3.2 Optimizing
Digestion Conditions
Before starting the H/D exchange, it is crucial to verify how the
protein is digested under HDX-MS compatible conditions. Different aspects to be considered are protein sequence coverage, peptide
length, and peptide redundancy but also whether the protein
remains soluble after quenching and freezing and how the presence
of DNA affects the whole procedure. The primary goal is to ideally
obtain full sequence coverage with peptides providing good spatial
resolution, i.e., not very short or long ones. Recommendations
regarding optimal length do vary slightly in the literature, but
fragments between 8 and 12 amino acids probably represent the
best standard. Besides the spatial resolution obtainable through the
generated peptides, one can also target nearly amino acid resolution
using suitable fragmentation techniques like electron transfer dissociation [19] or UV photodissociation [20] (see Note 11). In
addition, emphasis should also be put on the generation of large
numbers of overlapping peptides as these can further increase the
spatial resolution [21, 22] and/or provide higher confidence in the
observed changes (see Note 12). To fulfill all these goals, different
proteases, ideally immobilized and packed into a column, should be
tested [17, 23–26]. The columns can be used individually or combined in serial or parallel setting [27]. Column size as well as the
protease density on the POROS resin, which together dictate the
enzyme:protein ratio, can be varied. Other factors affecting the
digestion are flow, temperature, pressure, and the use of denaturing
agents. Time spent on the proteolytic column is primarily determined by the desalting LC flow. Therefore, higher flow leads to
faster digestion and produces longer fragments and higher redundancy (number of overlapping peptides). Slower flow, on the other
hand, produces more complete digestion, which however may at
times be even detrimental, if the peptides produced are too short.
Typical digestion temperature should be close to 0
C to minimize
deuterium-loss (back-exchange), but it can be raised to 15
C or
even 20
C locally to increase the digestion efficiency. This is usually
achieved either in a dedicated separately temperature-controlled
chamber for the protease column or by placing the column out of
the ice/water bath. However, when tweaking these parameters, one
should always keep in mind that optimal balance between efficient
digestion and minimal H/D back-exchange conditions must be
maintained (hence running the analysis as fast as possible and at
the lowest possible temperature). For proteins not offering satisfactory digestion under mild denaturing conditions provided by the
acidic environment of the quench buffer alone, addition of denaturing agents is suggested. Urea or guanidine can be used at quite
high concentrations with all the immobilized proteases; however,
besides rendering the target protein susceptible to digestion, these
agents also affect the protease itself as well as peptide binding to the
reversed phase resin in the trap column. While guanidine was
shown to have mainly a detrimental effect, urea can even enhance
HDX-MS of Protein-DNA Complexes
199
Digestion Conditions
Before starting the H/D exchange, it is crucial to verify how the
protein is digested under HDX-MS compatible conditions. Different aspects to be considered are protein sequence coverage, peptide
length, and peptide redundancy but also whether the protein
remains soluble after quenching and freezing and how the presence
of DNA affects the whole procedure. The primary goal is to ideally
obtain full sequence coverage with peptides providing good spatial
resolution, i.e., not very short or long ones. Recommendations
regarding optimal length do vary slightly in the literature, but
fragments between 8 and 12 amino acids probably represent the
best standard. Besides the spatial resolution obtainable through the
generated peptides, one can also target nearly amino acid resolution
using suitable fragmentation techniques like electron transfer dissociation [19] or UV photodissociation [20] (see Note 11). In
addition, emphasis should also be put on the generation of large
numbers of overlapping peptides as these can further increase the
spatial resolution [21, 22] and/or provide higher confidence in the
observed changes (see Note 12). To fulfill all these goals, different
proteases, ideally immobilized and packed into a column, should be
tested [17, 23–26]. The columns can be used individually or combined in serial or parallel setting [27]. Column size as well as the
protease density on the POROS resin, which together dictate the
enzyme:protein ratio, can be varied. Other factors affecting the
digestion are flow, temperature, pressure, and the use of denaturing
agents. Time spent on the proteolytic column is primarily determined by the desalting LC flow. Therefore, higher flow leads to
faster digestion and produces longer fragments and higher redundancy (number of overlapping peptides). Slower flow, on the other
hand, produces more complete digestion, which however may at
times be even detrimental, if the peptides produced are too short.
Typical digestion temperature should be close to 0
C to minimize
deuterium-loss (back-exchange), but it can be raised to 15
C or
even 20
C locally to increase the digestion efficiency. This is usually
achieved either in a dedicated separately temperature-controlled
chamber for the protease column or by placing the column out of
the ice/water bath. However, when tweaking these parameters, one
should always keep in mind that optimal balance between efficient
digestion and minimal H/D back-exchange conditions must be
maintained (hence running the analysis as fast as possible and at
the lowest possible temperature). For proteins not offering satisfactory digestion under mild denaturing conditions provided by the
acidic environment of the quench buffer alone, addition of denaturing agents is suggested. Urea or guanidine can be used at quite
high concentrations with all the immobilized proteases; however,
besides rendering the target protein susceptible to digestion, these
agents also affect the protease itself as well as peptide binding to the
reversed phase resin in the trap column. While guanidine was
shown to have mainly a detrimental effect, urea can even enhance
HDX-MS of Protein-DNA Complexes
199