Proteome-wide analysis of protein stability in Escherichia coli under acid stress.

Knowledge of protein acid sensitivity remains sparse and is largely derived from low-throughput, enzyme-specific assays. We used a scalable framework to map acid stability across the Escherichia coli proteome to assess the acid stability of 1,675 unique proteins, estimating pH50 values for over 90% of them. The parameter pH50 was defined as the pH value at which only 50% of the initial protein remains in solution following acid treatment. Proteome-wide pH50 values ranged from 2.28 to 6.33 (median 5.11). Approximately 9% of detected proteins remained stable across all tested pH conditions. Our results align with published data and the assay of citrate synthase (GltA) performed here. Protein acid stability differed significantly by subcellular localization: periplasmic proteins were relatively more abundant in the acid-stable group, cytoplasmic proteins were abundant at pH50 values 4.5-5.5, and inner membrane proteins at higher pH50 between 5.5 and 6.0. Outer membrane proteins were too few to draw strong conclusions regarding enrichment within specific pH50 groups. Notably, the periplasmic binding protein of the molybdate ABC transporter (ModA), was enriched after incubation at low pH. Estimated pH50 values showed no correlation with protein isoelectric point and molecular weight. Together, this work provides the first proteome-wide map of protein acid stability and establishes a general framework for studying different chemical stressors. One sentence summary This paper presents a quantitative map of acid stability of E. coli proteins and established a procedure for the study of protein acid sensitivity that is adaptable to other stressors and other organisms.