Technique

To prepare a biological target for Tethering, the target protein must contain a cys-teine residue near an area of interest (e.g. an enzyme active site or a "hot-spot" of a protein-protein interface). If no native cysteines are in a suitable location, a cysteine can be introduced at an appropriate spot via site-directed mutagenesis. One potential limitation of Tethering is this need to generate cysteine-containing mutant proteins, but with site-directed mutagenesis and protein expression, this is rarely a significant hurdle. Tethering does require sufficient knowledge about a protein's structure to inform where to place the cysteine; while a crystal structure is not required, a good model of the protein is essential. The most labor-intensive requirement for Tethering is the synthesis of a library of disulfide-containing fragments: very few disulfide-containing fragments are commercially available, and introducing a disulfide onto a fragment requires at least one additional chemical step. Sunesis Pharmaceuticals has synthesized a library of roughly 18 000 disulfide-containing fragments. They are pooled into groups of roughly ten fragments, each with a unique molecular weight in a given pool. The development and production of our Cys-mutants and disulfide-containing library are reviewed elsewhere [12].

In Tethering, the protein target is screened by mixing the target with each pool under reducing conditions that enhance disulfide exchange. 2-Mercaptoethanol (in the low millimolar concentration range) is used as the reductant due to its redox potential (—0.196 V at pH 7) [21]. Disulfide exchange allows a reduced cysteine on the protein to react with each disulfide-containing fragment. After equilibrium is established, the reaction mixture is injected into an LC/ESI-MS instrument, a system which is ideally suited for measuring protein modification by allowing both rapid on-line sample clean-up and the ability to accurately measure intact protein mass.

As we optimized Tethering we used a variety of mass spectrometers. In our experience, the sensitivity and high resolution of TOF analyzers has provided the most rapid and accurate analyses of intact proteins. An example of an ESI-TOF data set from a standard experiment is illustrated in Fig. 9.2. Figure 9.2A is the deconvoluted mass spectrum of a Cys-mutant target protein after equilibration

Fig. 9.2 (a) Deconvoluted ESI-TOF mass spectrum of a Cys-containing target protein equilibrated with a pool of ten disulfide-containing fragments with no hit discovered. Red. Reduced or unmodified protein; Ox. protein oxidized by cysteamine. (b) Deconvoluted ESI-TOF mass spectrum of the same protein representing a strong hit from a different pool of ten disulfide-containing fragments. The mass of the protein has shifted according to the mass of the fragment captured by the protein.

Fig. 9.2 (a) Deconvoluted ESI-TOF mass spectrum of a Cys-containing target protein equilibrated with a pool of ten disulfide-containing fragments with no hit discovered. Red. Reduced or unmodified protein; Ox. protein oxidized by cysteamine. (b) Deconvoluted ESI-TOF mass spectrum of the same protein representing a strong hit from a different pool of ten disulfide-containing fragments. The mass of the protein has shifted according to the mass of the fragment captured by the protein.

with a pool often disulfide-containing compounds from a single well in a 96-well plate. The two peaks represent the unmodified protein (17 355 Da) and the protein oxidized with either 2-mercaptoethanol or cysteamine, the solubilizing functionality that is common to all library members (17431 Da). The detection of cys-teamine modification indicates proper disulfide scrambling has occurred, and time-course experiments demonstrate when the reaction mixture is at equilibrium. In this pool, none of the ten disulfide-containing fragments had any detectable affinity for the target. In contrast, Fig. 9.2B is the deconvoluted mass spectrum representing a hit from a different pool of ten disulfide-containing compounds. The mass of the protein has shifted 373 Da, due to a shift in the complex equilibria towards a protein-fragment conjugate. The disulfide bond is stabilized due to contributions from the affinity between the protein and the fragment, and the covalently modified protein conjugate is easily identified by LC/ ESI-MS.

Tethering is also amenable to high throughput sample analysis, and analytical methods have been streamlined to routinely investigate thousands of protein-

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