title: Systematic comparison between SDS-PAGE/RPLC and high-/low-pH RPLC coupled tandem mass spectrometry strategies in a whole proteome analysis.
abstract: SDS-PAGE and high-pH RPLC are commonly used fractionation strategies in proteomics research. A comparative investigation of these two strategies would be meaningful to thoroughly understand their respective features. Here, we systematically compared the two methods by trying 4 SDS-PAGE/RPLC and 3 high-/low-pH RPLC different workflows for a higher sensitivity in protein identification. Totally 9793 proteins were identified in HepG2 cells, with 8581 proteins identified by high-/low-pH RPLC workflows and 7933 by SDS-PAGE/RPLC workflows. The results demonstrate that using high-pH RPLC in the first dimensional separation would favour a high-throughput proteome analysis but choosing SDS-PAGE could yield much better peptide coverage. We found that the SDS-PAGE fractionation method benefits the neutral pI peptides. We also analyzed unexpected modifications caused by the two strategies. Our results suggest that more pre-fractionation benefits protein identifications in both strategies and pooling of gel pieces according to their grey values increased the identification efficiency in SDS-PAGE/RPLC workflows.
authors.affiliation: Department of Chemistry and Institutes of Biomedical Science Shanghai Medical School, Fudan University, Shanghai 200032, China. shenhuali@fudan.edu.cn pyyang@fudan.edu.cn.
authors.forename: Xuefei
authors.initials: X
authors.lastname: Yin
authors.forename: Yang
authors.initials: Y
authors.lastname: Zhang
authors.forename: Xiaohui
authors.initials: X
authors.lastname: Liu
authors.forename: Chen
authors.initials: C
authors.lastname: Chen
authors.forename: Haojie
authors.initials: H
authors.lastname: Lu
authors.forename: Huali
authors.initials: H
authors.lastname: Shen
authors.forename: Pengyuan
authors.initials: P
authors.lastname: Yang
mesh_terms: D056148:Chromatography, Reverse-Phase; D004591:Electrophoresis, Polyacrylamide Gel; D056945:Hep G2 Cells; D006801:Humans; D020543:Proteome / Q000032:analysis* / Q000302:isolation & purification; D040901:Proteomics; D053719:Tandem Mass Spectrometry
chemical_list: D020543:Proteome