Validated hemoglobin-depletion approach for red blood cell lysate proteome analysis by means of 2D PAGE and Orbitrap MS
Deutscher übersetzter Titel: | Validierter Hämoglobinabbau gemäß der Proteomanalyse von Lysat roter Blutzellen mittels 2D-PAGE und Orbitrap MS |
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Autor: | Walpurgis, Katja; Kohler, Maxie; Thomas, Andreas; Wenzel, Folker; Geyer, Hans; Schänzer, Wilhelm; Thevis, Mario |
Erschienen in: | Electrophoresis : microfluidics & proteomics |
Veröffentlicht: | 33 (2012), 16, S. 2537-2545, Lit. |
Format: | Literatur (SPOLIT) |
Publikationstyp: | Zeitschriftenartikel |
Medienart: | Elektronische Ressource (online) Gedruckte Ressource |
Sprache: | Englisch |
ISSN: | 0173-0835, 1522-2683 |
DOI: | 10.1002/elps.201200151 |
Schlagworte: | |
Online Zugang: | |
Erfassungsnummer: | PU201410009950 |
Quelle: | BISp |
Abstract des Autors
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 2D PAGE analysis and Orbitrap MS. Hemoglobin removal was accomplished by using HemoVoid(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. Verf.-Referat