Updated project metadata. 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