Answers, PS12

CH908, Problem set 12 - Noncovalent complexes and protein
1. Explain two methods for generating unfolding (melting) curves for
proteins using mass spectrometry. What kind of resolution can be
First, deuterium labeling can be used in solution (for a short, fixed
time), followed by pepsin digestion and detection of the mass increase
for each peptic fragment peptide. If this experiment is repeated in a
temperature dependent manner, you will get unfolding curves for each
peptide as the mass increases dependent on the temperature.
Oxidation or any other labeling method can also be used. The
sequence resolution for the unfolding will be at the peptide level.
Second, Electron capture dissociation can be performed with the ions
equilibrated to different temperatures. Fragment ion intensities will
vary with temperature.
2. What are some advantages of using mass spectrometry to study
protein structure rather than crystallography? Disadvantages?
Any (clean) protein can be studied, protein structure stability is less
critical, and folding/unfolding dynamics can be studied. Flexible loops
and glycan moieties can be kept - unlike in crystallography where they
must be removed to get the protein to crystallize.
Resolution. Crystallography gives atomic resolution, mass
spectrometry can give amino acid resolution at best, but normally
peptide level resolution at worst.
Crystallography also distinguishes actual 3D positions of each atom,
MS just studies relative cleavages at particular linkages. MS cannot
usually distinguish subtle changes in conformers.
3. Labeling reactions, such as crosslinking, can be useful, but they
make for difficult MS/MS spectra. Why? As an example, consider the
fragments you'd expect if the following two peptides were crosslinked
from the aspartic acid on Peptide 1 to the central lysine on Peptide 2
(using a zero-length crosslinker).
Can you calculate the expected b/y fragments in this case?
Here's a cleavage diagram of the crosslinked peptide.
While some of the observed peaks will be similar to those expected
for a 1:1 mixture of the two peptides, others will be more complex.
The b1α - b9α, y1α - y2α, b1β - b9β, and y1β - y2β fragments will be the
same as normal. The rest will have a huge post-translational
modification mass shift that corresponds to the mass of the opposite
protein minus a water (for the crosslink).