In proteomics experiments the first critical step after sampling is certainly sample preparation. Multidimensional chromatography techniques have emerged as a powerful tool for the large-scale analysis of such complex samples as biological samples. In order to evaluate these separation techniques, microgram quantities of protein extracted from mouse heart tissue were fractionated by four different chromatographic methods. Regarding peptide-level fractionation, the first dimension of separation was performed with high-pH reversed-phase chromatography (pH-RP) and strong cation exchange chromatography (SCX). Regarding protein-level fractionation, C8 protein reversed-phase (C8-RP Prot) and high-recovery protein reversed-phase (hr-RP Prot) were used instead. The second dimension consisted of a reversed-phase nano-HPLC on-Chip coupled to an electrospray ionization quadrupole time-of-flight mass spectrometer for tandem mass spectrometric analysis. The performance and relative fractionation efficiencies of each technique were assessed by comparing the total number of proteins identified by each method. The peptide-level pH-RP and the hr-RP Prot protein-level separations were the best methods, identifying 1338 and 1303 proteins, respectively. The peptide-level SCX, with 509 proteins identified, was the worst method. Copyright © 2010 John Wiley & Sons, Ltd.