Evaluation on the effect of different in-gel peptide isoelectric focusing parameters in global proteomic profiling.

Peptide isoelectric focusing (IEF) is a common technique used in two-dimensional liquid chromatography tandem mass spectrometry (2D-LC-MS/MS) proteomic workflow, in which the tryptic peptide is first pre-fractionated based on pI values before being subjected to reverse phase LC-MS analysis. Although this method has been widely used by many research groups, a systemic study on the optimal conditions and fundamental parameters influencing the experimental outcomes has been lacking, including the effect of peptide extraction methods, the extent of pre-fractionation, and the choice of pH range. In this study, we compared the effect of different parameters on the numbers of peptides and proteins identified using two complex mouse proteomes. The results indicated that extraction of peptides from immobilized pH gradient (IPG) strips by sequential elution of increasingly organic solvents provided the highest number of peptide identification. In addition, we showed that approximately 45 more unique proteins were identified for every additional fraction collected during peptide IEF. Although narrow pH ranges provided higher resolution in peptide separation as expected, different pH ranges yielded similar numbers of peptide and protein identification. Overall, we demonstrated that the extraction solvent influenced the numbers of peptide and protein identification and quantitatively demonstrated the advantage of extensive fractionation and the performance of different pH ranges in practice.

Optimization of 2-dimensional gel electrophoresis for proteomic studies of solid tumor tissue samples.

The method of 2­dimensional gel electrophoresis (2-DE) has been widely used for the proteomic profiling of solid biological samples, however, the analytical conditions have not been optimized. The present study optimized the major conditions of 2­DE for determining the protein contents of solid tumor tissues, through enhancement of the separation efficiency and resolution. Three major analytical conditions of 2­DE analysis, namely protein extraction, focusing time for isoelectric focusing (IEF), and pre­reduction and alkylation prior to IEF, were carefully examined so that the optimal parameters and procedures were achieved. The use of a bead mill for protein extraction resulted in a higher protein yield in a minimal processing time. An optimal focusing time for IEF was established which improved the 2­DE image quality and reproducibility. Furthermore, reduction and alkylation of the protein sample prior to IEF reduced the horizontal streaking caused by oxidation and improved the resolution at the cathode. The optimized 2­DE analysis enabled the detection of 20% more protein spots compared with the previous reported conditions, with higher image quality and reproducibility. Accordingly, the optimized conditions may be used in the 2­DE analysis of tumor tissue samples, by which novel biomarkers of cancerous diseases and molecular targets of drugs are expected to be identified.