Updated publication reference for PubMed record(s): 30884231. We compared the performance of multidimensional protein identification (MudPIT) on Velos Pro Orbitrap (VPO) and Velos Orbitrap Elite (VOE) mass spectrometers to single dimension reversed phase (RP) chromatography on a Q Exactive Plus (QE+) and an Orbitrap Fusion™ Lumos™ (OFL). Using a digested HeLa cell protein extract, we carried out 16 different chromatography conditions on four different instrumentation platforms with three replicates of each condition, for a total of 48 proteomics analyses. We first compared selected chromatography conditions of the QE+ and OFL by varying column lengths, inner diameters, and C18-RP particle sizes. We next selected one chromatography condition on each system and varied the effective RP gradients lengths