Protein processing characterized by a gel-free proteomics approach

We describe a method for the specific isolation of representative N-terminal peptides of proteins and their proteolytic fragments. Their isolation is based on a gel-free, peptidecentric proteomics approach using the principle of diagonal chromatography. We will indicate that the introduction of an altered chemical property to internal peptides holding a free α-N-terminus results in altered column retention of these peptides, thereby enabling the isolation and further characterization by mass spectrometry of N-terminal peptides. Besides pointing to changes in protein expression levels when performing such proteome surveys in a differential modus, protease specificity and substrate repertoires can be allocated since both are specified by neo-N-termini generated after a protease cleavage event. As such, our gel-free proteomics technology is widely applicable and amenable for a variety of proteome-driven protease degradomics research

Size-Exclusion Chromatography-based isolation minimally alters Extracellular Vesicles' characteristics compared to precipitating agents

Extracellular vesicles (EVs) have become an attractive field among the scientific community. Yet, a major challenge is to define a consensus method for EVs isolation. Ultracentrifugation has been the most widely used methodology but rapid methods, including Size Exclusion Chromatography (SEC) and/or precipitating agents such as Polyethylene glycol (PEG) or PRotein Organic Solvent PRecipitation (PROSPR) have emerged. To evaluate the impact of these different methods on the resulting EV preparations, plasma EVs were isolated using SEC, PEG and PROSPR, and their total protein content, NTA and Cryo-electron microscopy profiles, and EV-markers were compared. Also, their effect on recipient cells was tested. Low protein content and Cryo-EM analysis showed that SEC removed most of the overabundant soluble plasma proteins, which were not removed using PEG and partially by PROSPR. Moreover, only SEC allowed the detection of the EV-markers CD9, CD63 and CD81, LGALS3BP and CD5L, suggesting a putative interference of the precipitating agents in the structure/composition of the EVs. Furthermore, PEG and PROSPR-based EV isolation resulted in reduced cell viability in vitro. These results stress that appropriate EV-isolation method should be considered depending on the forthcoming application of the purified EVs

A New method for the capture of surface proteins in <i>Plasmodium falciparum</i> parasitized erythrocyte

Introduction: We propose a new method for the selective labeling, isolation and electrophoretic analysis of the Plasmodium falciparum protein exposed on the erythrocyte cell surface. Historically, membrane surface proteins have been isolated using a surface biotinylation followed by capture of biotin-conjugated protein via an avidin/streptavidin-coated solid support. The major drawback of the standard methods has been the labeling of internal proteins due to fast internalization of biotin. Methodology: To solve this problem, we used a biotin label that does not permeate through the membrane. As a further precaution to avoid the purification of non surface exposed proteins, we directly challenged whole labeled cells with avidin coated beads and then solubilized them using non ionic detergents. Results: A marked enrichment of most of the RBC membrane proteins known to face the external surface of the membrane validated the specificity of the method; furthermore, only small amounts of haemoglobin and cytoskeletal proteins were detected. A wide range of P. falciparum proteins were additionally described to be exposed on the erythrocyte surface. Some of them have been previously observed and used as vaccine candidates while a number of newly described antigens have been presently identified. Those antigens require further characterization and validation with additional methods. Conclusion: Surface proteins preparations were very reproducible and identification of proteins by mass spectrometry has been demonstrated to be feasible and effective.</br

Effect of isolation conditions on structural properties and surface behavior of soy-whey proteins

In this study, the impact of isolation conditions on structural and surface properties at the air/water interface of soy-whey proteins (SWP) was assessed. SWP were obtained by precipitation of soy-whey (at pH 4.5 or 8.0) with acetone or ammonium sulfate. Despite the fact that all SWP samples exhibited similar electrophoretic patterns, they showed different protein content (from 54.2 to 98.2% w/w). When precipitation was performed at pH 4.5, SWP samples evidenced a decrease of protein solubility (SP) and thermal stability, while the precipitation with acetone promoted the enrichment in polysaccharides and minerals. For all samples, intrinsic fluorescence, surface hydrophobicity and Fourier transform infrared (FTIR) studies revealed structural changes correlated to protein unfolding and aggregation processes. However, the surface behavior can be predicted from these studies mainly due to differences in surface hydrophobicity and the differential contribution of insoluble aggregates. The heating of SWP samples enhanced the surface activity, regardless of the pH of the raw material and the isolation method. These results can be useful as a reference research and as a starting point for industrial exploitation of proteins from soy wastewater.Fil: Ingrassia, Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Física de Rosario. Universidad Nacional de Rosario. Instituto de Física de Rosario; ArgentinaFil: Sobral, Pablo Antonio. Universidad Nacional de La Plata. Facultad de Ciencias Exactas; ArgentinaFil: Risso, Patricia Hilda. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Física de Rosario. Universidad Nacional de Rosario. Instituto de Física de Rosario; ArgentinaFil: Palazolo, Gonzalo Gastón. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Investigación en Funcionalidad y Tecnología de Alimentos; ArgentinaFil: Wagner, Jorge Ricardo. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Investigación en Funcionalidad y Tecnología de Alimentos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Sites of Biosynthesis of Outer and Inner Membrane Proteins of Neurospora crassa Mitochondria

