The response to stress is crucial for the adaptation of cells and microorganisms to their environments. Research in this field has continued for more than 30 years, especially in terms of the heat shock response, focusing mainly on the bacterium Escherichia coli1,2. In the early stages of this research, researchers identified individual factors and analyzed the regulatory mechanisms, with a particular focus on heat shock factors. However, in the last decade, this research has come to be seen as a problem of cellular system responses, which have been investigated using “-omics” techniques, such as proteomics and transcriptomics3,4. A recent study focused on the process of heat shock recovery in mouse cells5. With a liquid chromatography–tandem mass spectrometry analysis using timsTOF Pro, the present study provides quantitative time-series data for 3,193 proteins of E. coli, which had been cultured at 30 °C, subjected to an extremely severe heat shock at 50 °C for 90 min, and then returned to culture at 30 °C (11 sampling points). As a control, we added proteome data from the period of the E. coli growth phase, when it was cultured at a constant temperature of 30 °C (seven sampling points).