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To investigate the role of ClpP-mediated proteolysis in the regulation of life stage specific proteins of Legionella pneumophila, the bacteria in the RP/TP of WT and ΔclpP were collected and analyzed by mass spectrometry. To screen the substrates of ClpP during the whole life cycle, bacterial whole-cell lysates from clpPwt and clpPtrap in the TP were prepared and His-tagged proteins were purified with Ni-NTA affinity column (GE Healthcare) following the manufacturer’s instructions. Substrates captured inside the proteolytic barrel were co-purified along with the His-tagged ClpP complex and identified by mass spectrometry to identify substrates of ClpP in the WT background. Each sample was analyzed in biological triplicates to allow for statistical tests and to improve consistency. Protein identification and quality criteria were very strict throughout the study.