Screening Differential Sub-Cellular Proteins Of Cultured Breast Cancer Cell Lines

Objective: To investigate available scheme for analyzing sub-cellular proteins of breast cancer cell lines by surface-enhanced laser desorption /ionization time of flight mass spectrometry (SELDI-TOF-MS). And to screen differential proteins by comparing mitochondrial, nuclear and membrane protein fingerprinting between breast cancer cells and normal breast epithelial cells, which may lay a foundation for screening protein biomarkers of breast cancer.Methods: 1. Molecular weight of SELDI-TOF-MS was calibrated by standard molecular weight materials including Insulin, Cyto C and Mb. 2. Human breast cancer cell line MCF-7 was cultured with PRMI1640, and cells of exponential growth phase were collected, Sub-cellular proteins were extracted according to manufacture instruction, following deposition of ACE, then the precipitation was dissolved with 4‰Triton X-114. 3. Extracted and pretreated sub-cellular proteins were measured by SELDI-TOF-MS with different laser intensities to establish optimal scheme for analysis of sub-cellular proteins. 4. Sub-cellular proteins of cells stored at -80℃more than two months were extracted and analyzed after ACE precipitation. The protein fingerprinting was compared with that of fresh cells to study if long time storage could influence sub-cellular protein analysis. 5. Mitochondrial, nuclear and membrane proteins of breast cancer cell line MDA-MB-231, MCF-7 and normal breast epithelial cell line HBL-100 were abstracted, deposited and dissolved with optimal scheme, and protein concentration of different sub-cellular components was analyzed by Olympus AU 2700 6. Sub-cellular proteins of same amount were added to proteinchip golden array and analyzed by SELDI-TOF-MS. Differential proteins were screened by Biomarker Wizard Software and Biomarker Patterns Software.Result: 1. The results showed that the deviation of molecular weight of SELDI-TOF-MS was less than 0.1% after three standard materials calibration, illustrating its accuracy in protein molecular weight detection. 2. Our study demonstrated that the optimal pretreatment scheme to measure sub-cellular protein by SELDI-TOF-MS was ACE precipitation and 4‰Triton X-114 dissolution. 3. Different sub-cellular protein fingerprinting was found after analyzing by different laser intensities, when the laser intensity was 190, the result showed more protein peaks, higher protein abundance and stable baseline. 4. Protein fingerprinting of sub-cellular showed no significant difference between fresh cells and cells stored in -80℃over two months, which meant that long time storage had little influence on sub-cellular protein fingerprinting detecting by SELDI-TOF-MS. 5. AU 2700 results showed that sub-cellular protein concentration of HBL-100, MDA-MB-231 and MCF-7 cell lines were following, mitochondrial: 82.23±21.70μg/ml, 78.76±26.45μg/ml and 85.07±19.39μg/ml;nuclear: 53.43±13.96μg/ml, 46.77±18.72μg/ml and 58.67±20.02μg/ml; membrane: 95.47±30.64μg/ml, 84.56±17.06μg/ml and 77.42±20.48μg/ml respectively. 6. When compared with normal breast cell line HBL-100, the sub-cellular protein fingerprinting of breast cancer cell lines MDA-MB-231 and MCF-7 showed significant difference after biomarker patterns software analysis. mitochondrial protein peaks at 9.2kD, 13.8kD, 14.0kD and 22.5kD were down-regulated in both two breast cancer cell lines, while protein peaks at 10.0kD and 11.6kD were up-regulated. nuclear protein peaks at 5.8kD, 8.3kD and 18.2kD over expressed in both two breast cancer cell lines, while protein peaks at 9.9kD and 15.7kD were under expressed, membrane protein peaks at 7.8kD, 8.4kD and 8.8kD increased in both two breast cancer cell lines, while protein peaks at 9.6kD decreased.Conclusion:1. The experiment provides a scheme to abstract, deposit and dissolve breast cancer mitochondrial, membrane and nuclear protein which is suitable for SELDI-TOF-MS. 2. Sub-cellular proteins of breast cancer cell lines MCF-7 and MDA-MB-231 show significant difference when compared with normal breast epithelial cell HBL-100. 3. In the study we obtain at least 10 differential sub-cellular proteins in breast cancer cell lines, which applies a new method for screening protein biomarkers of breast cancer.