Outer and inner membranes of Neurospora crassa mitochondria were separated by the combined swelling, shrinking, sonication procedure. Membranes were characterized by electron microscopy and by marker enzyme activities. A red carotenoid pigment was found to be concentrated in the outer membrane. The inner mitochondrial membrane was resolved into about 20 protein bands on polyacrylamide gel electrophoresis, whereas the outer membrane shows essentially one single protein band. Only negligible incorporation of radioactive amino acids occurs into outer membrane when isolated mitochondria are synthesizing polypeptide chains. In agreement with this observation labeling of outer membrane protein is almost entirely blocked, when whole Neurospora cells are incubated with radioactive amino acids in the presence of cycloheximide, an inhibitor of cytoplasmic protein synthesis. Finally, the essential electrophoretic protein band from outer membrane does not become labeled when mitochondria are incubated with radioactive amino acids either in vitro or in vivo in the presence of cycloheximide. It is concluded that the vast majority, if not all, of the outer membrane protein is synthesized by the cytoplasmic system and that polypeptide chains formed by the mitochondrial ribosomes are integrated into the inner mitochondrial membrane

A lymphoma plasma membrane-associated protein with ankyrin-like properties.

In this study we have used several complementary techniques to isolate and characterize a 72-kD polypeptide that is tightly associated with a major mouse T-lymphoma membrane glycoprotein, gp 85 (a wheat germ agglutinin-binding protein), in a 16 S complex. These two proteins do not separate in the presence of high salt but can be dissociated by treatment with 2 M urea. Further analysis indicates that the 72-kD protein has ankyrin-like properties based on the following criteria: (a) it cross-reacts with specific antibodies raised against erythrocyte and brain ankyrin; (b) it displays a peptide mapping pattern and a pI (between 6.5 and 6.8) similar to that of the 72-kD proteolytic fragment of erythrocyte ankyrin; (c) it competes with erythrocyte ghost membranes (spectrin-depleted preparations) for spectrin binding; and (d) it binds to purified spectrin and fodrin molecules. Most importantly, in intact lymphoma cells this ankyrin-like protein is localized directly underneath the plasma membrane and is found to be preferentially accumulated beneath receptor cap structures as well as associated with a membrane-cytoskeleton complex preparation. It is proposed that the ankyrin-like 72-kD protein may play an important role in linking certain surface glycoprotein(s) to fodrin which, in turn, binds to actin filaments required for lymphocyte cap formation

RECONSTITUTION OF ALLOPHYCOCYANIN FROM Mastigocladus laminosus WITH ISOLATED LINKER POLYPEPTIDE

The core linker polypeptide Lc 8.9 was isolated from Mastigocladus laminosus and purified on a preparative scale. A method for the reconstitution of allophycocyanin (AP)—linker complexes from isolated polypeptides was developed. The complex (αAP(βAP)3 Lc 8.9 was reconstituted and compared to (αAPβAP) and (αAPβAP)3 by sucrose density gradient ultracentrifugation, absorption, fluorescence emission and circular dichroism spectroscopy. Differences in the spectra of reconstituted and of directly isolated AP complexes are discussed

Isolation of oligomycin-sensitive adenosine triphosphatase from beef heart mitochondria and analysis of its fine structure

1. An oligomycin -sensitive ATPase was isolated and partially purified from beef heart mitochondria. The specific activity of ATPase sensitive to oligomycin of the fraction was five to eight times that of aged mitochondrial or of DNP-induced mitochondrial ATPase assayed under the same condition. 2. Electron micrographs of the partially purified oligomycin- sensitive ATPase reveal a structure in which headpieces are regularly attached by way of stalks to a thread-like structure derived from a superficial portion of base pieces. 3. A high concentration of the structured material coincided with a high activity of oligomycin-sensitive ATPase. When the headpieces were detached from the structure, the ATPase became insensitive to oligomycin. 4. The fraction of oligomycin -sensitive ATPase was essentially free of membrane structure and was contaminated with a small amount of cytochromes b and Cl but no cyt. a. Cytochrome concentrations of the preparations were indifferent to the activity of oligomycin sensitive ATPase. It follows that ATPase does not require cytochromes or membrane structure for its oligomycin sensitivity. 5. From these results it seems that the factor rendering ATPase sensitive to oligomycin should be contained in the stalks and/or the thread-like portion of basepieces of the structure. The structure is the simplest unit of oligomycinsensitive ATPase as yet obtained. 6. The structure was called &#34;oligomycin-sensitive ATPase particles&#34; (abbreviated as OSA particles). A unit of OSA particles consists of a headpiece attached by a stalk to a portion of base piece.</p