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Tethering is a registered service mark of Sunesis Pharmaceuticals, Inc., for its fragment-based drug discovery.
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Aurora A protein (Ref. 25, T217C) was adjusted to a final concentration of 5 μM in 50 mM Tris, pH 8, and 1 mM 2-mercaptoethanol. A 96-well plate format was used with a 40-μL total reaction volume per well. Each well contained the dynamic extender 1 at 50 μM, 10 unique disulfide containing fragments at 50 μM each, and the 5 μM protein/2-mercaptoethanol solution. After 4 h at room temperature each well was subjected to LC/MS analysis using a LCT time-of-flight mass spectrometer equipped with an eight-channel parallel multiplexed (MUX) ESI interface and (Waters Corp., Milford, MA) equipped with a Gilson 215/889 eight channel liquid handler (Gilson, Middleton, WI). The protein samples were desalted using reverse-phase Protein μTraps (Michrom BioResources Inc., Auburn CA) with a linear gradient of 5% acetonitrile (0.1% formic acid) to 95% acetonitrile (0.1% formic acid) over 0.3 min (held at 95% acetonitrile (0.1 % formic acid) for 2.3 min) at a total flow rate of 600 μL/min (75 μL/min per channel). Protein charge state distributions were deconvoluted to obtain the zero-charge spectrum using the MaxEnt algorithm (Waters Corp., Milford, MA).
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