Macromolecular Complexes
C Wan, B Borgeson, S Phanse, F Tu, K Drew, G Clark, et al. Panorama of ancient metazoan macromolecular complexes.Nature Volume:525, Pages:339–344 Date published:(17 September 2015). doi:10.1038/nature14877
Two of BSG products, NRicher™ 6 and HemogloBind™, were able to contribute to this rigorous examination of protein complexes. When our products were used as a pretreatment step in the overall workflow, about twice the number of observations and annotations became possible. This further validates that the sub-proteome bias characteristics of NRicher™ 6 can simplify complex proteomes into less complex sub-proteomes with efficiencies suitable for deep functional proteome characterization. Furthermore, this study demonstrated the importance of a key feature implicit to all of our products; that is the maintenance of functional and structural integrity after separations. Without that particular feature, these additional observations would not have been possible.
Human Peripheral Blood Mononuclear Cells (PBMCs)
Rubio-Navarro, Alfonso, et al."Hemoglobin induces monocyte recruitment and CD163-macrophage polarization in abdominal aortic aneurysm."International Journal of Cardiology (2015)."
A research article by authors Rubio-Navarro, Alfonso, et al in the journal International Journal of Cardiology (http://www.internationaljournalofcardiology.com/article/S0167-5273(15)30284-9/fulltext) cites Biotech Support Group’s HemogloBind™ sample preparation reagent to deplete hemoglobin (Hb) from conditioned medium from healthy aortas or abdominal aortic aneurysm. The article states:"Conditioned mediums from AAA were incubated with HemogloBind™ reagent for hemoglobin depletion"
In brief, authors describe high infiltration of CD163 monocytes surrounding micro-vesicles, low expression of CD14+ & CD16- monocytes and high CD163 mRNA/protein expression is a feature of abdominal aortic aneurysm (AAA) molecular pathology. Healthy aorta conditioned medium or complete or hemoglobin-depleted conditioned medium from abdominal aortic aneurysm were mixed with M-CSF macrophages to track CD163 and HLA-DR expression or hemoglobin uptake.
“We are pleased with this data on abdominal aortic aneuryism & hemoglobin proteomics. HemogloBind™ minimizes hemoglobin interference from blood samples and allows research on cardiovascular pathology” states Swapan Roy, Ph.D., President and Founder of Biotech Support Group.
Dried Blood Spot(DBS)/Whole Blood
Hakuna, Lovemore, et al. "A simple assay for glutathione in whole blood."Analyst (2015).
A research article in the journal Analyst ( http://pubs.rsc.org/en/content/articlelanding/2015/an/c5an00345h) cites Biotech Support Group’s HemogloBind™ sample preparation reagent to deplete hemoglobin (Hb) from whole blood samples containing glutathione to minimize interference from Hb in GSH fractions. Using a resorufin-acrylate fluorescent probe, GSH is quantitated in deproteinzed blood plasma and whole blood samples. The article states:
"Apart from dilution, Hb can be removed using a commercial product, HemogloBind™, which can isolate and remove up to 90% of blood Hb." Glutathione (GSH) is an antioxidant involved on nitric oxide regulation, covalent hemoglobin binding, DNA binding, leukotriene synthesis, protein synthesis and sepsis pathways. GSH is elevated in cancer tissues and proper minimally invasive GSH sample preparation from dried blood samples (DBS) allows research on neurodegenerative diseases, chronic respiratory diseases and diabetes. Authors Lovemore, et al cite a DBS method to fractionate GSH from blood components, deproteinization by filtration and depletion of hemoglobin enhances quantification & identification of GSH in whole blood samples. Then size exclusion chromatography or fluorescence imaging detection is performed for quantitative analysis and glutathione assay research.
Whole Blood
Snider, Thomas H., Christina M. Wilhelm, Michael C. Babin, Gennady E. Platoff Jr, and David T. Yeung. "Assessing the therapeutic efficacy of oxime therapies against percutaneous organophosphorus pesticide and nerve agent challenges in the Hartley guinea pig."The Journal of Toxicological Sciences 40, no. 6 (2015): 759-775.
