An understanding of the process of peptide fragmentation and what parameters are best to obtain the most useful information is important. This is especially true for large-scale proteomics where data collection and data analysis are most often automated, and manual interpretation of spectra is rare because of the vast amounts of data generated. We show herein that collisional cell peptide fragmentation, in this case higher collisional dissociation (HCD) in the Q Exactive, is significantly affected by the normalized energy applied. Both peptide sequence and energy applied determine what ion fragments are observed. However, by applying a stepped normalized collisional energy scheme and combining ions from low, medium, and high collision energies, we are able to increase the diversity of fragmentation ions generated. Application of stepped collision energy to HEK293T lysate demonstrated a minimal effect on peptide and protein identification in a large-scale proteomics dataset, but improved phospho site localization through increased sequence coverage. Stepped HCD is also beneficial for tandem mass tagged (TMT) experiments, increasing intensity of TMT reporters used for quantitation without adversely effecting peptide identification.