HemoVoid™ LC-MS On-Bead For RBC Proteomics

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;

Authors Lasonder et al published an article titled,"The Plasmodium falciparum schizont phospho-proteome reveals extensive phosphatidylinositol and cAMP-Protein Kinase A signalling" in the journal of Proteome Research describing an overview of the Plasmodium falciparum phosphoproteome. Researchers discovered phosphorylated P. falciparum proteins and phosphorylation sites at the schizont stage of parasite development. Scientists found phosphorylation regulated DNA replication, transcription, translation and mitotic events (DNA packaging, chromosome organization, actin cytoskeleton). Researchers also found the CAMP-PKA signaling pathway to be involved in these events. The phosphorylation/dephosphorylation steps are important regulatory process of egress from and invasion into erythrocytes by merozoites. Scientists found phosphorylation of enzymes in the inositol pathway and discovered role of phosphorylation in merozoite egress and red blood cell invasion. Scientists developed an in vitro kinase assay and established the correlation of cAMP-PKA signaling for motility. CDPK1, GAP45, Myosin A were identified substrates with the glideosome motor complex. The analysis of Plasmodium falciparum schizont phospho-proteome involved steps which depleted hemoglobin from the samples using Hemovoid™ from Biotech Support Group. Freezing/thawing lysed P. falciparum schizont-infected erythrocytes and uninfected RBCs. Halt phosphase inhibitors (100X concentrated solution of sodium fluoride, sodium orthovanadate, sodium pyrophosphate and β-glycerophosphate) and protease inhibitor cocktail (AEBSF, aprotinin, bestatin, E-64, leupeptin, pepstatin A and EDTA) was added to the sample followed by centrifugation to separate the soluble and pellet fractions. Hemovoid™ successfully depleted hemoglobin from the soluble fraction yielding a hemoglobin depleted soluble fraction. Through the science discovered at Biotech Support Group, HemoVoid™ remains one of the only products in the market today that can separate hemoglobin from a blood sample, without destroying the intrinsic bonds within the protein. Mild elution maintains tertiary structure and simple transfer to secondary analysis.

Red Blood Cell Lysate
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.

Biotech Support Group reports on a recent research article which describes the simplicity and efficiency of their proteomic sample preparation technology for depleting hemoglobin, and enriching the low abundance proteome from human erythrocyte lysates. The article states “…hemoglobin was depleted…using a commercial kit (HemoVoid™). This crude protein-level pre-fractionation proved helpful in identifying additional proteins and N-termini”. In brief, the article describes a goal of the Chromosome-centric Human Proteome Project to identify all human protein species. With 3,844 proteins annotated as “missing” this is challenging. Enucleated and largely void of internal membranes and organelles, 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 1369 human erythrocyte natural and neo-N-termini and 1234 proteins. From the HemoVoid™ treated, hemoglobin-depleted soluble fraction, 778 proteins were identified, 171of which were not represented in either the soluble non-depleted fraction or the membrane fraction. This study also establishes a general workflow suitable for the in-depth determination of the position and nature of human protein N termini in different tissues and disease states. The identification of 281 novel erythrocyte proteins and six missing proteins identified for the first time in the human proteome confirmed its utility. “While the authors acknowledged other low abundance enrichment methods, our HemoVoid™ product was chosen for protein level enrichment. I am pleased to see it proved exceedingly useful in this exciting new area of proteomic identification.” states Swapan Roy, Ph.D., President and Founder of Biotech Support Group.

