Top-down mass spectrometry and direct dissociation of gas phase intact proteins have been demonstrated to be a powerful platform for identifying proteins from complex mixtures and for elucidating post-translational modifications (PTMs). Fragmentation of proteins in the atmospheric pressure/vacuum interface of the electrospray ionization mass spectrometer is an effective dissociation technique that can be utilized for on-line HPLC top-down analysis. We demonstrate the capability to perform intact protein identifications in a single-stage time-of- flight (TOF) mass spectrometer in a data independent (DIA) acquisition fashion by rapidly switching the in-source dissociation (ISD) energy during protein elution from a liquid chromatography (LC) column. The intact protein and product ion masses obtained at low and high ISD energies, respectively, were measured using a TOF mass analyzer. By coupling on-line protein separations to dissociation in the atmospheric pressure/vacuum interface region of the mass spectrometer, we identified proteins in simple complexity mixtures, including subunits from the human 20S proteasome complex, and PTMs such as phosphorylation and N-terminal acetylation events. This proof-of-principle study demonstrates that a data-independent pseudo- MS/MS method could be a relatively in-expensive platform for top-down MS.