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.
State Key Laboratory of Quality Research in Chinese Medicine/Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Taipa, Macau, China.
Kin Ting R
KT
Kam
Xu
X
Liang
Jing-Rong
JR
Wang
Kam-Wai V
KW
Wong
Wen Luan
WL
Hsiao
Hua
H
Zhou
Zhi-Hong
ZH
Jiang
Liang
L
Liu
D000818:Animals; D018827:Carcinoma, Lewis Lung / Q000737:chemistry*; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D010446:Peptide Fragments / Q000302:isolation & purification*; D020543:Proteome / Q000032:analysis*; D018892:Proton-Motive Force; D052617:Solid Phase Microextraction; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000737:chemistry
D010446:Peptide Fragments; D020543:Proteome; D014357:Trypsin
Lung cancer; Peptide IEF; Proteomic profiling; Q-TOF MS