HemogloBind ™ Reference Applications –
Nguyen, Anthony T., et al.
"UBE2O remodels the proteome during terminal erythroid
differentiation." Science 357.6350
During reticulocyte maturation, the proteome is remodeled
through the programmed elimination of most generic constituents of the cell, in
parallel with abundant synthesis of hemoglobin. The study used multiplexed
quantitative proteomics to identify candidate substrates of UBE2O, an E2
(ubiquitin-conjugating) enzyme, in an unbiased and global manner. Because
of the overly abundant presence of Hemoglobin, selective depletion of
Hemoglobin was necessary. The article states “Reticulocytes were lysed
by vortexing for 5 minutes at room temperature… An additional 10 bed vol of
Hemoglobind™ suspension was added to the samples, which were then vortexed for
another 10 min at room temperature followed by 4 min of centrifugation at 10000
x g. The supernatants, which contain hemoglobin-depleted sample,
were … processed for TMT quantification.”.
Serum Exosome Analyses
Nishida-Aoki, Nao, et al.
"Disruption of Circulating Extracellular Vesicles as a Novel Therapeutic
Strategy against Cancer Metastasis." Molecular Therapy 25.1 (2017):
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
Complexes and Functional
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
Lahut, Suna, et al. "Blood RNA
biomarkers in prodromal PARK4 and REM sleep behavior disorder show role of
complexin-1 loss for risk of Parkinson's disease." Disease Models &
Mechanisms (2017): dmm-028035. http://dmm.biologists.org/lookup/doi/10.1242/dmm.028035
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™) following the
Katarína, et al. "Transketolase Activity but not Thiamine Membrane
Transport Change in Response to Hyperglycaemia and Kidney
Dysfunction." Experimental and Clinical Endocrinology &
Diabetes (2017). https://www.thieme-connect.com/products/ejournals/abstract/10.1055/s-0043-115009
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, t he 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…”.
Agnostic – Applications to Different Species
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.”
Craig, J. R., et al. "A comparison of the anatomical and gastrointestinal
functional development between gilt and sow progeny around birth and weaning." Journal
of animal science (2019).
Gilt progeny (GP) often have
restricted growth performance and health status in comparison to sow progeny
(SP) from birth. To better understand underlying mechanisms, the study aimed to
compare differences in growth and development between GP and SP in the first 24
h after birth and in the peri-weaning period. Because serum
samples were quite hemolysed after collection and processing, it became
necessary to use HemogloBind™ to allow for better detection of IgG by ELISA. The
article states “As per the manufacturer’s instructions, 250 μL of Hemoglobind
was added to 250 μL of hemolyzed serum…
Parvathi S. Kumar, Haree K. Pallera, Pamela S. Hair, Magdielis
Gregory Rivera, Tushar A. Shah, Alice L. Werner,Frank A. Lattanzio, Kenji M.
Cunnion, and Neel K. Krishna. Peptide
inhibitor of complement C1 modulates acute intravascular hemolysis of
mismatched red blood cells in rats.TRANSFUSION Volume 00,
May 2016. doi:10.1111/trf.13674.
In brief, the study evaluated the role of a peptide inhibitor of complement C1
(PIC1) in an animal model of acute intravascular hemolysis in both prevention
and rescue scenarios. The authors state "To remove free Hb that may cause
optical interference in bilirubin analysis, we treated all the samples with Hb
depletion from hemolyzed serum/plasma (HemogloBind, Biotech Support Group).
Bilirubin concentration was then measured with a Bilirubin Assay Kit
(Sigma-Aldrich, St. Louis, MO)."
Laing, Richard W., et al.
"The use of an acellular oxygen carrier in a human liver
model of normothermic machine perfusion." Transplantation 101.11
Red cell lysates
O’Connell, Grant C., et al. "Monocyte-lymphocyte cross-communication via soluble CD163
directly links innate immune system activation and adaptive immune system suppression
following ischemic stroke." Scientific
reports 7.1 (2017): 12940
Kyoungsook Park, Christopher D. Saudek, and Gerald W.
Expression of β-N-Acetylglucosamindase (O-GlcNAcase) in Erythrocytes from
Prediabetic and Diabetic Individuals.Diabetes.2010;59(7):1845-50.
Delobel J., Rubin O., Prudent M., Crettaz D., Tissot J.-D., Lion N.(2010) Biomarker Analysis of Stored Blood
Products: Emphasis on Pre-Analytical Issues. International
Journal of Molecular Sciences. 11(11):4601-4617
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.
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
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.