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;

This article states the HemoVoid™ process as a “…very efficient enrichment of low-abundant proteins by simultaneously reducing the hemoglobin concentration of the sample”, and that “…a two-dimensional reference map (pH 4-7) of the cytosolic red blood cell proteome was generated and a total of 189 different proteins were identified. Thus, the presented approach proved to be highly suitable to prepare reproducible high-resolution two dimensional protein maps of the RBC cytosol and provides a helpful tool for future studies investigating disease- or storage-induced changes of the cytosolic red blood cell proteome.” The identification of proteins in erythrocytes for proteomics studies is hampered by the huge abundance of hemoglobin (Hb) which hides other proteins and makes detection in 2-D gel-based separation difficult. It is therefore important to do pre-fractionation and depletion methods for the identification of low-abundant proteins. Authors Walpurgis et al demonstrate HemoVoid™’s application in the development of a protocol for proteomic analysis of hemoglobin-depleted RBC lysates in human blood from healthy donors. After using HemoVoid™, 2D-PAGE comparison of the unextracted hemolysate and the HemoVoid™ depleted hemolysate displays disappearance of the prominent and smeared hemoglobin ‘spot’ formerly containing high amount of hemoglobin on the gel is significantly reduced. Thus HemoVoid™ allows scientists to detect and study non-hemoglobin proteins using subsequent LC-MS/MS analysis for study of cytosolic proteome. Authors recorded that HemoVoid™ removed more than 98% of cellular hemoglobin, indirectly concentrated minor proteins and eliminated unbound hemoglobin. Upon comparison of the untreated and HemoVoid™ treated RBC lysates in the flowthrough and wash fractions by SDS-PAGE, scientists discovered the untreated RBC lysate showed two intense hemoglobin-derived bands (approximately 15 and 30 kDa), and the HemoVoid™-treated RBC lysate did show several bands which were not visible prior to hemoglobin depletion possibly representing non-hemoglobin proteins. Flow-through and three wash fractions contained some bands plus hemoglobin bands.

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.

The paper titled, "Cooperating G6PD mutations associated with severe neonatal hyperbilirubinemia and cholestasis" uses HemoVoid™ for performing native gel electrophoresis and immunoblotting on blood samples from the patient and control subjects that were lysed and depleted of hemoglobin. Subsequent to using HemoVoid™, non-bound protein was eluted at pH 9.8 and placed in native sample buffer, pH 6.8 and analyzed by native gel electrophoresis in polyacrylamide gradient gels of 4–15%. Separating hemoglobin from erythrocytes by contacting erythrocytes with a hypotonic buffer solution at a rate sufficient to render the release of hemoglobin from said erythrocytes without significant lysis. The hemoglobin is then separated from the erythrocytes. Hemovoid™ allows for the purification of hemoglobin solutions of DNA, endotoxins and phospholipids by contacting the hemoglobin solutions with an anion exchange medium.

Sudha Neelam, David G Kakhniashvili, Stephan Wilkens et al. Functional 20S proteasomes in mature human red blood cells Experimental Biology and Medicine.2011;236:580-591

Hemovoid™ is used to study the functional 20S and/or 26S proteasomes within red blood cells (RBCs; depleted of reticulocytes and leukocytes).Using methods such as double-immunofluorescence confocal microscopy to localize mature RBCs, proteasomes are isolated from mature RBC. After using Hemovoid™, a two-dimensional differential in-gel electrophoresis (2D-DIGE)approach was used to determine if proteasome-dependent protein degradation occurs within mature RBCs.

HemoVoid™: Hemoglobin Enrichment for Hemoglobin Variant Research

Biotech Support Group, LLC has been researching and creating innovative genomic and proteomic products for over 15 years. One of the new breakthroughs in our protein purification research is the novel method of separating and purifying hemoglobin from blood samples. Clinical researchers as well as lab technicians have been using Biotech Support Group's HemoVoid™ in various different applications in order to separate hemoglobin itself, from a milieu of proteins, in order to study the various antibodies, and biomarkers located on hemoglobin's surface. This is possible because hemoglobin retains its tertiary structure, and is not being denatured nor destroyed in the separation process. Through the science discovered at Biotech Support Group, HemoVoid™ remains one of the only products in the market today that can separate hemoglobin from a blood sample, without destroying the intrinsic bonds within the protein. Biotech Support Group's provides hemoglobin enrichment protocol from blood sample for hemoglobin variant research (HbS, HbF, HbA, HbA1c, Thalassemia, etc.)

