Question

protection factors provide information on the stability of individual amino acids in proteins. why is the determination of protection factors not possible using HDX-MS?

Answer 1

Ans . HDX (Hydrogen / deuterium exchange ) monitered by MS (mass spectrometry) is a promising technique for rapidly fingerprinting structural and dynamical properties of proteins. The time-dependent change in the mass of any fragment of the polypeptide chain depends uniquely on the rate of exchange of its amide hydrogens, but determining the latter from the former is generally not possible.

The key to interpreting HDX kinetics is the fact that exchange occurs faster for amides that are solvent exposed and / or not involved in hydrogen bonds.

HDX-MS relies on the measurable difference of mass between the deuterated and nondeuterated polypeptide chains. To obtain more specific information, the polypeptide is further fragmented by proteolysis at a low pH and a low temperature. Under MS analysis conditions, deuterium incorporated into exchangeable side chain groups and N - terminal amines is rapidly back exchanged , and as a consequence , HDX- MS is only sensitive to the backbone amide exchange.

HDX-MS yields the overall mass change over time for a whole peptide fragment ( usually presented as a centroid of the isotopic envelope ) but does not provide direct information on the exchange rate of individual residues.

The ratio of observed exchange rate to the maximal one ( i.e., that measured for a completely unstructured peptide ) is the so called protection factor. Protection factors contain both structural and dynamic information ; the degree of protection of amide hydrogen from solvent deuterium correlates to the degree of involvement in secondary and tertiary structure.