Thermophilic microorganisms that thrive in extreme environments are of great importance because they express heat-resistant enzymes with the potential to serve as biocatalysts in industrial applications. Thermal proteome profiling (TPP) is a multiplexed quantitative mass spectrometry method for analyses of structural information and melting behavior of thousands of proteins, simultaneously determining the thermal denaturation profiles of each protein. We report, in this study, TPP applied to a thermophilic bacterial proteome, a recently isolated strain ofGeobacillus thermoleovoransnamed as ARTRW1. The proteome was investigated in terms of thermostable enzymes that are relevant to industrial applications. In this study, we present the thermostability profiles of its 868 proteins. The majority ofG. thermoleovoransproteome was observed to melt between 62.5 degrees C and 72 degrees C, with melting point (T-m) mean value of 68.1 degrees C +/- 6.6 degrees C. Unfolding characteristics of several enzymes, including amylase, protease, and lipase, were demonstrated which are highly informative in terms of their applicability to specific industrial processes. A significant correlation was observed between protein melting temperature and the structural features such as molecular weight and abundance, whereas correlations were modest or weak in relation to the alpha-helix structure percentages. Taken together, we demonstrated a system-wide melting profile analysis of a thermal proteome and listed proteins with elevated T(m)values that are highly promising for applications in medicine, food engineering, and cosmetics in particular. The extracted T(m)values were found similar to those obtained by biophysical methods applied to purified proteins. TPP analysis has significant industrial and biomedical potentials to accelerate thermophilic enzyme research and innovation.