Research Article Cites HemoVoid™ in Fish Red Blood Cell Proteome Study

Biotech Support Group reports on a recent research article describing the simplicity and efficiency of their hemoglobin depletion technology for enriching the red cell sub-proteome, from trout red blood cells. Fish nucleated red blood cells (RBCs) generate a wide variety of immune-related gene transcripts when viruses replicate inside them and are their main target cell. However, the objective of this study not yet explored, was to determine the immune response and mechanisms of fish RBCs against viruses targeting other cells or tissues. The article states “a new proteomic analysis method was carried out that combines fractionation into cytosolic and membrane fractions, haemoglobin removal of the cytosolic fraction, protein digestion, pH reversed-phase peptide fractionation and finally LC ESI-MS/MS analysis of each of the fractions… . Briefly, the haemoglobin of the cytosolic fraction was removed using a column of HemoVoid™ kit (Biotech Support Group, Monmouth Junction, NJ), following the manufacturer instructions”. The authors conclude that their RBC Isobaric tag for relative and absolute quantification (iTRAQ) revealed Viral Haemorrhagic Septicaemia virus (VHSV) exposure can induce a global protein downregulation in trout RBCs, mainly related to RNA stability and proteasome pathways.

Biotech Support Group Presents Stroma Liquid Biopsy Cancer Data at HUPO World Congress 2017

Biotech Support Group described a panel of protein biomarkers dysregulated in cancer at the recent HUPO World Congress, that took place September 17-21 in Dublin, Ireland. This research shows that there are three common pathways which change in the bloodstream regardless of the primary tumor, stage, metastatic disease, or tumor burden. Five different cancers – lung, breast, pancreatic, lymph and ovary, were profiled along with normal/healthy individuals of approximate age and matched sex for comparison.

Research Article Cites HemogloBind™ In Diabetic Complications Study

Diabetic kidney disease, a common complication of both type 1 and type 2 diabetes, is associated with significant morbidity and mortality, and represents the most common cause of chronic kidney disease. The study hypothesized that protective pentose phosphate pathway action in diabetes might be compromised by limited intracellular availability of an active transketolase cofactor thiamine diphosphate (TDP). To evaluate the levels of thiamine tranporter proteins in whole blood, the article states “For protein isolation, whole blood aliquots were lysed with water and haemoglobin was removed using HemogloBind™ (Biotech Support Group) according to manufacturer’s instructions…”. The article concludes that both in vitro and human experiments showed decrease or unchanged expression, respectively, of thiamine transporters induced by hyperglycaemia while transketolase activity in parallel with intracellular TDP was increased in chronic kidney disease patients with or without diabetes.

Research Article Cites HemoVoid™ in Functional Proteome Study

In brief, the authors aimed to investigate whether RBCs carry a functional soluble guanylate cyclase (sGC) signalling pathway and to address whether this pathway is compromised in coronary artery disease. The article states “Using a commercial resin (HemoVoid™), which removes hemoglobin… and allows enrichment of soluble cytoplasmic proteins, we established a procedure that allows fast and reliable preparation of hemoglobin-free cell lysates from as little as 1-2 ml blood. In those samples, expression and activity of the cGMP-generating sGC, cGMP-hydrolyzing PDE5 and cGMP-transducing PKG was assessed by enzymatic assays and Western blot analysis”. The authors conclude that human and murine RBCs carry a catalytically active α1β1-sGC (isoform 1), which converts 32P-GTP into 32P-cGMP, as well as PDE5 and PKG. Collectively, the article demonstrates that an intact sGC/PDE5/PKG-dependent signaling pathway exists in RBCs, which remains fully responsive to NO and sGC stimulators/activators in patients with endothelial dysfunction.

Research Article Cites Cleanascite™ for Analyzing Membrane Interaction Proteins

In brief, the article’s authors aimed to identify a receptor of the exocyst, an essential component of the secretory pathway required for delivery of basolateral proteins to the plasma membranes of epithelial cells. To determine if phospholipids were essential for the interaction, a lipid-binding resin was used to remove lipids. The article states “Lipids from cells and bacterial lysates were removed using Cleanascite™…”. The authors conclude that Par3 is an exocyst receptor required for targeted membrane- protein delivery.

Journal Article Highlights AlbuVoid™ in Cancer Functional Proteomic Analysis

In this study, a functional proteomics technology was used to systematically monitor metabolic enzyme and protease activities from resolved serum proteins produced by a modified 2-D gel separation and subsequent Protein Elution Plate, a method collectively called PEP. AlbuVoid™ was used to remove Albumin and enrich the low abundance serum proteome. The article states “The most dramatic difference for enzyme activity detection in using the AlbuVoid™ for serum protein enrichment was demonstrated. … Compared with the direct serum proteinase measurement, both the levels and species of proteases were increased significantly in the enriched serum sample. …protease activity in the direct serum analysis suggested that the protease levels in the serum were below the detection threshold of protease activity…,and it is necessary to use AlbuVoid™ to enrich these low level proteases to bring them to a high enough level to be detected.” Both qualitative and quantitative differences in the metabolic enzyme and protease activity were detected between breast cancer patient and control group, providing excellent biomarker candidates for breast cancer diagnosis and drug development.

