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Fig. 9.3 Flowchart illustrating how data is automatically handled and processed to yield the chemical structure of a hit. The LC/MS systems have been integrated with software to automatically deconvolute each protein charge state distribution and determine potential protein conjugation and obtain the structure of a hit by database searching.

Fig. 9.3 Flowchart illustrating how data is automatically handled and processed to yield the chemical structure of a hit. The LC/MS systems have been integrated with software to automatically deconvolute each protein charge state distribution and determine potential protein conjugation and obtain the structure of a hit by database searching.

fragment interactions on a daily basis. The reaction mixtures are set up in 96 well plates and standard autosamplers inject the equilibrated reaction mixtures into the LC/MS system. Ballistic HPLC gradients are used to rapidly load, desalt and elute the proteins using C4 guard columns with a total run time of 2-3 min per analysis. To expedite data handling, each LC/MS system has been integrated with customized software to automatically deconvolute each spectrum, determine potential protein conjugation, and obtain the structure of a hit by database searching (Fig. 9.3). After the raw data set is collected, it is transferred to a processing computer where the charge state distribution of the protein is located in the total ion count (TIC), the spectra are averaged and each averaged charge state distribution spectrum is deconvoluted to a zero charge state spectrum, revealing the accurate mass of the protein and any potential modifications.

Next, custom software is used to interrogate the deconvoluted data set to identify the protein's mass and the intensity of the peak, determine any potential modification above a user-defined intensity threshold and, if there is a hit, calculate the mass and the relative conjugation of the fragment. In fact, the percent conjugation is used as a measure of relative affinities of the fragment hits. Since the library is mass encoded (all compounds in a well have a unique mass), the calculated mass of any hits are queried into a database to identify their structures.

Step 2

Locate Peak in TIC Average ESI-Spectra Automated Deconvolution

Step 3

Locate Reduced Protein Mass Locate Modified Protein Mass Calculate Hit Mass Calculate % Conjugation

310 | 9 Tethering: Fragment-based Drug Discovery by Mass Spectrometry 9.2.2

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