotis sections from ApoE-/- mice transplanted with WT or APN-/- PVAT 12 weeks after atherosclerosis established. F, Quantitative analysis of intimal surface area in arteria carotis with WT or APN-/- PVAT (n = 6 per group). *P<0.05 versus (WT) PVAT. G, Bar graph shows quantification of lumen stenosis in arteria carotis with WT or APN-/- PVAT (n = 6 per group). *P<0.05 versus (WT) PVAT. H, HE staining of atherosclerotic plaques from the indicated mice. I, plaque disruption rate of he indicated mice (n = 6 per group). *P<0.05 versus surgery control.</p

DataSheet1_Physicochemical and adsorptive properties of biochar derived from municipal sludge: sulfamethoxazole adsorption and underlying mechanism.docx

Municipal sludge waste could be transformed into useful biochar through pyrolysis process. In this study, municipal sludge-derived biochar (SBC) was successfully synthesized via the one-pot pyrolysis method, and the yield of sludge biochar gradually decreased with the pyrolysis temperature increased from 300°C to 800°C. The sludge biochar exhibited an alkaline surface due to the gradual accumulation of ash and the formation of carbonate and organic anion during high-temperature pyrolysis process. Moreover, the prepared samples were analyzed by different characterization techniques including BET, SEM, and XPS. Adsorption experiments using the optimized biochar sample of SBC800 resulted in a 95% sulfamethoxazole (SMX) removal efficiency and the maximum adsorption capacity of 7033.4 mg/kg, which was 47.5 times higher than that of SBC300. The adsorption process of SBC800 for SMX was more in line with the Freundlich and D-A isotherm model, the whole process was an exothermic reaction. SBC800 could effectively remove SMX through pore filling effect, electrostatic attraction, hydrogen bonding, hydrophobic effect, and π-π EDA interaction. Site energy distribution analysis showed that SMX preferentially occupied the high-energy adsorption site of SBC800, and then gradually diffused to the low-energy adsorption site. This study proposed a sustainable method for recycling municipal sludge for organic pollutant removal.</p

APN secreted by PVAT aggravates autophagy in plaque.

<p>A, Representative western blot of LC3 expression in arteria carotis transplanted with WT or APN^-/- PVAT 4 weeks after atherosclerosis (n = 6 per group). B, Quantitative analysis of LC3 protein expression in various groups. *P<0.05 versus (WT) PVAT. C, Immunofluorescence of p62, another marker of autophagy, in the arteria carotis with WT or APN^-/- PVAT (n = 6 per group). D, Histogram shows p62 positive cells per 100 cells. *P<0.05 versus (WT) PVAT.</p

APN exacerbates macrophage autophagy in vitro.

<p>A and B respectively show the representative western blot and quantitative analysis of LC3 protein level in VSMC and macrophage stimulated with or without APN (5 μg/ml). n = 6 per group. *P<0.05 versus macrophage without APN. C and D respectively show the western blot and quantitative analysis of P62 and Beclin 1 protein level in macrophage treated with or without APN (5 μg/ml). n = 6 per group. *P<0.05 versus macrophage without APN.</p

Toward a Global Analysis of the Human Pituitary Proteome by Multiple Gel-Based Technology

We describe a comprehensive method for the analysis of complex proteomes, multiple gel-based technology (MGT), which combines in-gel IEF-LC-MS/MS and SDS-PAGE-LC-MS/MS strategies. MGT was applied to the analysis of the proteome of human pituitary tissue. A total of 1449 proteins were uniquely identified. To our knowledge, this is the most comprehensive proteomic analysis of human pituitary tissue to date. Categorization of the identified proteins revealed that MGT provides an excellent and largely unbiased access to proteins with diverse characteristics, including low-abundance proteins, membrane proteins, and proteins with extremes in pI and MW

Effect of Natural Osmolytes on Recombinant Tau Monomer: Propensity of Oligomerization and Aggregation

The pathological misfolding and aggregation of the microtubule associated protein tau (MAPT), a full length Tau2N4R with 441aa, is considered the principal disease relevant constituent in tauopathies including Alzheimer’s disease (AD) with an imbalanced ratio in 3R/4R isoforms. The exact cellular fluid composition, properties, and changes that coincide with tau misfolding, seed formation, and propagation events remain obscure. The proteostasis network, along with the associated osmolytes, is responsible for maintaining the presence of tau in its native structure or dealing with misfolding. In this study, for the first time, the roles of natural brain osmolytes are being investigated for their potential effects on regulating the conformational stability of the tau monomer (tauM) and its propensity to aggregate or disaggregate. Herein, the effects of physiological osmolytes myo-inositol, taurine, trimethyl amine oxide (TMAO), betaine, sorbitol, glycerophosphocholine (GPC), and citrulline on tau’s aggregation state were investigated. The overall results indicate the ability of sorbitol and GPC to maintain the monomeric form and prevent aggregation of tau, whereas myo-inositol, taurine, TMAO, betaine, and citrulline promote tau aggregation to different degrees, as revealed by protein morphology in atomic force microscopy images. Biochemical and biophysical methods also revealed that tau proteins adopt different conformations under the influence of these osmolytes. TauM in the presence of all osmolytes expressed no toxicity when tested by a lactate dehydrogenase assay. Investigating the conformational stability of tau in the presence of osmolytes may provide a better understanding of the complex nature of tau aggregation in AD and the protective and/or chaotropic nature of osmolytes

APN induces autophagy in macrophage through Akt-FOXO3a pathway.

<p>A, Western blot shows the protein level of p-Akt, Akt, p-FOXO3a, FOXO3a in macrophages stimulated with phosphate buffered saline, APN, Akt agonist (740Y-P) or with APN in combination with 740Y-P. B, Western blot shows the protein level of PTEN, p-mTOR, mTOR in macrophages stimulated with phosphate buffered saline, APN, Akt agonist (740Y-P) or with APN in combination with 740Y-P. C, Quantitative analysis of p-Akt/Akt ratio, p-FOXO3a/FOXO3a ratio and p-mTOR/mTOR ratio in macrophages stimulated with phosphate buffered saline, APN, and APN in combination with 740Y-P, respectively. n = 6 per group. *P<0.05 versus macrophage treated with saline. D, Quantification of the optical density of PTEN in each groups. n = 6 per group. *P<0.05 versus macrophage treated with saline.</p

NSAF values for every protein see Additional file are shown graphically

NSAF values of identified proteins fall within the range from 1.7 × 10to 1.8 × 10.<p><b>Copyright information:</b></p><p>Taken from "Comprehensive analysis of the mouse renal cortex using two-dimensional HPLC – tandem mass spectrometry"</p><p>http://www.proteomesci.com/content/6/1/15</p><p>Proteome Science 2008;6():15-15.</p><p>Published online 23 May 2008</p><p>PMCID:PMC2412861.</p><p></p