Acetylcholine is an essential neurotransmitter, and inhibitors of cholinesterases(ChEs) are potent toxins. A primary component of anti-organophosphorus therapy is an oxime reactivator to rescue inhibited acetylcholinesterases. For this, clinical signs of toxicity can be measured from blood cholinesterase [Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE)] activity utilizing a modified Ellman's method. Biotech Support Group’s unique solid-phase polymer for hemoglobin depletion, was used for pretreatment. The article states “Briefly, whole blood samples were treated with HemogloBind™ which interferes with the ChE activity assay due to spectral overlap.”
Brittain, Matthew K., Kevin G. McGarry, Robert A. Moyer, Michael C. Babin, David A. Jett, Gennady E. Platoff, and David T. Yeung. "Efficacy of Recommended Prehospital Human Equivalent Doses of Atropine and Pralidoxime Against the Toxic Effects of Carbamate Poisoning in the Hartley Guinea Pig." International journal of toxicology (2016): 1091581816638086. The article states “Whole blood samples were processed and analyzed as described by McGarry et al.10 Briefly, whole blood samples were treated with HemogloBind to remove hemoglobin, which interferes with the ChE activity assay due to spectral overlap. To prepare the HemogloBind treated blood samples for ChE activity analysis, samples were diluted 2-fold in assay buffer (1 PBS). Subsequently, samples were diluted an additional 2-fold into the test plate by adding 100 mL of sample to a total volume of 200 mL in each well of a 96-well plate. Cholinesterase activity was assessed using a spectrophotometric assay conducted in a manner similar to Ellman et al,11 as described in the in vitro reactivation section above. The relative AChE activity level for each animal (RAAChE) was defined as the ATC turnover rate in the terminal blood sample divided by that in the same animal’s baseline blood sample. A similar calculation was performed using butyrylthiocholine (BTC) turnover rates to determine RABChE.”
Blood Plasma
Johns, Michael, et al. "SR-135, a peroxynitrite decomposing catalyst, enhances β-cell function and survival in B6D2F1 mice fed a high fat diet." Archives of Biochemistry and Biophysics (2015).
A research article in the journal Archives of Biochemistry & Biophysics(http://www.sciencedirect.com/science/article/pii/S0003986115001988) cites Biotech Support Group's HemogloBind™ sample preparation reagent to deplete hemoglobin (Hb) from lysed red blood cells. Authors cite peroxynitrite decomposing catalysts such as Mn(III) bis(hydroxyphenyl)-dipyrromethene complexes as important molecules in obesity sample preparation & development of anti-diabetic agents. SR-135 and it's analogs are synthesized to decompose peroxynitirie. In addition, authors provide experiment data on prevention of nitration assay, rat islet uptake, quantitation of tyrosine nitration, beta-cell area quantitation, glucose-stimulated insulin secretion and plasma insulin level detection. The article states: "Blood plasma (50 µl) was mixed with HemogloBind (50 µl) to remove hemoglobin from lysed red blood cells."Plasma hemoglobin is depleted using Biotech Support Group's HemogloBind™ to extract hemoglobin from lysed red blood cells. Plasma concentration of high-density lipoprotein (HDL), total cholesterol and triacylglycerol (TAG) is obtained. Precipitation using polyethylene glycol (PEG) of beta-lipoproteins, very low-density lipoprotein (VLDL) and low-density lipoprotein(LDL) from HDL fractions is performed."We are pleased with this research on HemogloBind™ as interference is minimized and concentration of cholesterol proteins is obtained." states Swapan Roy, Ph.D., President and Founder of Biotech Support Group.
Lung Tissue Specimens & Pulmonary Research (Acute Respiratory Distress Syndrome & Acute Hypoxic Failure)
Bhargava, Maneesh""Proteomic Studies in Acute Hypoxic Respiratory Failure." PhD diss., UNIVERSITY OF MINNESOTA. (2015).
Protein expression in the bronchoalveolar lavage fluid (BALF) from subjects with acute respiratory distress syndrome (ARDS) was evaluated. The BALF samples were processed by desalting, concentration and removal of high abundance proteins. The protein fractions were trypsin digested and labeled with the iTRAQ reagent for mass spectrometry (MS). The thesis states “BALF samples containing at least 1.2 mg of proteins were processed for LCMS/MS…concentrated and desalted using Amicon 3-MWCO filters. Hemoglobin depletion was performed with HemogloBind™ (Biotech Support Group LLC, Monmouth Junction, NJ) per the manufacturer's instructions.”
