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Full text of DOI:10.1038/nm.2436

Supplementary Materials
Chemicals and biochemicals : [αS] 2-oxo-clopidogrel (SR121883), 3’-methoxyphenacyl-
cis-thiol (SAR206251) and 4’-bromophenacyl-endo-thiol (SAR195539) were gifts of Sanofi-
Aventis. All other products including enzymes were from Sigma-Aldrich (St Quentin
Fallavier, France).
Microsomal Incubations: Human microsomes (pool, 10 mg protein/mL) were obtained from
BD-Gentest (Le Pont de Claix, France). Typical incubations were performed in potassium
phosphate buffer (0.1M, pH 7.4) containing 2 mM CaCl , 100 mM KF, microsomes (1 mg
2
protein/mL), 2-oxo-clopidogrel (100 µM) and a reducing agent (20 mM ascorbic acid, or 0.5
mM dithiothreitol, or 5 mM glutathione (GSH)) with or without NADPH generating system
(1 mM NADP, 15 mM glucose-6-phosphate, 2 u/mL of glucose-6-phosphate dehydrogenase)
at 37°C for 30 min. Reactions were stopped by adding one half volume of CH CN:
3
CH COOH (9:1) and proteins were removed by centrifugation at 13000g.
3
HPLC-MS studies were performed on a Surveyor HPLC instrument coupled to an LCQ
Advantage ion trap mass spectrometer (Thermo, Les Ulis, France), using a Gemini C18
column (100 x 2 mm, 3 µm; Phenomenex), and a gradient starting at 40% B for 1 min then
increasing linearly to 100% B in 15 min (A= ammonium acetate buffer (10 mM, pH 4.6) and
B= CH CN: CH OH: H O (7:2:1)) at 200 µL/min. Mass spectra were obtained by
3
3
2
electrospray ionization (ESI) in positive ionization mode detection under the following
conditions : source parameters: sheeth gas 20, auxiliary gas 5, spray voltage 4.5 kV, capillary
temperature 200°C, capillary voltage 15V, m/z range for MS recorded generally between 300
2
and 700 (except for exploratory experiments with a wider range 300-800). MS energy were
tested between 20 and 40 eV and were generally 35 eV.
2
Figure S1 : MS spectra of endo and cis thiols
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Panel 1 shows the MS spectra of the thiols at m/z = 356 (1A endo, 1B and 1C cis thiols).
2
Panel 2 shows the MS spectra of the 3’-methoxyphenacyl derivatized thiols at m/z = 504:
(2A endo and 2B and 2C cis thiols).
Nature Medicine doi:10.1038/nm.2436