CH908, Problem set 12 - Noncovalent complexes and protein conformation. 1. Explain two methods for generating unfolding (melting) curves for proteins using mass spectrometry. What kind of resolution can be achieved? 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? Advantages: 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. Disadvantages: 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). Peptide 1: RGAVIVWYSDGK Peptide 2: QQLMGPKGAVLK 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).