A new protocol published in Methods in Molecular Biology aims at preparing red blood cells (RBCs) for analysis of cytosolic and membrane sub-proteomes.1 Using an approach developed by Alvarez-Llamas et al. (2013), 188 unique proteins were identified in RBCs. A review of previous strategies used to characterize RBCs2 identified a total of 751 proteins found within human RBCs. Unlike other cell types, RBCs contain predominately hemoglobin and few internal structures or organelles. This specialized protocol is necessary to characterize the RBCs (and maximize the number of proteins identified) due to the large amount of hemoglobin present, which can cause interferences and prevent the discovery of smaller, less prolific proteins.
The surface of an RBC is home to plasma membrane proteins that allow the RBC to change shape as needed, in order to flow through the smallest of microcapillaries. Surface membrane characterization of RBCs is of particular interest in blood pathologies such as for sickle cell disease, which distorts RBCs into a rigid “sickle” shape and prevents them from properly flowing through blood vessels. The protocol developed by Alvarez-Llamas et al. allows both membrane and cytosolic RBC proteins to be characterized.
In the report describing the researchers’ experimental procedures,1 whole blood samples were collected in tubes containing ethylenediaminetetraacetic acid. After allowing the tubes to sit for three days at 4 degrees C, samples were centrifuged and washed with wash buffer. Next, cells were repeatedly lysed and spun using a centrifuge to release hemoglobin. HemogloBind™ (Biotech Support Group LLC) was used to bind to latent hemoglobin still present in the suspended cells. The cytosolic suspension was placed on a vortex for 20 seconds before being spun with the centrifuge. The solution was then added to desalting column in preparation for Two-Dimensional Electrophoresis (2-DE). Desalting was necessary to prevent an interference between the HemogloBind™ and the 2-DE. The protocol up to this point was nearly identical for membrane proteins, the exception being that before 2-DE, membrane proteins required additional spinning via centrifuge, which helped to remove particulate matter.
Following 2-DE, gels were stained with silver. Membrane proteins were digested with modified porcine trypsin in preparation for liquid chromatography and tandem mass spectrometry (LC-MS/MS) on an LTQ-Orbitrap mass spectrometer (Thermo Scientific) coupled to a C-18 reverse phase nano-column. Hemoglobin was depleted from fractions of cytosolic proteins using TCA/acetone. Desalted peptides were trapped and separated on a C-18 column prior to analysis on an LTQ linear ion trap mass spectrometer.
Following LC-MS on both cytosolic and membrane proteins, mass spectra searches were performed using an MSDB database with MASCOT (Swiss-Prot database) and SEQUEST (NCBI database) programs for protein identification and post-translational modification characterization. Proteome Discoverer 1.0 software, and Bioworks 3.2 software (Thermo Scientific) were also used to search the IPI human protein database. This straightforward method of preparing RBCs for LC-MS/MS analysis will likely improve protein identification and lead to a greater understanding of blood pathologies.
1. Alvarez-Llamas, G., et al. (2013) “Characterization of membrane and cytosolic proteins of erythrocytes,” Methods in Molecular Biology, 1000 (pp. 71–80).
2. Goodman, S.R., et al. (2007) “The human red blood cell proteome and interactome,” Experimental Biology and Medicine (Maywood, NJ), 232(11) (pp. 1391–1408).