Research Article Cites HemogloBind™ in Parkinson’s Disease Study

In this study, Parkinson’s disease progression is investigated through the accumulation and aggregation of the lipid-binding SNARE complex component alpha-synuclein (SNCA) which underlies vulnerability and defines its stages. The authors studied blood samples from a new large pedigree with SNCA gene duplication (PARK4 mutation), to identify effects of SNCA gain-of-function as potential disease biomarkers. The article states “For protein extraction from the EDTA tubes, 300 μl blood were lysed with equal amount of 1% SDS-RIPA buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Igepal CA-630 (Sigma), 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF and one tablet Complete Protease Inhibitor Cocktail (Roche)] and sonicated for 10 sec. The blood lysates were rotated at 4 °C for 30 min and centrifuged at 4 °C for 30 min. The supernatants were depleted in hemoglobin content using a commercial kit (HemogloBind, Biotech) following the manufacturer’s instructions”. After hemoglobin depletion, immunoblot analysis identified that PARK4 blood showed upregulation of alpha-synuclein monomer, with no high molecular weight aggregates.

Research Article Cites HemoVoid™ in Red Blood Cell Proteome Study

In brief, using proteomics-based evaluation of red blood cells (RBC), the authors identified differentially abundant proteins associated with Obstructive Sleep Apnea Syndrome (OSA). Proteome variations between various time points were assessed. The article states “RBC cytoplasmic fraction depleted of hemoglobin, using Hemovoid™ system, were analyzed by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE), the 2D image software-based analyzed and relevant differentially abundant proteins identified by mass spectrometry (MS)”. The authors conclude that 31protein spots were differentially abundant, corresponding to 21 unique proteins possibly due to the existence of post-translational modifications.

Patent Cites HemogloBind™ in Complement Mediation

The invention describes synthetic peptide compounds and uses for therapy and diagnostics of complement-mediated diseases, such as inflammatory diseases, autoimmune diseases, and microbial infections; and non-complement-mediated diseases, such cystic fibrosis and various acute diseases. The patent states “ Due to large amounts of hemolysis in the latter time points and the associated optical interference in bilirubin analysis, all the samples were pre-treated with HemogloBind™ (Biotech, N.J.) prior to analysis with the Bilirubin Assay Kit”.

Research Article Cites HemogloBind™ in Cancer Derived Exosome Study

This study considers that therapeutic strategies targeting cancer-derived extracellular vesicles (EVs) hold great promise because of the possibility they reposition microenvironments to accommodate metastasis. The researchers report on a novel strategy of therapeutic antibody treatment to target cancer-derived EVs and inhibit the metastasis of breast cancer in a mouse model. The article states “Hemoglobin was accumulated with HemogloBind beads (Biotech support group, Monmouth Junction NJ, USA) followed by 0.22 μm filtration. Then, the EVs in the sera were concentrated by ultracentrifugation…”. The authors conclude that therapeutic antibody administration effectively suppresses EV-triggered metastasis and that the elimination of cancer-exosome derived EVs could be a novel strategy for therapy.

Research Article Cites Cleanascite™ for Mass Spectrometric Proteome Analysis of Human Bile Fluid

In brief, the article’s authors report methods to overcome the biological variability of analyzing a high number of bile samples. They advance that easy sample preparation protocols are demanded representing a compromise between proteome coverage and simplicity in this study. For this, they evaluated the performance of simple workflows allowing for "one sample, one shot" experiments to identify biomarker candidates for various diseases of the hepatobiliary system. In detail, sixteen different protocols with modifications at the stages of desalting, delipidation, deglycosylation and tryptic digestion were examined. The article states “For delipidation, the Cleanascite™ Lipid Removal Reagent and Clarification Kit (BSG, NJ 08852, USA) was used following manufacturer's instructions.”. The authors concluded that delipidation yielded a considerable number of complementary protein identifications and that Cleanascite™ treatment was indispensable for in-solution digestion methods.

Proteomics Research Article Cites HemogloBind™ in Profiling Lung Injury

The authors consider pulmonary complications due to infection and idiopathic pneumonia syndrome (IPS) in hematopoietic stem cell transplant (HSCT) recipients. For this, a proteomic characterization of global bronchoalveolar lavage fluid (BALF) was studied to identify proteins and pathways that differentiate IPS from infectious lung injury after HSCT. Because the BALF samples appeared tinged with blood, the authors tested HemogloBind™ to determine whether the removal of hemoglobin (in addition to high-abundance proteins) would improve the depth of coverage. The article states “Hemoglobin removal improved protein identification to 845 proteins at 1% global FDR compared with 496 proteins with high-abundance protein depletion alone”. The authors conclude that the protein expression differences provide insights into mechanisms activated in lung injury and suggest potential therapeutic targets to augment lung repair.