Quantitative Analysis of Human Red Blood Cell Proteome

Red blood cells (RBCs)
are the most abundant cell type in the human
body. RBCs and, in particular, their plasma membrane composition have
been extensively studied for many years. During the past decade proteomics
studies have extended our knowledge on RBC composition; however, these
studies did not provide quantitative insights. Here we report a large-scale
proteomics investigation of RBCs and their “white ghost”
membrane fraction. Samples were processed using the multienzyme digestion
filter-aided sample preparation (MED-FASP) and analyzed using Q-Exactive
mass spectrometer. Protein abundances were computed using the total
protein approach (TPA). The validation of the data with stable isotope-labeled
peptide-based protein quantification followed. Our in-depth analysis
resulted in the identification of 2650 proteins, of which 1890 occurred
at more than 100 copies per cell. We quantified 41 membrane transporter
proteins spanning an abundance range of five orders of magnitude.
Some of these, including the drug transporter ABCA7 and choline transporters
SLC44A1 and SLC44A2, have not previously been identified in RBC membranes.
Comparison of protein copy numbers assessed by proteomics showed a
good correlation with literature data; however, abundances of several
proteins were not consistent with the classical references. Because
we validated our findings by a targeted analysis using labeled standards,
our data suggest that some older reference data from a variety of
biochemical approaches are inaccurate. Our study provides the first
“in-depth” quantitative analysis of the RBC proteome
and will promote future studies of erythrocyte structure, functions,
and disease.