[Two-dimensional polyacrylamide gel electrophoresis-based serum protein separation: comparison of two sample preparation methods].
OBJECTIVE
To evaluate the effects of two sample preparation methods on two-dimensional polyacrylamide gel electrophoresis (2-DE)-based serum protein separation, and produce high-resolution and reproducible 2-DE images for identifying disease-related serum protein.


METHODS
Direct solubilization and hot SDS methods were used separately to extract and handle the total proteins of serum samples from patients with colorectal carcinoma. Immobilized pH gradient 2-DE was used to separate the total proteins. After image analysis of silver-stained 2-D gels, 3 differential protein spots were identified by matrix-assisted laser desorption/time-of-flight mass spectrometry.


RESULTS
The total proteins treated with hot SDS method were used to perform 2-DE. 2-DE patterns with high resolution and reproducibility were obtained for human serum samples. 2-DE was performed 3 times for the samples treated by direct solubilization and hot SDS methods, respectively, resulting in the average number of spots of 675-/+46 and 702-/+49, respectively. The average matching protein spots were 573-/+42 and 623-/+52, with average matching rate of 85.3% and 89.6%, respectively. The average position deviation of matched spots in different gels was 0.85-/+0.30 mm and 0.81-/+0.28 mm in IEF direction, and 1.02-/+0.18 mm and 0.97-/+0.12 mm in SDS-PAGE direction. Mass spectrometry of the 2-D gels treated with hot SDS method generated high-quality mass spectra, and the sample preparation method allowed detection of relatively low abundance protein.


CONCLUSIONS
Hot SDS method is more effective for human serum protein sample preparation and well-resolved, reproducible 2-DE profiles of human serum have been established in this study.
Zhao
Liang
L
Department of Pathology, Southern Medical University, Guangzhou 510515, China. liangzhao_gd@ yahoo.com.cn
Ding
Yan-Qing
YQ
Liang
Li
L
Li
Xin
X
Li
Xue-Hua
XH
Wu
Li-Sha
LS
D001797:Blood Protein Electrophoresis; D001798:Blood Proteins / Q000032:analysis* / Q000302:isolation & purification*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006801:Humans; D010449:Peptide Mapping; D025941:Protein Interaction Mapping; D015203:Reproducibility of Results
D001798:Blood Proteins