Measuring precise concentrations of proteins can provide insights into biological processes. Here, we use efficient protein extraction and sample fractionation and state-of-the-art quantitative mass spectrometry techniques to generate a comprehensive, condition-dependent protein abundance map of Escherichia coli. We measure cellular protein concentrations for 55% of predicted E. coli genes (>2300 proteins) under 22 different experimental conditions and identify methylation and N-terminal protein acetylations previously not known to be prevalent in bacteria. We uncover system-wide proteome allocation, expression regulation, and post-translational adaptations. These data provide a valuable resource for the systems biology and broader E. coli research communities.

While persisters are a health threat due to their transient antibiotic tolerance, little is known about their phenotype and what actually causes persistence. Using a new method for persister generation and high‐throughput methods, we comprehensively mapped the molecular phenotype of Escherichia coli during the entry and in the state of persistence in nutrient‐rich conditions. The persister proteome is characterized by σS‐mediated stress response and a shift to catabolism, a proteome that starved cells tried to but could not reach due to absence of a carbon and energy source. Metabolism of persisters is geared toward energy production, with depleted metabolite pools. We developed and experimentally verified a model, in which persistence is established through a system‐level feedback: Strong perturbations of metabolic homeostasis cause metabolic fluxes to collapse, prohibiting adjustments toward restoring homeostasis. This vicious cycle is stabilized and modulated by high ppGpp levels, toxin/anti‐toxin systems, and the σS‐mediated stress response. Our system‐level model consistently integrates past findings with our new data, thereby providing an important basis for future research on persisters.

Mesothelin is a tumor differentiation antigen expressed by epithelial tumors, including pancreatic cancer. Currently, mesothelin is being targeted with an antibody-drug conjugate (ADC) consisting of a mesothelin-specific antibody coupled to a highly potent chemotherapeutic drug. Considering the toxicity of the ADC and reduced accessibility of pancreatic tumors, non-invasive imaging could provide necessary information. We therefore developed a zirconium-89 (89Zr) labeled anti-mesothelin antibody (89Zr-AMA) to study its biodistribution in human pancreatic tumor bearing mice. Biodistribution and dose-finding of 89Zr-AMA were studied 144 h after tracer injection in mice with subcutaneously xenografted HPAC. MicroPET imaging was performed 24, 72 and 144 h after tracer injection in mice bearing HPAC or Capan-2. Tumor uptake and organ distribution of 89Zr-AMA were compared with nonspecific 111In-IgG. Biodistribution analyses revealed a dose-dependent 89Zr-AMA tumor uptake. Tumor uptake of 89Zr-AMA was higher than 111In-IgG using the lowest tracer dose. MicroPET showed increased tumor uptake over 6 days, whereas activity in blood pool and other tissues decreased. Immunohistochemistry showed that mesothelin was expressed by the HPAC and CAPAN-2 tumors and fluorescence microscopy revealed that AMA-800CW was present in tumor cell cytoplasm. 89Zr-AMA tumor uptake is antigen-specific in mesothelin-expressing tumors. 89Zr-AMA PET provides non-invasive, real-time information about AMA distribution and tumor targeting.

Transforming growth factor-b (TGF-b) promotes cancer invasion and metastasis and is therefore a potential drug target for cancer treatment. Fresolimumab, which neutralizes all mammalian active isoforms of TGF-b, was radiolabeled with 89 Zr for PET to analyze TGF-b expression, antibody tumor uptake, and organ distribution. Methods: 89 Zr was conjugated to fresolimumab using the chelator N-succinyldesferrioxamine-B-tetrafluorphenol. 89 Zr-fresolimumab was analyzed for conjugation ratio, aggregation, radiochemical purity, stability, and immunoreactivity. 89 Zr-fresolimumab tumor uptake and organ distribution were assessed using 3 protein doses (10, 50, and 100 mg) and compared with 111 In-IgG in a human TGF-b-transfected Chinese hamster ovary xenograft model, human breast cancer MDA-MB-231 xenograft, and metastatic model. Latent and active TGF-b1 expression was analyzed in tissue homogenates with enzyme-linked immunosorbent assay. Results: 89 Zr was labeled to fresolimumab with high specific activity (.1 GBq/ mg), high yield, and high purity. In vitro validation of 89 Zr-fresolimumab showed a fully preserved immunoreactivity and long (.1 wk) stability in solution and in human serum. In vivo validation showed an 89 Zr-fresolimumab distribution similar to IgG in most organs, except for a higher uptake in the liver in all mice and higher kidney uptake in the 10-mg group. 89 Zr-fresolimumab induced no toxicity in mice; it accumulated in primary tumors and metastases in a manner similar to IgG. Both latent and active TGF-b was detected in tumor homogenates, whereas only latent TGF-b could be detected in liver homogenates. Remarkably high 89 Zr-fresolimumab uptake was seen in sites of tumor ulceration and in scar tissue, processes in which TGF-b is known to be highly active. Conclusion: Fresolimumab tumor uptake and organ distribution can be visualized and quantified with 89 Zr-fresolimumab PET. This technique will be used to guide further clinical development of fresolimumab and could possibly identify patients most likely to benefit.