Sample preparation freeze-quench

The freeze quench sample preparation can allow characterization of reaction intermediates. 

The first semiautomated HX-MS system was described by Virgil Woods in 2001 and was based around the decoupled experimental paradigm [44], Woods outlined an end-to-end HX workflow, titled DXMS, that began with (manual) sample preparation and freezing of samples in quench solution. When ready, samples were placed in a modified HPLC autosampler able to perform the thawing, digestion, and LC-MS analysis. Although this automation was never commercialized, it was in use for over a decade and was demonstrated to be extremely productive. 

The rapid-quench method [78] was used in Ref. 83 to analyze the mechanism of a bacterial phenylalanine hydroxylase, a mononuclear nonheme iron protein that uses tetrahydropterin as the source of the two electrons needed to activate O2 for the hydroxylation of phenylalanine to tyrosine. Mossbauer spectra of samples prepared by freeze-quenching the reaction of the complex enzyme— Fe(ll)-phenylalanine-6-methyltetrahydropterin with O2 revealed the accumulation of an intermediate at short reaction times (20-100 ms). The Mossbauer parameters of the intermediate (3 = 0.28mms, A q= l.26mms ) suggested it to be a high-spin Fe(IV) complex similar to those that have previously been detected in the reactions of other mononuclear Fe(ll) hydroxylases. 

Bray who has pioneered the rapid freeze method for studying fast kinetics, has recently used this method in conjunction with XAS The procedure involves quenching reacting solutions by squirting them into a cold imiscible solvent and allows the preparation of a series of samples, each frozen at a particular reaction time (from about 3 ms upwards). Bray and co-workers used this method to Study the structural... 

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