Red Cell Lysates
Kyoungsook Park, Christopher D. Saudek, and Gerald W. Hart Increased Expression of β-N-Acetylglucosamindase (O-GlcNAcase) in Erythrocytes from Prediabetic and Diabetic Individuals. Diabetes.2010;59(7):1845-50.
Erythrocyte proteins are highly O-GlcNAcylated. In individual with pre-diabetes and diabetes, the level of O-GlcNAcase expressed significantly increases. From serum samples, erythrocyte proteins were extracted and hemoglobin was depleted followed by sonication and centrifugation. From the red blood cell lysates hemoglobin is efficiently depleted using HemogloBind™ from Biotech Support Group. Because HemogloBind™ is engineered for a high degree of selectivity and does not cross react with most common serum components, subsequent analysis of O-GlcNAcylation process in erythrocyte proteins is done by Western blotting using an O-GlcNAc specific antibody. Finally, the study of O-GlcNAcase allows for developing, validating, and qualifying biological markers that are compared with the level of A1C.
Stored Blood Products
Delobel J., Rubin O., Prudent M., Crettaz D., Tissot J.-D., Lion N. Biomarker Analysis of Stored Blood Products: Emphasis on Pre-Analytical Issues. International Journal of Molecular Sciences.2010;11(11):4601-4617
Authors Delobel et al cited Hemoglobind™'s application for hemoglobin depletion from the paper by Alvarez-Llamas et al, A novel methodology for the analysis of membrane and cytosolic sub-proteomes of erythrocytes by 2-DE. Electrophoresis, 30: 4095–4108. For biomarker discovery from erythrocyte proteome samples of erythrocyte concentrates, platelet concentrates and fresh frozen plasma blood products are used in proteomic analysis. This paper reviews the importance of standardizing sample preparation steps and controlling pre-analytical factors to identify proteins from cytosolic or membrane fractions. After using Hemoglobind™, one dimensional (sodium dodecyl sulphate polyacrylamide gel electrophoresis, SDS-PAGE) and 2D electrophoresis are implemented to identify unique proteins from by MALDI-TOF MS analysis.
Red Blood Cells(RBC)/Forensic Research
Danielson, Phillip B. "Isolation of Highly Specific Protein Markers for the Identification of Biological Stains: Adapting Comparative Proteomics to Forensics." (2011).
Biotech Support Group reports on a technical report prepared by the U.S. Department of Justice. The central goal of the project was to isolate and identify candidate protein biomarkers that are highly specific to individual types of biological stains of forensic utility (i.e., saliva, semen, peripheral blood, menstrual blood, vaginal secretions, and urine).
The report is entitled “Isolation of Highly Specific Protein Markers for the Identification of Biological Stains:Adapting Comparative Proteomics to Forensics". The National Criminal Justice Reference Service (NCJRS) has made this federally funded grant final report available electronically at: https://www.ncjrs.gov/App/Publications/abstract.aspx?ID=258706 The report states:
“Serum obtained from menstrual blood samples was typically contaminated with erythrocyte cellular components due to the lysing of fragile red blood cells that are abundant in the endometrial lining during menses. As hemoglobin comprises 32-36% of all the proteins found in red blood cells the serum from menstrual blood samples contained large quantities of hemoglobin which served to mask the detection of less abundant menstrual blood specific proteins. For this reason, hemoglobin was removed from collected serum prior to proteome fractionation through use of HemogloBind™ (Biotech Support Group, Monmouth Junction, NJ). This hemoglobin capture reagent is a solid-phase, non-ionic adsorbent product that binds
specifically to hemoglobin allowing for the removal of 80-90% of hemoglobin from serum or red cell lysates. HemogloBind™ does not cross react with most common serum components, making it suitable for the proteomic applications of this research project.”“Here we find another use for HemogloBind™, further validating our unique surface technology approach, not based on biologicals, as being highly selective and an efficient method for the depletion of hemoglobin” states Swapan Roy, Ph.D., President and Founder of Biotech Support Group.
