Systematic comparison between SDS-PAGE/RPLC and high-/low-pH RPLC coupled tandem mass spectrometry strategies in a whole proteome analysis

SDS- PAGEand 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 workflowsfor a higher sensitivity in protein identification. Totally 9793 proteins were identified in HepG2 cells, with 8581 proteins identified by high-/low-pH RPLC workflowsand 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- PAGEcould yield much better peptide coverage. We found that the SDS- PAGEfractionation 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.