Supplementary Figure 1: Evaluation and recovery of endogenous SPA-tagged E. coli cell envelope proteins (CEPs).

(a) Detergent solubilization of SPA-tagged CEPs. Immunoblots showing representative set of inner membrane proteins (IMPs), outer membrane proteins (OMPs), and periplasmic proteins (PEPs) in various indicated detergents. Detergent extraction efficiency was estimated from the immunoblot results by comparing total recovery (anti-FLAG antibody used to detect tagged bait CEPs) of SPA-tagged CEP with (i) and without (w/o; ii) detergent, and the relative band signal intensity was reported in terms of percentile (iii). Triton, C12E8 and DDM highlighted by a dashed rectangular box (i) was deemed to be effective for the CEPs tested in a pilot optimization study. Detailed description of the detergents are shown in Supplementary Table 5 and Supplementary Note 1. (b) Silver-stained SDS polyacrylamide gels (i) portraying bacterial proteins identified by mass spectrometry (MS) after solubilization and affinity purification in the presence of detergents indicated for a TonB energy transducing (ExbB) SPA-tagged CEP. Distinct gel bands representing putative polypeptide subunits of the purified ExbB protein were analyzed by in-gel trypsin digestion followed by MALDI-TOF MS; protein identities are indicated with arrows. Asterisk indicates tagged CEP bait recovery (as detected by MALDI-TOF MS) in the presence of 3 different detergents is shown along with a control, emphasizing that the ExbB bait was not recovered in the absence of detergent. Known interactions of ExbB, including ExbD, FecA, FepE and FhuA that were captured in at least one of the 3 detergents are shown. Additional previously known ExbB interactions missed by MALDI-TOF MS but identified in our LC-MS/MS (ii) are shown with their corresponding spectral count (SPC) from SEQUEST and MS ID probability score (%) from STATQUEST algorithms. (c) Immunoprecipitations (IP) on detergent-solubilized extracts isolated from the indicated SPA-tagged CEPs. Anti-FLAG antibody was used to detect the tagged bait CEPs. Untagged or wild-type (WT) DY330 parental strain (1,2) served as negative control; wash, protein extract not bound to the beads after 3rd washing with and w/o the detergent; and EB, protein bound to the beads after elution. Asterisk indicates the bait recovery with strong signal intensity in the presence of DDM solubilized extracts. Molecular masses (kDa) of the marker (M) proteins are indicated. (d) Tagged CEPs (annotated and orphan) vs all E. coli soluble proteins (SPs) showing links to drug sensitivity from the large-scale phenomics screen (Nichols et al., 2011).