Blood, Cytosolic Red Blood Cell Proteome
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).

Transfusion-associated graft-versus-host disease is prevented by gamma or X-ray irradiation of blood products. Radiation-induced dose-dependent erythrocytic damage causing hemolysis, storage/structural alterations, protein structure modifications, red blood cell (RBC) deformability and membrane leakage of extracellular K+ concentration. Sample preparation techniques involving hemoglobin depletion in addition to optimal irradiation dose and post-irradiation storage period is important for preventing transfusion-associated graft-versus-host disease and preserving blood product quality.

Cytosolic RBC proteome is researched using 2D-DIGE and nano-LC high-resolution/ high-accuracy Orbitrap MS. Sample preparation using HemoVoid™* for hemoglobin depletion ensures proper red cell concentrate preparation, storage and transfusion. Authors Walpurgis et al published an article in the journal Proteomics Clinical Applications titled, 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. Authors describe proteomic workflows and cite Biotech Support Group's HemoVoid™ for sample preparation. Whole blood samples containing citrate-phosphate-dextrose anticoagulant was collected. A leukocyte filter was used for leukoreduction. A Heraeus Cryofuge 6000i centrifugation was used for RBC, buffy coat and plasma separation.

A MacoPress blood component extractor. A colorimetric assay determined protein concentration of purified samples. Cytosolic RBC proteomic sample preparation involved cell lysis, hemoglobin depletion and protein determination. Washed RBCs were lysed, centrifuged and HemoVoid™ hemoglobin depletion protocol was performed. Upon removing hemoglobin, cytosolic proteins bound to HemoVoid™ matrix could be enriched. The elution fraction is centrifuged and concentrated. The retentate is washed with lysis buffer. 2D-DIGE was performed after gamma irradiation and ex vivo storage of the cytosolic RBC proteome.

Protein spots were identified via De-Cyder DIA module and ISTD images via BVA module. Altered protein spots and protein abundances were identified. The spotmaps of the untreated and unstored samples were compared with the samples stored following irradiation. In gel tryptic digestion and high-resolution/high-accuracy Orbitrap MS identified protein composition.Changes in protein abundances of the different samples was measured. Cytosolic RBC proteins, including TGase 2 or acylaminoacid-releasing enzyme, and the total number of identified peptides, sequence coverage, protein score was noted. 1D and 2D Western blotting in order to confirm their identity and validate the observed irradiation and storage-induced changes was performed. The 1D and 2D Western blots were validated against an antibody and the DDB1, VCP, and TGase 2 proteins were identified as sensitive markers for storage and irradiation-induced RBC lesions. Ionizing radiation or increased ex vivo storage which caused erythrocyte damage could affect RBC membrane. Potassium leakage, deformability and hemolysis are examples of RBC membrane defects. Moreover, gamma or x-ray radiation, irradiation dose, whole or RBC concentrate, pre-irradiation and storage before or after radiation was measured. Observed declines in protein abundance was measured. Irradiation-induced generation ROS was measured in RBC lysates as oxidation increased the intracellular proteases and insoluble protein aggregates also increased. Lactate dehydrogenase and hemoglobin were increased enhanced leakage from irradiated RBCs.

A decrease in cytosolic concentrations could be attributed to altered proteins codepleted from the cells. A screening assay which monitored RBC quality during ex vivo storage for lesions is developed using identified proteins as validated biomarkers. Engraftment and expansion of residual donor leukocytes could lead to transfusion-associated graft-versus-host disease. In blood transfusion recipients, irradiation of red blood cell (RBC) and ex vivo storage could benefit from hemoglobin depletion as proper sample preparation reduces variability in proteomic results. The development of a validated biomarker screening assay for quality of screening assays is an important field of research to prevent, detect and treat transfusion-associated graft-versus-host disease in blood transfusion patients.