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 instruments. 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.