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
quantification of glycated Cu-Zn superoxide dismutase in erythrocytes by
electrospray ionization mass spectrometry,
Biochimica et Biophysica Acta. 1999.1426(3):483-90
HemoVoid™ Reference Applications
Human Red Blood Cells (RBC)
Bollenbach, Alexander, et al. "GC-MS and
LC-MS/MS pilot studies on the guanidine (NG)-dimethylation in native,
asymmetrically and symmetrically NG-dimethylated arginine-vasopressin peptides
and proteins in human red blood cells." Journal of
Chromatography B (2020):
Previous studies showed that human
red blood cells are rich in large (> 50 kDa) asymmetric dimethylarginine -containing
proteins of unknown identity. The study aimed to report the identity,
biological activity and concentration of NG-methylated proteins
by using GC-MS and LCMS/MS approaches. The article states “we included in our
method the use of HemoVoid™ to remove specifically most erythrocytic hemoglobin
and to improve the SDS-PAGE separation of proteins for further processing. The
HemoVoid™, … allowed removal of erythrocytic hemoglobin to a large extent from
the hemolysate. … removal of hemoglobin by this technique enabled an effective
separation by SDS-PAGE and isolation of bands, presumably by avoiding
overloading of the gels by hemoglobin.”.
Kitao, Akihito, et al. "Band 3 ectopic expression in
colorectal cancer induces an increase in erythrocyte membrane-bound IgG and may
cause immune-related anemia." International Journal of Hematology (2020):
Autoimmune hemolytic anemia (AIHA) is a rare comorbidity in
colorectal cancer (CRC) and has an unknown etiology. To better understand
cancer-related anemia, the authors’ investigated ectopic band 3 expression and
erythrocyte membrane-bound IgG in a CRC cohort. To reduce the interference from
Hemoglobin, the article states “Erythrocytes were lysed … and hemoglobin was
depleted using HemoVoid (Biotech Support Group, NJ, USA, Cat. No.
Rosin-Arbesfeld, Rina, and Ronen
SIMAN-TOV. "Article of manufacture and methods for
increasing survival of red blood cells." U.S. Patent Application
patent application describes an ex - vivo method of increasing survival of red
blood cells (RBCs). The method comprises contacting the RBCs with an activator
of the non - canonical Wnt pathway, which results in actin polymerization,
thereby increasing survival of RBCs. The invention’s description states “The
Haemolysates were enriched with over 95 % hemoglobin. For hemoglobin depletion,
the hemoglobin depletion kit of HemoVoid … was used”. Upon depletion of
hemoglobin, a reduction in cytoplasmic actin levels was observed.
Nemkov, Travis, et al. "Hypoxia modulates the purine salvage pathway and decreases red blood
cell and supernatant levels of hypoxanthine during refrigerated storage." haematologica 103.2 (2018): 361-372.
The goal of this study
was to use proteomics in part to understand hypoxanthine catabolism in vivo for stored red blood cells. It is still
unclear whether accumulation of hypoxanthine in stored red blood cell units is
clinically relevant for transfused recipients. The
article states “Leukocyte-reduced
human RBC from healthy donor volunteers were washed five times in
phosphate-buffered saline prior to lysis in distilled water with sonication.
Proteomic analyses of RBC membranes and cytosols were performed…RBC cytosolic
proteins were depleted of hemoglobin using Hemovoid™ (Biotech Support Group,
Monmouth Junction, NJ, USA), prior to high-pH reversed phase fractionation”.
Miriam M., et al. "Identification of a soluble guanylate cyclase in RBCs:
preserved activity in patients with coronary artery disease." Redox Biology (2017).
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”.
Amélia, et al. "Evening and morning alterations in Obstructive Sleep Apnea
red blood cell proteome." Data in
Brief (2017). http://dx.doi.org/10.1016/j.dib.2017.01.005
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)”.
Philipp F Lange, Pitter F Huesgen, Karen Nguyen, and Christopher M
Overall. ” Annotating N termini for the
Human Proteome Project: N termini and Nα-acetylation status differentiate
stable cleaved protein species from degradation remnants in the human
erythrocyte proteome”, J. Proteome Research., Just
Accepted Manuscript • DOI: 10.1021/pr401191w • 21 Feb 2014
article describes a goal of the Chromosome-centric Human Proteome Project to
identify all human protein species. Enucleated, erythrocytes are simple yet
proteomically challenging cells due to the high hemoglobin content (about 97%
by mass) and wide dynamic range of protein concentrations that impedes protein
identification. Using a N-terminomics procedure called TAILS, the authors
identified from the HemoVoid™ treated, soluble fraction, 778 proteins were
identified, 171 of which were not represented in either the soluble
non-depleted fraction or the membrane fraction.
