Short Communication An easy preparative gel electrophoretic method for targeted depletion of hemoglobin in erythrocyte cytosolic samples Hemoglobin (Hb) approximately constitutes 98% of the protein composition of a red blood cell (RBC), thus masking the remaining 2% which has still to be discovered completely due to the difficulty in its analysis. Here, we proposed a large-scale native gel electrophoresis that effectively tackles this limitation through a novel sample preparation strategy able to concentrate low-abundance species by removing Hb by means of electrophoretic instru- ments. Clear native PAGE was performed in a gel electrophoresis tube where the run was intermittently interrupted and different fractions were recovered in liquid phase into a collection chamber placed at the end of the tube. In this way, fractions containing multi- protein complexes with different molecular weights were collected in the native form by a simple elution. Red fraction containing Hb multi-protein complexes can be excluded from subsequent analyses, or rather be analyzed separately, reducing therefore the dynamic range of erythrocyte cytosolic protein concentrations and increasing the number of protein identifications. In particular, 838 protein spots in total were detected when fractions were analyzed by 2-D IEF-SDS-PAGE. This depletion method is inexpensive, simple to perform, reproducible and makes it possible to process large amounts of sample (up to 150 mg), thus making it suitable for in-depth proteome investigations. Furthermore, this strategy has the potential to be applied both to native and denatured proteomes of different biological samples. Keywords: Hemoglobin depletion / Prefractionation / Preparative electrophoretic meth- odologies / Red blood cell DOI 10.1002/elps.201000659 One of the main obstacles hindering proteomic research is the dramatic dynamic range of protein concentrations in biological samples. This statement holds significant pitfalls in the field of protein biomarker discovery, especially when handling blood and blood components [1]. Both in native and non-native separations, the presence of high-abundance proteins (HAPs) reduces the possibility to investigate the so- called hidden proteome composed of a large number of low- abundance proteins (LAPs). Interestingly, it is just in the low-abundance fraction of blood proteins that the potential biomarker-rich population is currently thought to reside [1]. To solve this problem, several pre-fractionation strategies have been developed in the preparation for two-dimensional electrophoresis (2-DE) or/and mass spectrometry (MS) analysis [2]. In the particular case of red blood cells (RBCs), inter- ference of hemoglobin (Hb) when detecting cytoplasmic proteome has been well documented. RBC cytosol proteome was investigated in different studies by 1-D SDS-PAGE [3], cation exchange chromatography [4], in-solution isoelec- trofocusing (IEF) [5] and Hb bind TM reagent [6]. These approaches allowed the identification of some hundreds of proteins (e.g. Pasini et al. were able to identify 252 proteins) but they were limited in sample loading capacity in the order of some tens of milligram. In 2008, Ringrose et al. [7] proposed a double depletion strategy to remove Hb by exploiting its affinity for Ni(II), and carbonic anhydrase-1 (CA-1) through ion exchange (IEX) chromatography. Using this approach, the authors were able to identify a total of 699 unique proteins. By a different technique based on combi- natorial peptide ligand library (CPLL) technology, Roux-Dalvai et al. [8] identified 1570 proteins (of which 950 by Gian Maria D’Amici Sara Rinalducci Lello Zolla Department of Environmental Sciences, Tuscia University, Viterbo, Italy Received December 10, 2010 Revised January 12, 2011 Accepted January 26, 2011 Abbreviations: CA, carbonic anhydrase; CA-1, carbonic anhydrase-1; CN, clear native; CPLL, combinatorial peptide ligand library; HAP, high-abundance proteins; Hb, hemoglobin; LAP, low-abundance proteins; MAPs, medium- abundance proteins; MPCs, multi-protein complexes; MW, molecular weight; RBC, red blood cell Correspondence: Professor Lello Zolla, Tuscia University, Largo dell’ Universita ` snc, 01100 Viterbo, Italy E-mail: zolla@unitus.it Fax: 139-0761-357-179 & 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com Electrophoresis 2011, 32, 1319–1322 1319