Amide HX in Unstructured Polypeptides

The initial event in the proton transfer reaction (Eq. 1.1) is the diffusion-limited collision of a proton donor (AH) with a proton acceptor (B) leading to the formation of a hydrogen-bonded complex which is followed by a rapid proton redistribution across the hydrogen bond and finally dissociation of the proton donor and acceptor complex [23]  

The proton transfer is successful when the proton is transferred to the proton acceptor (dissociation to the right) and unsuccessful if the proton remains with the proton donor (dissociation to the left) [24]. The overall forward rate constant for the proton transfer reaction is given by Equation 1.2  

The pH dependence of amide HX forms the essential basis of the application of LC-MS to measure protein HX. By reducing pH, the exchange reaction can be sufficiently quenched to allow time for mass spectrometric detection of deutaium incorporation. For unstractured peptides in solution, the chemical HX rate constant of an amide is given by Equation 1.3  

The HX rates are also dependent on temperature. An increase in temperature affects HX rates primarily hy altering the water ionization constant, K, and thus increasing the concentration of OH . Further, some evidence suggests that temperature may also affect the collisional rate constant, k, in Equation 1.2 hy altering buffer viscosity and thus the diffusional collisional rate constant [24, 25]. A more recent study, however, has indicated that the effect of bulk viscosity on HX is negligible [30]. Theoretical HX rates can be determined as a function of temperature by a modified form of the Arrhenius equation (Eq. 1.4) and reference HX rate constants determined experimentally at 20°C  

The relationship between the chemical exchange rate and the temperature is exponential a temperature decrease from 25 to 0°C, which represents the typical conditions used during the labeling and quench step of an HX-MS experiment, respectively, leads to an approximate 14-fold 

---