Tandem affinity purification (TAP) coupled with mass spectrometry has become the technique of choice for characterization of multicomponent protein complexes. While current TAP protocols routinely provide high yield and specificity for proteins expressed under physiologically relevant conditions, analytical figures of merit required for efficient and in-depth LC-MS analysis remain unresolved. Here we implement a multidimensional chromatography platform, based on two stages of reversed-phase (RP) separation operated at high and low pH, respectively. We compare performance metrics for RP-RP and SCX-RP for the analysis of complex peptide mixtures derived from cell lysate, as well as protein complexes purified via TAP. Our data reveal that RP-RP fractionation outperforms SCX-RP primarily due to increased peak capacity in the first dimension separation. We integrate this system with miniaturized LC assemblies to achieve true online fractionation at low (≤5 nL/min) effluent flow rates. Stable isotope labeling is used to monitor the dynamics of the multicomponent Ku protein complex in response to DNA damage induced by γ radiation.