Red blood cell storage in SAGM and AS3: a comparison through the membrane two-dimensional electrophoresis proteome.

Background: SAGM is currently the standard additive solution used in Europe, while AS-3 is the third additive solution that has been licensed in the USA, and is also the one used in part of Canada. Although AS-3 is based on a saline-adenine-glucose solution, it also contains citrate and phosphate. Storage of red blood cell concentrates in CPD-SAGM is known to lead to the accumulation of a wide series of storage lesions, including membrane protein fragmentation and vesiculation, as we could previously determine through 2-dimensional gel electrophoresis.

Combinatorial peptide ligand libraries to discover liver disease biomarkers in plasma samples.

High-abundance proteins present in blood plasma make the detection of low-abundance proteins extremely difficult by proteomics technology. Hexapeptide combinatorial ligand libraries can be used to investigate the hidden proteome in depth. Here we describe how liver disease biomarkers can be successfully discovered in blood plasma by two main steps: preparative methods that reduce the dynamic range of protein concentration, and analytic methods that allow resolution of proteins.

Analysis of the cattle liver proteome by high-sensitive liquid chromatography coupled with mass spectrometry method.

The present chapter describes methods for the separation and identification of proteins in liver metabolism through a comparison of the protein expression profiles of the two breeds taken into account as a model: Holstein Friesian and Chianina cattle. The liver has received special attention, containing as it does, enzymes involved in energy generation, carbohydrate, lipid, amino acid, and xenobiotic metabolism, as well as proteins involved in polypeptide synthesis, folding, and cell structure. The first step in the procedure is the preparation of purified protein fractions from liver tissues, followed by sample preparation for 2-DE analysis in order to identify proteins which could be differentially expressed in the livers of the two breeds and relate them to different liver functions.

Red blood cell subpopulations in freshly drawn blood: application of proteomics and metabolomics to a decades-long biological issue.

Background: It has long been known that red blood cells comprise various subpopulations, which can be separated through Percoll density gradients.

Materials And Methods: In this study, we performed integrated flow cytometry, proteomic and metabolomic analyses on five distinct red blood cell subpopulations obtained by Percoll density gradient separation of freshly drawn leucocyte-depleted erythrocyte concentrates. The relation of density gradient fractions to cell age was confirmed through band 4.

Native analysis of plasma-derived clotting factor VIII concentrates: "sponge effect" and contaminants.

In the present study, we analysed two commercially available plasma-derived FVIII preparations, Beriate and Emoclot, through native gel-based approaches (CN-PAGE). The rationale behind this study was to assess whether protein complexes from plasma resisted the aggressive manufacturing processes. A preliminary analysis of plasma complexes was performed focusing on the molecular weight range between 45 kDa and 1 MDa.

Red blood cell processing for cryopreservation: from fresh blood to deglycerolization.

Background: Cryostorage of red blood cells (RBCs) represents a valid alternative to liquid storage, since units can be preserved safely for at least a decade while conserving RBC viability. While cryostorage has attracted a great deal of attention clinically, little is known about the biochemistry and physiology of cryostored erythrocyte concentrates.

Study Design And Method: In the present study, we investigated cryostorage of RBCs through monitoring of cell processing steps (from fresh blood, to glycerolization, thawing and deglycerolization/washing) through repeated assays of standard parameters (MCV, RDW-SD) and scanning electron microscopy.

Depletion of hemoglobin and carbonic anhydrase from erythrocyte cytosolic samples by preparative clear native electrophoresis.

Proteomic analysis of red cells is compromised by the presence of high-abundance proteins (hemoglobin and carbonic anhydrase-1), which completely obscure low-abundance species. The depletion method presented here involves performing native gel electrophoresis in a polyacrylamide gel tube using a modified electroelution cell. The electrophoretic run is interrupted intermittently to allow the recovery of at least three different liquid fractions, which can be analyzed by both native PAGE and 2D isoelectric focusing SDS-PAGE, or by shotgun mass spectrometry analysis after trypsin in-solution protein digestion.

Time-course investigation of SAGM-stored leukocyte-filtered red bood cell concentrates: from metabolism to proteomics.

Background: Results from recent, highly debated, retrospective studies raised concerns and prompted considerations about further testing the quality of long stored red blood cells from a biochemical standpoint.

Design And Methods: We performed an integrated mass spectrometry-based metabolomics and proteomics time-course investigation on SAGM-stored red blood cells. In parallel, structural changes during storage were monitored through scanning electron microscopy.