Red Blood Cells(RBC)/Blood
Christina M. Wilhelm, Thomas H. Snider, Michael C. Babin, David A. Jett, Gennady E. Platoff Jr., David T. Yeung. A comprehensive evaluation of the efficacy of leading oxime therapies in guinea pigs exposed to organophosphorus chemical warfare agents or pesticides. Toxicology and Applied Pharmacology Available online 31 October 2014. doi:10.1016/j.taap.2014.10.009
Cholinesterase Analysis for Evaluating Oxime Therapies. Acetylcholine is an essential neurotransmitter, and inhibitors of cholinesterases(ChEs) are potent toxins. The objective of the present study is to identify an oxime antidote, under standardized and comparable conditions, that offers protection against chemical warfare agents or pesticides. Clinical signs of toxicity were observed for 24 h post challenge and blood cholinesterase [AChE and butyrylcholinesterase (BChE)] activity was analyzed utilizing a modified Ellman's method. In the modified Ellman's enzymatic assay for evaluating ChE activity, HemogloBind™, Biotech Support Group's unique solid-phase polymer for hemoglobin depletion, was used for pre-treatment. The article states "Terminal blood samples were collected and processed for all survivors using HemogloBind™".
Barasa, Benjamin, and Monique Slijper. " Challenges for red blood cell biomarker discovery through proteomics." Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics 1844.5 (2014): 1003-1010
Biotech Support Group reports on a recent review article which describes the simplicity and efficiency of their proteomic sample preparation technology for selectively depleting hemoglobin, to help solve the dynamic range problem for comprehensive erythrocyte proteome analysis. The citation is: Barasa, Benjamin, and Monique Slijper. " Challenges for red blood cell biomarker discovery through proteomics." Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics 1844.5 (2014): 1003-1010.In brief, this review describes the many challenges to generate in-depth RBC proteome analysis, such as to obtain pure red blood cells, and to acquire an in-depth proteome, despite the dynamic range problem due to a few highly over-represented RBC proteins – especially hemoglobin which accounts for approximately 97% of the cytosolic mass. The article states "Hemoglobin can also be depleted from an RBC lysate by employing Hemoglobind- [39] or HemoVoid [40] affinity systems. Hemoglobind consist of an elastomeric poly-electrolytic surface that has been optimized to bind Hb from serum samples with high affinity, and can as well be used to remove Hb from RBC lysates [39]. Walpurgis et al. used a complex matrix to deplete the RBC sample for Hb, named HemoVoid, which is made of a library of different ligand combinations, consisting of several kinds of ionic, aromatic, and polymer ligands [40]. Low abundance proteins in the RBC lysate are captured and enriched by the HemoVoid ligand library, while the high abundance proteins such as Hb and CA-I are thought to quickly saturate the system, and they primarily end up in the flow-through. The high abundance proteins in an RBC lysate can thus be easily separated from the low abundance protein fraction. The chosen Hb-depletion approaches by both Alvarez-Llamas et al. [39] and Walpurgis et al. [40] are well compatible with analysis of the RBC protein fractions by 1D or 2D gel electrophoresis, followed by protein identification through mass spectrometry."
"It is worthwhile to note that the authors describe both our strategies for hemoglobin depletion, as the correct choice will vary with the application. With our own experience and with users such as those referenced in this article, we have gained the necessary knowledge to guide our users to the best option for hemoglobin depletion and/or low abundance enrichment" states Swapan Roy, Ph.D., President and Founder of Biotech Support Group.
References Acknowledged in the Review
1.[39] G. Alvarez-Llamas, F. de la Cuesta, M.G. Barderas, V.M. Darde, I. Zubiri, C. Caramelo, F. Vivanco A novel methodology for the analysis of membrane and cytosolic sub-proteomes of erythrocytes by 2-DE Electrophoresis, 30 (2009), pp. 4095–4108.
[40] K. Walpurgis, M. Kohler, A. Thomas, F. Wenzel, H. Geyer, W. Schanzer, M. Thevis Validated hemoglobin-depletion approach for red blood cell lysate proteome analysis by means of 2D PAGE and Orbitrap MS Electrophoresis, 33 (2012), pp. 2537–2545
Hikosaka, Keisuke, et al. " Deficiency of Nicotinamide Mononucleotide Adenylyltransferase 3 (Nmnat3) Causes Hemolytic Anemia by Altering the Glycolytic Flow in Mature Erythrocyte" Journal of Biological Chemistry(2014): jbc-M114.
