Investigating protein structure, conformation, and activity under hydrostatic pressure is a critical challenge, particularly since a sizable portion of life is marine or deep subsurface. There is a perception that most proteins experience limited changes under pressures environmentally relevant on Earth; this conclusion is biased as studies focus on pressure sensitivity within a select few individual molecules, which tend to be “well-behaved”, stable proteins. More high-throughput biophysical techniques must be employed to identify promising model systems with measurable pressure effects to expand the investigation for piezo-tolerance and -sensitivity origins. Limited proteolysis (LiP) is a technique that uses differential non-specific protease susceptibility to gauge protein structure and conformational changes. When combined with mass spectrometry (MS), this method permits the profiling of subtle structural alterations across entire proteomes. The impressive throughput of LiP-MS offers a unique opportunity to survey hydrostatic pressure effects en masse. Here, we introduce the first high-pressure structural proteomics study with novel, purpose-built equipment allowing LiP-MS to be completed under ocean-bottom pressures (100 MPa). Results in a piezo-sensitive model species, Thermus thermophilus¸ show whole-proteome structural changes are rapid and more extensive than anticipated, demonstrating the capacity of high-pressure LiP-MS (HiP-LiP-MS) to profile pressure-sensitive proteins and provide a rich source of new targets for future studies.