Challenges for red blood cell biomarker discovery through proteomics.

Red blood cells are rather unique body cells, since they have lost all organelles when mature, which results in lack of potential to replace proteins that have lost their function. They maintain only a few pathways for obtaining energy and reducing power for the key functions they need to fulfill. This makes RBCs highly sensitive to any aberration. If so, these RBCs are quickly removed from circulation, but if the RBC levels reduce extremely fast, this results in hemolytic anemia. Several causes of HA exist, and proteome analysis is the most straightforward way to obtain deeper insight into RBC functioning under the stress of disease. This should result in discovery of biomarkers, typical for each source of anemia. In this review, several challenges to generate in-depth RBC proteomes are described, like to obtain pure RBCs, to overcome the wide dynamic range in protein expression, and to establish which of the identified/quantified proteins are active in RBCs. The final challenge is to acquire and validate suited biomarkers unique for the changes that occur for each of the clinical questions; in red blood cell aging (also important for transfusion medicine), for thalassemias or sickle cell disease. Biomarkers for other hemolytic anemias that are caused by dysfunction of RBC membrane proteins (the RBC membrane defects) or RBC cytosolic proteins (the enzymopathies) are sometimes even harder to discover, in particular for the patients with RBC rare diseases with unknown cause. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.

Proteomics reveals reduced expression of transketolase in pyrimidine 5'-nucleotidase deficient patients.

PURPOSE: To date, it remains a challenge to correctly and timely diagnose red blood cell (RBC) enzymopathies that result in hereditary nonspherocytic hemolytic anemia (HNSHA), the third most common of which is pyrimidine 5'-nucleotidase (P5N) deficiency with just over 100 cases recognized and confirmed worldwide. EXPERIMENTAL DESIGN: We have investigated the RBC proteome of a patient with P5N deficiency due to a homozygous frameshift mutation in the NT5C3A gene. Protein expression levels were analyzed against healthy controls and against patients with hemolytic anemia of different origin, to account for the patient's elevated reticulocyte versus RBC ratio. RESULTS: Stringent relative quantification of the patient's protein levels revealed reduced levels of P5N, and unexpectedly, also decreased levels of transketolase, an enzyme involved in the nonoxidative phase of the pentose phosphate pathway, one of the few key pathways active in RBCs. Immunoblotting of whole blood samples from this and other P5N-deficient patients with dissimilar mutations indicated that P5N deficiency was correlated with reduced transketolase levels. CONCLUSIONS AND CLINICAL RELEVANCE: Consequently, insight into patient RBC proteomes illustrates potential benefit of coupling quantitative proteomics strategies with routine HNSHA diagnostic procedures. Proteomics facilitates finding novel biomarkers for HNSHA patients, for example, suffering from P5N deficiency, providing new prospects for future diagnosis and therapy.