-- pH CONTROL AND A SIMPLE EXPERIMENT
pH IS THE SECRET....(WELL PART OF IT)
The secret to making hydrogen exchange measurements with
mass spectrometry is in controlling the pH. The rate of hydrogen exchange is very
sensitive to pH -- a change in one pH unit equals a ten-fold change in the exchange rate!!
(see FIGURE 1).
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Let's use FIGURES 1 and 2 for
if the exchange rate of an average amide hydrogen in a completely unstructured peptide at
pH=7 equals 10 per second, the same average amide hydrogen in the same unstructured
peptide at pH=5 would have an exchange rate of 0.1 per second. The actual time
involved to make the exchange in this example is far less than 1 second (see half-life in
So in the simplest case scenario, one would
want to do the exchange reaction at a higher pH and then reduce the pH to stop the
exchange from occurring. This is essential to do a proper mass spectrometry
analysis, since it takes some time to carry out the mass spectral analysis.
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And don't forget, proteins are not just unstructured
peptides! Their exchange rates will be slowed by:
1. Hydrogen bonding that creates the
secondary structure, primarily alpha helices and beta sheets.
2. Protection from the solvent, primarily
due to being buried in the hydrophobic core of the protein.
3. Hydrogen bonding to water in the solvent,
a much smaller effect than #'s 1 and 2.
The pH effect can be used in a number of ways in addition
to the simplest one mentioned above. For example, one could do protein folding at pH
7 and then pulse quickly to pH=10 to "flash-label" any amide hydrogen not
involved in hydrogen bonds with other parts of the protein.
THE REST OF THE SECRET....
Another problem remains! Considering the example in
FIGURE 2, even if you do an experiment at pH 7.0 and reduce the pH to 2.5 (the minimum of
the curve in FIGURE 1), there will only be about 11 minutes (half-life) for you to do the
mass spectral analysis before you loose half the deuterium label!. To further slow
the exchange rates, the temperature must be lowered to zero degrees celsius (see FIGURE
2). At pH=2.5 and temp=0, the average half-life of exchange for the average amide
hydrogen is 30-60 minutes. These conditions give enough time to analyze the sample.
AND A MINOR COMPLICATION....
Even in unstructured peptides, every sequence will not
exchange at the same rate. That means that the loss of deuterium from a labeled
protein will not be the same. There are influences from the neighboring amino acids,
which depends of course on sequence. The amino acid at the N-terminus will also
exchange its backbone amide hydrogen much faster than ones further down the line.
These issues have been addressed in the literature. To read more, see:
For sequence effects:
Connelly GP, Bai Y, Jeng MF, Englander SW. (1993). Isotope effects in peptide group
hydrogen exchange. Proteins 17(1), 87-92.
Bai Y, Milne JS, Mayne L, Englander SW. (1993). Primary structure effects on peptide group
hydrogen exchange. Proteins 17(1), 75-86.
For calculating the average deuterium loss for an
Zhang, Z., and Smith, D.L. (1993). Determination of amide hydrogen exchange by mass
spectrometry: A new tool for protein structure elucidation. Protein Sci. 2,
A SIMPLE EXPERIMENT
|For the simplest type of experiment, called
continuous labeling, a protein is labeled with deuterium at pH=7.0. The reaction is
quenched by lowering the pH to 2.5 and the temperature to 0 deg. C. Its VERY
important that one control the pH change precisely. The best way to do that is to
use a weak (25 mM) phosphate buffer for the labeling step at pH =7.0 and then use a strong
(100 mM) phosphate buffer for the quench step. This ensure that the pH will drop to
2.5 and stay there. See the section on "Controls & Calculations".
To do electrospray (see the section on "mass
spectrometry") mass spectral analysis, you must desalt the sample before it goes into
the machine. Desalting serves a second, and very important purpose - it washes away
the deuterium from amino acid sidechains. The exchange rate of the amide hydrogens
in side chains (for example in arginine, or histidine) is very-very fast compared to the
exchange rate of the amide hydrogens in the backbone. When desalting is performed
with standard HPLC buffers (which have a pH of 2.5 and are made of H2O), the hydrogen in
the water of the HPLC buffers replaces the deuterium that exchanged into the side
chains. This leaves only deuterium label at the backbone amide positions.
Having only these amide hydrogens to analyze is a convenient situation:
1. it gives an individual exchange-rate
sensor for every amino acid (expect proline).
2. it eliminates messy details of exchange into side chains, which can be
influenced by things other than secondary structure and solvent shielding.
FIGURE 3 shows an HPLC column, injector and associated
tubing in a bath of ice water to maintain the temperature at 0 deg. C. Using this
set-up reduces the losses of deuterium during analysis, whether it be from a whole protein
or from peptides.
One can analyze whole proteins, or chop a protein into
smaller pieces with an enzyme. The enzyme that seems to work the best is porcine
pepsin. It likes working at pH 2.5 and can tolerate being at 0 deg. C. When
the sample is ready for analysis, it is digested for 5 minutes at 0 deg. C with
pepsin. After digestion, the peptides are separated quickly at 0 deg. C in 5-6
minutes. The column, solvent lines and injector are kept in an ice bath (see FIGURE
3). FIGURE 4 is a brief flow-chart of a a continuous labeling experiment.
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OTHER EXPERIMENTS AND MORE READING
There are many variations one can do to the general type
of experiment explained above. Whether analysis if done with MALDI rather than
electrospray, whether a pH pulse is used, whether denaturants are thrown in.... all
experiment must take into account the pH factor and the temperature factor. Without
doing so, the results of such analyses can be meaningless.
It is also critical to USE PROPER CONTROL samples.
The details of this are discussed in the section titled "Controls and
Other recommended reading on the basics of pH and
setting up experiments:
Engen, J.R. & Smith, D.L. (2000).
Investigating the higher order structure of proteins: Hydrogen exchange, proteolytic
fragmentation & mass spectrometry. In "Protein and Peptide Analysis: New Mass
Spectrometric Applications" (J. Chapmann, ed.). Meth. Mol. Biol. Vol. 146.