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Validated hemoglobin‐depletion approach for red blood cell lysate proteome analysis by means of 2 D PAGE and O rbitrap MS
Author(s) -
Walpurgis Katja,
Kohler Maxie,
Thomas Andreas,
Wenzel Folker,
Geyer Hans,
Schänzer Wilhelm,
Thevis Mario
Publication year - 2012
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201200151
Subject(s) - proteome , hemoglobin , cytosol , lysis , chemistry , red blood cell , intracellular , biochemistry , reagent , chromatography , enzyme
The analysis of the cytosolic red blood cell (RBC) proteome is negatively affected by the high intracellular amount of hemoglobin complicating the detection of low‐abundant cytosolic proteins. In this study, an alternative approach for the preparation of hemoglobin‐depleted RBC lysates is presented, which was established in combination with downstream 2 D PAGE analysis and O rbitrap MS . Hemoglobin removal was accomplished by using H emo V oid TM depletion reagent, which enabled a very efficient enrichment of low‐abundant proteins by simultaneously reducing the hemoglobin concentration of the sample. After defining selected sample preparation protocol characteristics including specificity/selectivity, precision and linearity, a 2D reference map (pH 4–7) of the cytosolic RBC proteome was generated and a total of 189 different proteins were identified. Thus, the presented approach proved to be highly suitable to prepare reproducible high‐resolution 2D protein maps of the RBC cytosol and provides a helpful tool for future studies investigating disease‐ or storage‐induced changes of the cytosolic RBC proteome.