In-depth analysis of the membrane and cytosolic proteome of red blood cells

Abstract

In addition to transporting oxygen and carbon dioxide to and from the tissues, a range of other functions are attributed to red blood cells (RBCs) of vertebrates. Diseases compromising RBC performance in any of these functions warrant in-depth study. Furthermore, the human RBC is a vital host cell for the malaria parasite. Much has been learned from classical biochemical approaches about RBC composition and membrane organization. Here, we use mass spectrometry (MS)-based proteomics to characterize the normal RBC protein profile. The aim of this study was to obtain the most complete and informative human RBC proteome possible by combining high-accuracy, high-sensitivity protein identification technology (quadrupole time of flight and Fourier transform MS) with selected biochemical procedures for sample preparation. A total of 340 membrane proteins and 252 soluble proteins were identified, validated, and categorized in terms of subcellular localization, protein family, and function. Splice isoforms of proteins were identified, and polypeptides that migrated with anomalously high or low apparent molecular weights could be grouped into either ubiquitinylated, partially degraded, or ester-linked complexes. Our data reveal unexpected complexity of the RBC proteome, provide a wealth of data on its composition, shed light on several open issues in RBC biology, and form a departure point for comprehensive understanding of RBC functions.

Oxidation of proteins: Basic principles and perspectives for blood proteomics

“…The RBC proteome has been the subject of extensive efforts, and more and more data accumulate through time [131][132][133][134][135][136][137][138][139][140][141], providing a high quality dictionary of red blood cell proteins. Red blood cells are inherently under continuous oxidative stress, as they pass the lungs once a minute; they contain high levels of O 2 and haemoglobin which autooxidises to produce O 2 2 and H 2 O 2 .…”

Characterization of the Vitreous Proteome in Diabetes without Diabetic Retinopathy and Diabetes with Proliferative Diabetic Retinopathy

“…Intracellular proteins were increased in the vitreous from noDR and PDR patients according to both quantitative and bioinformatic analysis. Many of the proteins exclusively detected in noDR and PDR are annotated as intracellular proteins and abundant in erythrocytes, 28 including carbonic anhydrase-I (CA-I), hemoglobin-R1, -δ, peroxiredoxin-1, and catalase. The detection of CA-I and hemoglobin chains in the noDR group was primarily due to their appearance in a single sample, whereas all 7 PDR samples contained these proteins.…”

Vascular proteomics

“…Mature RBCs lack internal cell structures and consist of cytoplasm within a plasma membrane envelope. To maximize the number of identified proteins, total RBC protein was divided into two fractions: membrane associated proteins, including the membrane skeleton, and cytoplasmic proteins [178]. The two fractions were further divided into subfractions and the proteins were identified in each subfraction as tryptic fragments.…”

“…The tryptic peptides were separated by RP-HPLC and identified by IT MS/MS. A total of 181 unique protein sequences were identified: 91 in the membrane fractions and 91 in the cytoplasmic fractions [178]. Three major types of blood products are used for transfusions: red cell concentrates, platelet concentrates, and fresh frozen plasma.…”