Authors Hikosaka et al describe research on nicotinamide mononucleotide adenylyltransferase 3 (Nmnat3) from red blood cells and its regulation of nicotinamide adenine dinucleotide. The cytoplasmic fraction consisted of hemolyzed samples obtained from the supernatant and ghost for membrane fraction. An alpha cellulose & microcrystalline cellulose column for depleting platelets and leukocytes from heparinized whole blood for an assay of RBC metabolic enzymes is described. Extraction of metabolites from RBCs and NAD related metabolites from whole blood by perchloric acid method for LC-MS/MS measurement is done. Measurement of metabolite levels involved using MS and HPLC, quantitative analysis software and separations using a column. The article states "...Each sample was normalized by hemoglobin concentration at 20 µg/µl, and then hemoglobin was depleted using HemogloBind™".
McGarry, Kevin G., et al. " Evaluation of HemogloBind™ treatment for preparation of samples for cholinesterase analysis." (2013). Advances in Bioscience and Biotechnology, 2013, 4, 1020-1023
In this article, measurement of cholinesterase activity prior to depletion and after removing hemoglobin is performed. A comparison of total cholinesterase activity with Ellman method and after Hemoglobind™ treatment prior to Ellman method did not display a statistical difference in mean ChE activity. Ellman's assay invovled measuring the sum of RBC membrane ChE activity and plasma ChE activity. Total cholinesterase activity of whole blood samples with Hemoglobind™ treatment prior to Ellman method is also consistent. Moreover, the Hemoglobind™ protocol is simpler with one incubation and short, low speed centrifugation.This article further validates our unique surface technology approach, not based on antibodies or engineered bio-ligands, as being highly selective and an efficient method for the depletion of hemoglobin concurrent with the recovery of functional activity.
Alvarez-Llamas, Gloria, Fernando de la Cuesta, Maria G. Barderas, Irene Zubiri, Maria Posada-Ayala, and Fernando Vivanco. " Characterization of Membrane and Cytosolic Proteins of Erythrocytes." In Vascular Proteomics, pp. 71-80. Humana Press, 2013.
Proteomic profiling of erythrocyte proteins to identify novel proteins linked to diseases is an evolving field of clinical proteomics. Cytosolic proteins could contribute to pathology of diseased erythrocytes. Hemoglobin interferes with LC-MS/MS analysis of low abundance cytosolic proteins. Hemoglobin depletion of cytosolic proteins is essential for proteomic sample preparation. Authors Gloria Alvarez-Llamas et al published a chapter in the book Vascular Proteomics titled, Characterization of membrane and cytosolic proteins of erythrocytes, which cites HemogloBind™ from Biotech Support Group for hemoglobin depletion of erythrocyte cells from the cytosolic fraction. A simple method of hemoglobin depletion using HemogloBind™ protocol allows for subsequent downstream proteomic analysis using 2-DE as it reduces major interference of hemoglobin from samples of red blood cells (RBCs) and identifies proteins.
Alvarez-Llamas, G., de la Cuesta, F., Barderas, M. G., Darde, V. M., Zubiri, I., Caramelo, C., Vivanco, F. A novel methodology for the analysis of membrane and cytosolic sub-proteomes of erythrocytes by 2-DE. Electrophoresis.2009;30:4095-4108
Authors in this study focused on the analysis of human cytosolic and membrane sub-proteomes. Hemoglobin from samples of red blood cells was studied using different strategies for isolation of the membrane and cytosolic fractions to determine the influence it has on proteome profiling by 2-DE and hemoglobin removal. Biotech Support Group's hemoglobin depletion reagent, HemogloBind™ was successfully used to erythrocyte cells. The results showed that Hemoglobind™ does have a high degree of specificity for hemoglobin and minimal interference. Particularly interesting is how authors developed a novel combined strategy based on hypotonic lysis isolation for identification of high molecular weight proteins (i.e. spectrin, ankyrin) by nano-LC coupled to an LTQ-Orbitrap mass spectrometer.
