Subcellular fractionation of stored red blood cells reveals a compartment-based protein carbonylation evolution ☆ Julien Delobel, Michel Prudent, Olivier Rubin, David Crettaz, Jean-Daniel Tissot, Niels Lion ⁎ Service Régional Vaudois de Transfusion Sanguine, route de la Corniche 2, CH-1066 Epalinges, Switzerland ARTICLE INFO ABSTRACT Available online 10 May 2012 During blood banking, erythrocytes undergo storage lesions, altering or degrading their metabolism, rheological properties, and protein content. Carbonylation is a hallmark of protein oxidative lesions, thus of red blood cell oxidative stress. In order to improve global erythrocyte protein carbonylation assessment, subcellular fractionation has been established, allowing us to work on four different protein populations, namely soluble hemoglobin, hemoglobin-depleted soluble fraction, integral membrane and cytoskeleton membrane protein fractions. Carbonylation in erythrocyte-derived microparticles has also been investigated. Carbonylated proteins were derivatized with 2,4-dinitrophenylhydrazine (2,4-DNPH) and quantified by western blot analyses. In particular, carbonylation in the cytoskeletal membrane fraction increased remarkably between day 29 and day 43 (P < 0.01). Moreover, protein carbonylation within microparticles released during storage showed a two-fold increase along the storage period (P <0.01). As a result, carbonylation of cytoplasmic and membrane protein fractions differs along storage, and the present study allows explaining two distinct steps in global erythrocyte protein carbonylation evolution during blood banking. This article is part of a Special Issue entitled: Integrated omics. © 2012 Elsevier B.V. All rights reserved. Keywords: Aging Erythrocyte Membrane Microparticles Protein carbonylation Storage lesions 1. Introduction Transfusion medicine is a very important field in human health, concerning millions of lives worldwide. Red blood cells (RBCs) processed from whole blood donation or from apheresis are stored as erythrocyte concentrates (ECs) at 4 °C during 42 to 49 days, depending on the additive solution used (saline– adenine–glucose–mannitol, SAGM, or phosphate–adenine– glucose–guanosine–saline–mannitol, PAGGSM, respectively). This cold storage allows slowing down the RBC metabolism in order to improve the storage duration, and preserve the product from bacterial contamination. In spite of the undeniable bene- fits of transfusion in human health, some unknown counter- parts have to be considered, due to lesions accumulating in labile blood products during their storage [1–6], even under standardized optimal storage conditions [7,8]. Such storage lesions occur in ECs, altering biochemical [9] and biomechanical [10] properties of erythrocytes, as well as their protein content. Understanding the mechanisms altering RBC quality during storage is of great interest since increasing post-chirurgical complications and reducing patient survival have been associ- ated with the age of received blood products [11]. More recently, a meta-analysis of studies comparing transfusion outcomes with blood storage-times revealed that the risk of death significantly increased when using older blood products [12]. Such studies make the transfusion medicine community in- vestigating new storage conditions in order to improve blood quality. In particular, new additive solutions have been pro- posed during the last decade [13–15], as well as special banking conditions such as anaerobic storage [16–18]. JOURNAL OF PROTEOMICS 76 (2012) 181 – 193 ☆ This article is part of a Special Issue entitled: Integrated omics. ⁎ Corresponding author. Tel.: +41 21 314 65 68; fax: +41 21 314 65 78. E-mail address: niels.lion@mavietonsang.ch (N. Lion). 1874-3919/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jprot.2012.05.004 Available online at www.sciencedirect.com www.elsevier.com/locate/jprot