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In this study, we used multiplex tandem mass tag labeling combined with nano-LC-MS/MS to compare the proteome of adult and cord RBCs and reticulocytes.2838 unique proteins were identified, providing the most comprehensive compendium of RBC proteins to date.
Other differences have been reported (8, 9), including expression of i rather than I antigen and weak expression of ABH antigens on cord cells.Cells were washed twice in cold PBS, followed by two washes with iso-osmotic buffer (Na PMSF (Sigma-Aldrich, Poole, UK)) at 50x packed cell volume, followed by centrifugation at 15,000 rpm for 10 min at 4 °C.The first supernatant was collected and kept as a cytoplasmic fraction.Transduction of erythroblasts was performed as described by Satchwell RNA (400 ng) was reverse transcribed into c DNA using Super Script II reverse transcriptase (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA).Globin expression was analyzed using primers to β-globin 5′ CTTTAGTGATGGCCTGGCTC and 5′ GGCAGAATCCAGATGCTCAA, and γ-globin 5′ GGGCAAGGTGAATGTGGAAGAT and 5′ GGGTCCATGGGTAGACAACCA as described previously (15).Cytoplasmic and nuclear fractions were prepared in the same way as samples for mass spectrometry (above).
Proteins were resolved by SDS-PAGE and transferred to PVDF by Western blotting.
Membranes were blocked with 10% milk powder for 1 h, followed by incubation with primary antibodies overnight at 4 °C.
Primary antibodies used were aquaporin I (CHIP28) and glycophorin A (CVDP) 00 dilution, ankyrin 1 (BRIC274) 0 dilution, and band 3 (BRIC170) 00 dilution, all validated and supplied by IBGRL ( Products/Res Prod Home.htm).
Overall, these data suggest that, with respect to the proteome, there is no barrier to the use of cord progenitors for the –6).
Cells from adult PB and cord can be expanded and induced to differentiate efficiently down the erythroid pathway to generate significant numbers of enucleated reticulocytes as an end point (1).
In addition cord stem cell banks are generally more representative of blood group diversity in the population compared with adult donor blood banks.