Zihao Wang, Kyoungsook Park, Frank Comer1, Linda C. Hsieh-Wilson, Christopher D. Saudek, Gerald W. Hart. Site-Specific GlcNAcylation of Human Erythrocyte Proteins: Potential Biomarker(s) for Diabetes Mellitus. Diabetes.2008;58, 309-317.
O-GlcNAc actively cycles on erythrocyte, regulates insulin signaling and is a mediator of glucose toxicity. Therefore studying it may reveal potential biomarker for diagnoses of diabetes. Highly efficient enrichment methods based Hemoglobind™ overcome the challenges of low stoichiometry, suppressed ionization efficiency in presence of unmodified peptides, and intrinsic lability in gas phase mass spectrometric methods. In this paper, authors used Hemoglobind™ to study erythrocyte proteins and compared it with their abundance between normal and diabetic samples proteins. Blood samples were obtained from normal and diabetic patients collected into a vial containing EDTA and OGlcNAcase inhibitor PUGNAc. Next the researchers fractionated the blood cells to isolate erythrocytes. After erythrocytes were lysed and centrifuged, the supernatant containing hemoglobin was partially depleted by HemogloBind™ from Biotech Support Group.
Datta, Pradip. Effect of Hemolysis, High Bilirubin, Lipemia, Paraproteins, and System Factors on Therapeutic Drug Monitoring. Handbook of Drug Monitoring Methods.2008; 97-109.
Bilirubin, hemoglobin, lipids, paraproteins are endogenous interferents of immunoassays used in clinical laboratories which affect therapeutic drug monitoring (TDM), drugs of abuse (DAU) testing, and toxicology assays. Hemoglobin interference is caused by its absorption, fluorescence and chemiluminescence properties. Often assays are repeated with different methods or by removing the interferent from the sample. In this book chapter, authors Datta et al cited HemogloBind™ the synthetic solid phase anionic polyelectrolyte from Biotech Support Group for hemoglobin depletion and reduction of matrix effects.
Yuichi Miki, Tomoki Tazawa, Kazuya Hirano, Hideki Matsushima, Shoko Kumamoto, Naotaka Hamasaki, Tomohiro Yamaguchi, Masatoshi Beppu. Clearance of oxidized erythrocytes by macrophages: Involvement of caspases in the generation of clearance signal at band 3 glycoprotein. Biochemical and Biophysical Research Communications.2007; 363(1):57-62
Reduction of erythrocyte clearance by macrophages happens when oxidative stress is decreased either by pretreatment with Hemoglobind™ or enzymes inhibiting caspases causing decreased band 3 aggregation. Band 3 aggregation increased by actions of caspases and was reduced by treatment with caspase inhibitors Z-VAD-fmk or Z-DQMD-fmk (caspase 3 selective) prior to oxidation. Pretreatment of erythrocytes exposed to H2O2 have increased propensity to bind and get phagocyted by macrophages. In this paper authors used anti-band 3 serum to reduce binding and pretreatment of erythrocytes with Hemoglobind™ & polylactosamine-cleaving enzyme.
Sarawathi,et al., Relative quantification of glycated Cu-Zn superoxide dismutase in erythrocytes by electrospray ionization mass spectrometry, Biochim Biophys Acta. 1999 Feb2; 1426(3):483-90.
Electrospray ionization mass spectrometry (ESIMS) was used for relative quantification of glycated Cu-Zn superoxide dismutase (SOD-1) in human erythrocytes. SOD-1 samples were prepared from erythrocytes by removing hemoglobin using Hemoglobind™ gel followed by ethanol and chloroform extraction. The reproducibility in measurement of the relative percentage of glycated protein was good, and the standard deviation of each measurement was 4.0%. From the mass spectral analysis of a mixture of commercial SOD-1 and in vitro partially glycated SOD-1 in several ratios, it was found that free and glycated SOD-1 have the same ionization efficiencies. The percentage of glycation on SOD-1 was measured in 30 individuals, including patients with diabetes mellitus. The glycation levels ranged from 4.5% to below the detection limit. The SOD-1 sample extracted from erythrocytes was fractionated by Glyco-Gel B chromatography, and the separated fractions were analyzed by MS. The mass spectra of absorbed fraction showed significant amounts of non-specific binding of non-glycated proteins to Glyco-Gel B.
