Volume 23, Issue 2, Pages 290-298 (February 2016) Systematic Survey of Serine Hydrolase Activity in Mycobacterium tuberculosis Defines Changes Associated with Persistence  Corrie Ortega, Lindsey N. Anderson, Andrew Frando, Natalie C. Sadler, Robert W. Brown, Richard D. Smith, Aaron T. Wright, Christoph Grundner  Cell Chemical Biology  Volume 23, Issue 2, Pages 290-298 (February 2016) DOI: 10.1016/j.chembiol.2016.01.003 Copyright © 2016 Elsevier Ltd Terms and Conditions

Cell Chemical Biology 2016 23, 290-298DOI: (10. 1016/j. chembiol. 2016 Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 Fluorophosphonate (FP) Activity Probe and Labeling of Mtb SHs (A) Structure of the FP probe with the fluorophosphonate reactive group (red) and an alkyne for the conjugation of tags by click chemistry (green). (B) Gel visualization of Mtb lysate shows many labeled SHs, some of which are sensitive to PMSF inhibition. (C) Labeling of the known serine protease Rv3671c shows efficient labeling of recombinant protein. Cell Chemical Biology 2016 23, 290-298DOI: (10.1016/j.chembiol.2016.01.003) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 Global Identification of SHs by LC-MS/MS Heatmap representation of Z scores (difference from the mean in SDs) of the quantitative mass spectrometry data. The map shows labeling of 78 proteins, robust separation of the labeled and the DMSO-labeled control group and three distinct clusters with similar activity changes. Table S1 provides the data for all replicates. Cell Chemical Biology 2016 23, 290-298DOI: (10.1016/j.chembiol.2016.01.003) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 Selectivity and Pathway Distribution of SHs in the Mtb Proteome (A) The pie chart shows the functional classification of 78 proteins identified as SHs or hypothetical proteins according to current annotation. (B) The relative representation of SH hits in specific pathways compared to all Mtb genes. Cell Chemical Biology 2016 23, 290-298DOI: (10.1016/j.chembiol.2016.01.003) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 Characterization of Hypothetical Proteins Rv0525, Rv0613c, Rv1192, and Rv3311 have protease activity against β-casein. Asterisks indicate the input recombinant protein, angle bracket indicates position of undigested β-casein. Rv3671c is MarP protease positive control. Cell Chemical Biology 2016 23, 290-298DOI: (10.1016/j.chembiol.2016.01.003) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 5 Conditionally Active SHs in Mtb Replication and Non-replication (A) The SH activity in Mtb lysate from cultures grown in air and in hypoxia were compared by gel analysis using a fluorescent probe. (B) Venn diagram shows the number of conditionally active and SH activities shared in replication and non-replication. Cell Chemical Biology 2016 23, 290-298DOI: (10.1016/j.chembiol.2016.01.003) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 6 Correlation Between SH Abundance and Activity Changes SH activity and overall abundance of SHs was determined by quantitative mass spectrometry in aerated and hypoxic culture and the fold change of activity plotted against abundance. To obtain a fold change for activity measurements when peptides were undetectable in one condition, missing values were arbitrarily set to seven. Cell Chemical Biology 2016 23, 290-298DOI: (10.1016/j.chembiol.2016.01.003) Copyright © 2016 Elsevier Ltd Terms and Conditions