Antigen-subtracted 2-DE/MS strategy, a novel proteomic analysis platform

Abstract

In the present study, we developed a novel proteomic research strategy named antigen-subtracted 2-DE/MS strategy and applied it to comparative proteomic analysis of anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide-transformed human bronchial epithelial cell line (16HBE-C) and its parental cell line (16HBE) G0/G1 cells. Following pre-purification by ammonium sulfate precipitation, rabbit antibodies against 16HBE G0/G1 cells were coupled with protein A/G PLUS-agarose under the maximal coupling rate of about 50%. The agarose-antibody complex was then used in immunoprecipitation known as antigen subtraction. When the mass ratio of antibody to the sample was 2.5–3:1, the subtraction rates for 16HBE and 16HBE-C G0/G1 cell proteins were 44 and 34%, respectively. Both subtracted and unsubtracted samples were then subjected to the 2-DE resolution. In 16HBE-subtracted 2-DE maps, 315 protein spots were subtracted and 49 new protein spots were detected, whereas in 16HBE-C-subtracted 2-DE maps, 287 protein spots were subtracted and 33 new protein spots were detected. Taken together, antigen subtraction results in 65 new protein spots being allowed to be detected, therefore, makes it possible to get more information of the samples. Finally, 4 protein spots only detected in 16HBE-C-subtracted 2-DE maps were analyzed by the Q-TOF MS/MS, and successfully identified as U6 snRNA-associated Sm-like protein LSm3, 60S acidic ribosomal protein P1, Peroxiredoxin-6 and 60S acidic ribosomal protein P2. These proteins are involved in carcinogenesis, oxidation stress and protein synthesis. In conclusion, the antigen-subtracted 2-DE/MS strategy could reduce the complexities of protein samples and therefore, improve the resolution for the sample analysis.