Proteomic analysis of plasma membrane lipid rafts of HL-60 cells

Neutrophils acquire phagocytic activity as they differentiate. Recently, plasma membrane lipid rafts have been shown to play important roles in the process of phagocytosis in neutrophils. To characterize the proteins involved in phagocytosis and to elucidate the process by which they acquire phagocytic activity; we investigated by nano-LC-MS/MS analysis the changes in protein composition of plasma membrane lipid rafts during DMSO-induced differentiation of the human leukemia cell line HL-60 cells into neutrophilic lineage. Based on the spectrum counts of 147 proteins identified, 25 proteins were upregulated and 49 were downregulated by DMSO treatment. CD11b/CD18 subunits of beta(2)-integrin Mac-1, CD35, and GPI-80, which are known to be upregulated during differentiation, were dominantly detected in the lipid rafts of DMSO-treated cells. Many known membrane proteins, G proteins, and cytoskeletal proteins were also detected and they showed characteristic distributions. Absolute quantification of nine proteins in the lipid rafts using internal standard peptides labeled with stable isotopes showed that the amount of protein almost corresponded to the results obtained by spectrum count. Identified proteins, expression of which was altered by DMSO treatment, are expected to be candidate proteins involved in differentiation and functions of neutrophils.