Journal of the American Chemical Society
ARTICLE
was activated for this process, with a repeat count of one and exclusion
duration of 30 s. For CID, the activation Q was set at 0.25, isolation width
(m/z) 2.0, activation time 30 ms, and normalized collision energy 35%.
The Extract_Msn (version 4.0) program found in Bioworks Browser
3.3 (Thermo Electron, Germany) was used to extract tandem MS
spectra in the dta format from the raw data of the LTQ-FT ultra. These
dta files were then converted into MASCOT generic file format using an
in-house program. Intensity values and fragment ion m/z ratios were not
manipulated. These data were used to obtain protein identities by
searching against the corresponding database by means of an in-house
MASCOT server (version 2.2.03, Matrix Science, Boston, MA). The
search was limited to a maximum of two missed trypsin cleavages, #13C
of 2, mass tolerances of 10 ppm for peptide precursors and 0.8 Da for
fragment ions. Fixed modification was carbamidomethyl at the Cys
residue, whereas variable modifications were oxidation at the Met
residue and phosphorylation at the Ser, Thr, or Tyr residue. Only pro-
teins with a MOWSE score higher than 70, corresponding to p < 0.05,
were considered significant. The peptide/protein lists obtained were
exported to an html file.
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’ ASSOCIATED CONTENT
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S
Supporting Information. Experimental details, chemical
b
synthesis and characterizations, complete protocols for array
fabrication, KD binding analysis, IC50 analysis, and additional
biochemical experiments. This material is available free of charge
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’ AUTHOR INFORMATION
Corresponding Author
’ ACKNOWLEDGMENT
Funding support was provided by the Agency for Science,
Technology and Research (R-143-000-391-305) and the Minis-
try of Education (R-143-000-394-112). We thank Prof. McKer-
row (UCSF) for providing proteases cruzain and rhodesain, and
the groups of Kevin Tan and Cynthia He (NUS) for donating
parasites P. falciparum and T. brucei (BSF), respectively.
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