M. Rothmann et al. / Bioorg. Med. Chem. 20 (2012) 667–671
671
at 37 °C for 1 h. Background fluorescence was removed by washing
with 1 M NaCl (3 Â 200 L).
samples, are present in all 3 samples, have >3 fold change over
the average counts in the apo-control, and <5 counts in the nega-
tive control. The identification of partner enzymes specific for C8
or C12 ACP were defined as having >15 spectral counts and >2 fold
enrichment over both apo and holo ACP samples and a T test value
60.05.
l
4.6. Culturing conditions
E. coli K12 or Shewanella oneidensis MR-1 was streaked on LB-
agar and incubated overnight at 37 °C. A single colony was picked
and used to inoculate a 5 mL liquid LB starter culture and rotated
overnight at 37 °C. 2 mL of this starter culture was then used to
inoculate 1 L of autoclaved LB media. This culture was allowed to
grow until stationary phase (OD600 ꢀ1.0). At this point the cells
were harvested and centrifuged (8000g, 20 min, 4 °C) and cell pel-
lets were frozen overnight.
Acknowledgments
This work was funded by NIH GM094924. Special thanks to H.
Mori from the Japanese E. coli consortium for providing genes from
the ASKA plasmid ORF library22 for providing the GFP fusion clone
of FabF and J. J. La Clair (UC San Diego) for helpful discussions.
4.7. Lysate preparation
A. Supplementary data
Cells were resuspended in lysis buffer (25 mM potassium phos-
phate, pH 7.4, 100 mM NaCl) and cell lysis performed by two passes
through a French pressure cell. Soluble cell extract were cleared by
centrifugation (20,000g, 30 min, 4 °C) and quantified to 1 mg/mL.
Supplementary data associated with this article can be found, in
References and notes
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8.0 (200
lL) All samples were reduced by treatment with 10 mM
TCEP, alkylated by treatment with 12 mM iodoacetamide. At this
point, the solution was diluted with 50 mM Tris pH 8.0 to a final con-
centration of 2 M Urea (400
lL). This was followed by the addition of
trypsin (1 g) and 2 mM CaCl2. Samples were allowed to digest 12 h
l
at 37 °C. At this point, digestion of samples was halted by the addi-
tion of formic acid to a final concentration of 5% and frozen at
À80 °C until analysis. Tryptic peptides enriched by ACP loaded with
probes as well as apo and holo ACPs, were loaded onto a biphasic
strong cation exchange/reverse phase capillary column (Agilent)
and analyzed by 2D-LC separation in combination with tandem
MS. Peptides were eluted in a five-step MudPIT experiment and data
were collected in an ion trap mass spectrometer (ThermoFisher LTQ)
set in a data-dependent acquisition mode with dynamic exclusion
turned on (60 s). Spray voltage was set to 2.75 kV and the flow rate
through the column was 0.25 L/min.
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4.9. Filtering of MudPIT results
MudPIT filtering conditions were set to annotate proteins as en-
riched if they have >15 spectral counts when averaged over three