Docosohaexanoic acid-supplemented PACA44 cell lines and over-activation of Krebs cycle: an integrated proteomic, metabolomic and interactomic overview.

Recent investigations have pointed out the ability of fatty acids, in particular of docosohaexanoic acid (DHA), to induce growth inhibition and apoptosis in the human PaCa-44 pancreatic cancer cell line through a series of mechanisms which has been hypothesized to mimic apoptosis. While preliminary evidences indicated the involvement of lipid-targeting oxidative stress in DHA-induced apoptotic processes, mainly through the alteration of the glutathione (GSH) homeostasis and oxidized-glutathione (GSSG) turn-over through their extra-cellular extrusion, no further molecular data have been hitherto accumulated. To this end, we hereby propose simultaneous protein-targeting and metabolite-oriented analyses, which have been integrated through the auxilium of in silico elaboration of those protein-protein interaction pathways and enrichment of biological/molecular functions.

An easy preparative gel electrophoretic method for targeted depletion of hemoglobin in erythrocyte cytosolic samples.

Hemoglobin (Hb) approximately constitutes 98% of the protein composition of a red blood cell (RBC), thus masking the remaining 2% which has still to be discovered completely due to the difficulty in its analysis. Here, we proposed a large-scale native gel electrophoresis that effectively tackles this limitation through a novel sample preparation strategy able to concentrate low-abundance species by removing Hb by means of electrophoretic instruments. Clear native PAGE was performed in a gel electrophoresis tube where the run was intermittently interrupted and different fractions were recovered in liquid phase into a collection chamber placed at the end of the tube.

The photosynthetic membrane proteome of Rhodobacter sphaeroides R-26.1 exposed to cobalt.

Cells of the carotenoidless strain R-26.1 of Rhodobacter sphaeroides were grown in the presence of a high concentration (5 mM) of cobalt ions. The photosynthetic intracytoplasmic membranes were isolated and investigated by proteomic analysis using non-denaturating blue native electrophoresis in combination with LC-ESI-MS/MS.

Oxidative stress-dependent oligomeric status of erythrocyte peroxiredoxin II (PrxII) during storage under standard blood banking conditions.

Although biochemical properties of 2-Cys peroxiredoxins have been extensively studied in various cell lines and organisms, redox-induced structural transitions of peroxiredoxin II (PrxII) in human erythrocytes certainly warrant further investigation. In this work, cytosol and membrane ghosts of both fresh erythrocytes (cells obtained just after blood collection) and 28-day stored erythrocytes were analyzed by proteomics tools. We demonstrated that in fresh red blood cells PrxII exhibits four different oligomeric states in cytosol, whereas no PrxII complexes are in the membrane.

Recombinant clotting factor VIII concentrates: Heterogeneity and high-purity evaluation.

Factor VIII is an important glycoprotein involved in hemostasis. Insertion of expression vectors containing either the full-length cDNA sequence of human factor VIII (FLrFVIII) or B-domain deleted (BDDrFVIII) into mammalian cell lines results in the production of recombinant factor VIII (rFVIII) for therapeutic usage. Three commercially available rFVIII concentrates (Advate, Helixate NexGen and Refacto), either FLrFVIII or BDDrFVIII, were investigated by 1- and 2-DE and MS.

Plasma gelsolin protein: a candidate biomarker for hepatitis B-associated liver cirrhosis identified by proteomic approach.

Background: Despite the significant improvement in internal medicine and supportive therapy in recent years, liver fibrosis/cirrhosis remains a serious health issue in hepatitis B virus (HBV) infected patients. Invasive liver biopsy is presently the best means of diagnosing cirrhosis, but it carries a significant risk and has well recognised limitations such as sampling error, hence the importance in developing early diagnosis biomarkers. With this aim, we performed a pilot proteomic study to assess this as a strategy for plasma marker detection in patients suffering from HBV-associated liver cirrhosis.

Iron stabilizes thylakoid protein-pigment complexes in Indian mustard during Cd-phytoremediation as revealed by BN-SDS-PAGE and ESI-MS/MS.

Two-dimensional BN-SDS-PAGE, ESI-MS/MS and electron microscopy (EM) were used to study the role of iron (Fe) under cadmium (Cd) stress in retention of thylakoidal multiprotein complexes (MPCs) and chloroplast ultrastructure of Indian mustard, a moderate hyperaccumulator plant. Mustard was grown hydroponically with or without iron for 17 days and then exposed to CdCl2 for 3 days. Fe deficiency led to an increase in oxidative stress and damage to chloroplast/thylakoids accompanied by a decrease in chlorophyll content; exposure of plants to Cd further enhanced the oxidative stress and Cd accumulation (more in -Fe plants).

