We evaluated the performance of the Orbitrap Ascend in comparison to the Orbitrap Eclipse in RTS-SPS-MS3-based analysis of human proteomes using fractionated and unfractionated samples labeled with TMT and TMTpro reagents. We did this comparison in the context of different sample input amounts; we analyzed differences in quantitative precision; we compared the effectiveness in identifying protein abundance changes induced by treatment with covalent inhibitors of PIN1.