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.