and membrane concentrations (aggregation, instability, etc).
Protein amounts should be be maintained within a range that
yields adequate signal on the MS and good coverage (usually
strive for coverage above 90%), while avoiding overloading the
LC system. This range is generally found between 5 and
150 pmol per sample. However, the appropriate protein
amount will be completely dependent on the sensitivity of the
mass spectrometer being used, protease efficiency, size of the
protein, and interaction kinetics. Membrane concentration
must also be optimized as proteins may sometimes become
unstable when in the presence of membranes at high concentration, with this being tested before setup of HDX samples.
This issue can be surmounted by minimizing membrane concentration in the initial protein–membrane incubation, or by
incorporating membrane within the D 2 O buffer, thereby initiating membrane–protein incubation and deuteration
simultaneously [3].
3. Care must be taken to not load excess lipids or detergents onto
the LC-MS system during the HDX-MS experiment. Detergents and lipids may accumulate on the analytical or trap
columns resulting in increased back pressure and decreased
column performance. Lipids and detergents can also lead to
significant ion suppression during MS analysis. One solution to
this problem is to incorporate additional washing of the reverse
phase system in organic solvent, sending the wash product to
waste rather than injecting it onto the MS system. It is important to note that if this approach is taken, one must carefully
monitor the performance of the LC system (i.e., pressure and
retention times) to verify that there is no degradation over
time [3].
4. The quenching conditions must be optimized prior to
performing an HDX. The nature of HDX experiments requires
that the hydrogen–deuterium exchange be dramatically
decreased, or “quenched,” so as to monitor exchange rates
accurately. The quenching solution generally consists of a chaotropic agent, a reducing agent, and an acid. The chaotropic
agent, such as guanidine hydrochloride, will denature the protein(s) post hydrogen–deuterium exchange, thus facilitating
proteolysis by the acid functional protease. If incomplete digestion is obtained (<80% coverage in initial MS/MS analysis),
the amount of denaturant should be optimized, with either
increased denaturant amount or increased time of the quench
buffer sitting with protein before freezing in liquid nitrogen.
The reducing agent should only be incorporated within the
quenching solution if the protein contains disulfide bonds.
Disulfide bonds will prevent the protein from properly unfolding, therefore reducing protease access to the protein. The
Probing Protein-Membrane Interactions Using HDX-MS
481
Protein amounts should be be maintained within a range that
yields adequate signal on the MS and good coverage (usually
strive for coverage above 90%), while avoiding overloading the
LC system. This range is generally found between 5 and
150 pmol per sample. However, the appropriate protein
amount will be completely dependent on the sensitivity of the
mass spectrometer being used, protease efficiency, size of the
protein, and interaction kinetics. Membrane concentration
must also be optimized as proteins may sometimes become
unstable when in the presence of membranes at high concentration, with this being tested before setup of HDX samples.
This issue can be surmounted by minimizing membrane concentration in the initial protein–membrane incubation, or by
incorporating membrane within the D 2 O buffer, thereby initiating membrane–protein incubation and deuteration
simultaneously [3].
3. Care must be taken to not load excess lipids or detergents onto
the LC-MS system during the HDX-MS experiment. Detergents and lipids may accumulate on the analytical or trap
columns resulting in increased back pressure and decreased
column performance. Lipids and detergents can also lead to
significant ion suppression during MS analysis. One solution to
this problem is to incorporate additional washing of the reverse
phase system in organic solvent, sending the wash product to
waste rather than injecting it onto the MS system. It is important to note that if this approach is taken, one must carefully
monitor the performance of the LC system (i.e., pressure and
retention times) to verify that there is no degradation over
time [3].
4. The quenching conditions must be optimized prior to
performing an HDX. The nature of HDX experiments requires
that the hydrogen–deuterium exchange be dramatically
decreased, or “quenched,” so as to monitor exchange rates
accurately. The quenching solution generally consists of a chaotropic agent, a reducing agent, and an acid. The chaotropic
agent, such as guanidine hydrochloride, will denature the protein(s) post hydrogen–deuterium exchange, thus facilitating
proteolysis by the acid functional protease. If incomplete digestion is obtained (<80% coverage in initial MS/MS analysis),
the amount of denaturant should be optimized, with either
increased denaturant amount or increased time of the quench
buffer sitting with protein before freezing in liquid nitrogen.
The reducing agent should only be incorporated within the
quenching solution if the protein contains disulfide bonds.
Disulfide bonds will prevent the protein from properly unfolding, therefore reducing protease access to the protein. The
Probing Protein-Membrane Interactions Using HDX-MS
481