The in-depth analysis of complex proteome samples requires fractionation of the 
sample into subsamples prior to LC-MS/MS in shotgun proteomics experiments. We 
have established a 3D workflow for shotgun proteomics that relies on protein 
separation by 1D PAGE, gel fractionation, trypsin digestion, and peptide 
separation by in-gel IEF, prior to RP-HPLC-MS/MS. Our results show that applying 
peptide IEF can significantly increase the number of proteins identified from 
PAGE subfractionation. This method delivers deeper proteome coverage and 
provides a large degree of flexibility in experimentally approaching highly 
complex mixtures by still relying on protein separation according to molecular 
weight in the first dimension.