Major proteins in normal human lymphocyte subpopulations separated by fluorescence-activated cell sorting and analyzed by two-dimensional gel electrophoresis.
We have compared the overall patterns of protein synthesis of normal human lymphocyte subpopulations taken from five volunteers using high resolution two-dimensional gel electrophoresis. The lymphocytes were isolated using density gradient centrifugation, labeled with subtype-specific MoAbs, and separated to a high degree of homogeneity by FACS into CD4+ helper T cells, CD8+ suppressor T cells, CD20+ B cells, and N901 (NHK-1)+ NK cells. The four lymphocyte subpopulations were labeled with [35S]methionine for 14 hr, solubilized in lysis buffer, and analyzed by two-dimensional gel electrophoresis (IEF). Of about 1000 proteins resolved in each case, most were found to be common to all subpopulations. However, eight putative markers for B1+ (proteins 5525, Mr = 63,700; 5621, Mr = 63,700; 8311, Mr = 36,900; 2202, Mr = 36,300; 6121, Mr = 30,300; 106, Mr = 29,300; 5009, Mr = 23,000; 8012, Mr = 11,600) and one for N901+ (protein 8129, Mr = 30,400) were identified. In contrast, no major protein markers were found that could differentiate T4+ and T8+ cells from each other or from B cells and NK cells. With the exception of two B1+ markers (proteins 5525 and 5621), lower but variable levels of the other markers were observed in all cell types. All the putative protein markers have been identified in the protein database of human peripheral blood mononuclear cells (PBMCs) (see accompanying article by Celis et al.). Comparison of the overall patterns of protein synthesis of the unsorted PBMCs with those of the four subpopulations showed that the synthesis of some major PBMC proteins decreased substantially in the sorted subsets. These proteins are most likely not of monocyte origin, as these cells constituted only about 15% of the total PBMCs. Also, the inhibition does not seem to be due to the addition of the single MoAbs or to cell cycle differences. Taken together, the data provide a background for further studies of protein profiles in normal (resting or activated) and malignant hematopoietic cells.
Madsen
P S
PS
University Department of Medicine and Haematology, Aarhus Amtssygehus, Denmark.
Hokland
M
M
Ellegaard
J
J
Hokland
P
P
Ratz
G P
GP
Celis
A
A
Celis
J E
JE
D000943:Antigens, Differentiation / Q000032:analysis; D001798:Blood Proteins / Q000378:metabolism*; D002469:Cell Separation; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005434:Flow Cytometry; D006801:Humans; D008214:Lymphocytes / Q000145:classification* / Q000276:immunology / Q000378:metabolism; D009000:Monocytes / Q000378:metabolism; D010641:Phenotype
D000943:Antigens, Differentiation; D001798:Blood Proteins