Automated 16-Plex Plasma Proteomics with Real-Time Search and Ion Mobility Mass Spectrometry Enables Large-Scale Profiling in Naked Mole-Rats and Mice

Performing large-scale plasma proteome profiling is challenging due to limitations imposed by lengthy preparation and instrument time. We present a fully automated multiplexed proteome profiling platform (AutoMP3) using the Hamilton Vantage liquid handling robot capable of preparing hundreds to thousands of samples. To maximize protein depth in single-shot runs, we combined 16-plex Tandem Mass Tags (TMTpro) with high-field asymmetric waveform ion mobility spectrometry (FAIMS Pro) and real-time search (RTS). We quantified over 40 proteins/min/sample, doubling the previously published rates. We applied AutoMP3 to investigate the naked mole-rat plasma proteome both as a function of the circadian cycle and in response to ultraviolet (UV) treatment. In keeping with the lack of synchronized circadian rhythms in naked mole-rats, we find few circadian patterns in plasma proteins over the course of 48 h. Furthermore, we quantify many disparate changes between mice and naked mole-rats at both 48 h and one week after UV exposure. These species differences in plasma protein temporal responses could contribute to the pronounced cancer resistance observed in naked mole-rats. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [1] partner repository with the dataset identifier PXD022891.