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.
Katja Walpurgis, Maxie Kohler, Andreas Thomas et al. Validated hemoglobin-depletion approach for red blood
cell lysate proteome analysis by means of 2D-PAGE and Orbitrap MS.Electrophoresis.2012;
Mizukawa, B., George, A., Pushkaran,
S. et al. Cooperating G6PD mutations associated with severe
neonatal hyperbilirubinemia and cholestasis.Pediatric Blood Cancer.2011;56: 840-842.
Sudha Neelam, David G Kakhniashvili,
Stephan Wilkens et al. Functional 20S proteasomes in mature human red blood
cells Experimental Biology and
HemoVoid™ On Bead Digestion Application Work On RBC by Irene Granlund, Umeå University
RBCs in Parkinson’s Disease
Stephan, et al. "Optimizing red blood cell protein extraction for
biomarker quantitation with mass spectrometry." Analytical and Bioanalytical Chemistry (2020): 1-14.
article describes the advantage of HemoVoid™ in detection of low abundance
proteins when comparing their amounts (in percent) between four alternative extraction
conditions, stating “…
Most peptides, following HemoVoid™ extraction, showed ion abundances ranging between
1.00E+5 and 1.00E+6 (31%). In comparison to this, fewer peptides (10–23%) were within this range following extraction with all
other protocols”. With respect to potential biomarkers for Parkinson’s
Disease, the article states “For example, PRDX6 accounts for 0.4% of the total
ion abundance after DOC (deoxycholate) extraction, whereas following HV
(HemoVoid™) extraction, this increases to 8%, a 20-fold enrichment”. The
authors conclude that the HemoVoid™ method significantly reduces the
concentration of hemoglobin, resulting in an increased signal-to noise of the
remaining red cell proteins. The article describes methods to digest the
HemoVoid™ bead-bound proteome, greatly simplifying the workflow for LC-MS/MS
Elhadi, Suaad Abd, et al. "α‐Synuclein in blood cells differentiates Parkinson’s
disease from healthy controls." Annals of Clinical and Translational Neurology.
goal of this study was to determine
whether blood cells expressing α-Synuclein can differentiate Parkinson’s
disease (PD) from healthy controls. Two proteoforms
- PSer129 a-Syn (phosphorylated
pathological form in Lewy bodies) and Oxidized a-Syn levels are observed in blood cells, but both at considerably lower
concentration than total a-Syn,
so the extremely high abundance of hemoglobin interferes with their analysis.
To compensate, the article states for PSer129 α -Syn & Oxidized α -Syn
detection by immunoassay, “followed from hemoglobin clearance with HemoVoid kit
(Biotech Support Group LLC, NJ, US)”.
Red Blood Cells,
Machado, Patrícia Isabel Pires. Pyruvate kinase and
glucose-6-phosphate dehydrogenase deficiencies and their association with
malaria–population genetics and proteomic studies. Diss. Universidade do Porto, 2013.
Walpurgis, Katja, et al. "Effects of gamma irradiation and 15 days of subsequent ex
vivo storage on the cytosolic red blood cell proteome analyzed by 2D DIGE and
Orbitrap MS." PROTEOMICS-Clinical Applications (2013).
P. Falciparum Clone 3D7
Cultured In Human Erythrocytes
Lasonder E, Green JL, Camarda G, Talabani H, Holder AA, Langsley G, Alano P. The Plasmodium falciparum schizont phospho-proteome
reveals extensive phosphatidylinositol and cAMP-Protein Kinase A signalling. J Proteome Research. 2012;
Species Agnostic – Applications
in non-human samples
Puente-Marin, Sara, et al. "In Silico Functional Networks Identified in Fish Nucleated Red Blood
Cells by Means of Transcriptomic and Proteomic Profiling." Genes 9.4 (2018): 202.
Nucleated red blood
cells (RBCs) of fish have, in the last decade, been implicated in several
immune-related functions, such as antiviral response, phagocytosis or
cytokine-mediated signaling. Label-free shotgun proteomic analyses were carried
out for in silico functional pathway profiling of rainbow trout RBCs. The
article states “The
cytosolic fraction, approximately 300 μL, was depleted of hemoglobin using
HemoVoidTM kit (Biotech Support Group, Monmouth
Junction, NJ, USA), in accordance with the manufacturer’s instructions”.
Puente-Marin S, Chico V et al. Identification of diverse defense mechanisms
in trout red blood cells in response to VHSV halted viral replication [version
1; referees: awaiting peer review].
F1000Research 2017, 6:1958 (doi: 10.12688/f1000research.12985.1)
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