Oligomeric characterization of the photosynthetic apparatus of Rhodobacter sphaeroides R26.1 by nondenaturing electrophoresis methods.

Blue and colorless native gel electrophoresis in combination with LC-ESI-MS/MS are powerful tools in the analysis of protein networks in biological membranes. We used these techniques in the present study to generate a comprehensive overview on a proteome-wide scale of intracytoplasmic membrane (ICM) associated proteins in order to investigate the native supramolecular organization of Rhodobacter sphaeroides R26.1 photosynthetic apparatus.

Comparative proteomics and transcriptomics analyses of livers from two different Bos taurus breeds: "Chianina and Holstein Friesian".

The Holstein Friesian and Chianina cattle breeds are representative of extreme selection for milk and meat traits, respectively, with significant changes in metabolism resulting from human selection over the past centuries. In the present study, we wanted to assess whether selection for different purposes has had a measurable effect on liver metabolism through a comparison of the protein and gene expression profiles of the two breeds. We applied 2-DE in order to identify proteins which were differentially expressed in the livers of the two breeds and relate them to different liver functions.

Separation of thylakoid membrane proteins by sucrose gradient ultracentrifugation or blue native-SDS-PAGE two-dimensional electrophoresis.

Generally, a combination of two or more chromatographic and/or electrophoretic methods is conducted to separate membrane protein complexes. Here we describe how thylakoid membrane protein complexes from the photosynthetic apparatus can be successfully separated by two main steps: preparative methods that enable purification of membrane protein complexes in the native (intact) form, and analytical methods that allow resolution of each membrane protein. Thus, separation of intact supercomplexes was achieved by solubilisation of the sample using mild detergents followed either by sucrose gradient ultracentrifugation or by blue native gel (BNG) electrophoresis.

Transfusion medicine in the era of proteomics.

Blood components (BCs) are highly complex mixtures of plasma proteins and cells. At present, BC and blood derivatives (BDs) quality control is mainly focused on standardized quantitative assessment, providing relatively limited information about products. Unfortunately, during the production, inactivation, and storage processes there is the risk of changes in their integrity, especially at the protein level, which could cause negative effects on transfusion.

Coupling of native liquid phase isoelectrofocusing and blue native polyacrylamide gel electrophoresis: a potent tool for native membrane multiprotein complex separation.

In this study, a new 3D native electrophoretic protocol is proposed for an exhaustive separation and identification of membrane proteins. It is based on native liquid phase isoelectrofocusing (N-LP-IEF) of protein complexes in the first dimension, followed by blue native polyacrylamide gel electrophoresis (BN-PAGE) in the second dimension, where both the pI and the molecular masses of protein complexes (2D N-LP-IEF-BN) were used to separate them in their native form. Finally, each single component can be resolved using denaturing electrophoresis (3D N-LP-IEF-BN-SDS-PAGE).

Proteomics, pigment composition, and organization of thylakoid membranes in iron-deficient spinach leaves.

The changes induced in the photosynthetic apparatus of spinach (Spinacia oleracea L.) seedlings exposed to iron deficiency shortly after germination were characterized with two proteomic approaches coupled with chlorophyll and xanthophyll analysis and in vivo measurements of photosynthesis. During the first 10 d of iron deficiency the concentrations of chlorophyll b and violaxanthin were greatly reduced, but all xanthophylls recovered after 13-17 d of iron deficiency, when both chlorophylls were negatively affected.

Proteomic analysis of RBC membrane protein degradation during blood storage.

Two-dimensional gel electrophoresis and mass spectrometry were used to identify protein profile changes in red blood cell membranes stored over time under atmospheric oxygen, in the presence or absence of protease inhibitors. New spots with lower molecular masses, ranging between 7 and 15 kDa were observed during the first 7 days storage, while over time, further fragments and high-molecular-mass aggregates appeared, seen as a smearing in the upper part of the gel. Some of the protein changes turned out to be shifts in isoelectric point, as a consequence of chemical oxidations.

Purification and characterization of phycocyanin from the blue-green alga Aphanizomenon flos-aquae.

Aphanizomenon flos-aquae (AFA) is a blue-green alga and represents a nutrient-dense food source. In this study the presence of phycocyanin (PC), a blue protein belonging to the photosynthetic apparatus, has been demonstrated in AFA. An efficient method for its separation has been set up: PC can be purified by a simple single step chromatographic run using a hydroxyapatite column (ratio A620/A280 of 4.