Temperature Jump and FPOP Probe Submillisecond Protein Folding
Fast photochemical oxidation of proteins (FPOP), developed in this MS resource, has proved to be a fast footprinting method that probes the solvent accessible residues of a protein 1, 2. The high speed of this chemical approach (~1 µs) makes feasible an MS-based means of studying protein folding/unfolding on that time scale. Thus, we developed a “pump-probe” laser method using a Nd:YAG to provide a temperature jump (T jump) triggering protein folding and a second laser to generate OH· to footprint the consequences of the T jump3.
The schematic of the device is shown in figure 1. A Nd:YAG laser driving a Raman cell produced a 1900 nm pulse that was absorbed by water molecules in a flowing solution held in a small capillary to cause the T jump (~20 °C). A 248 nm laser flash from an excimer laser photolyzed endogeneous H2O2 to give OH· in a 14 ns pulse. The frequency of the lasers was controlled by a function generator, and the time delay between two lasers (µs to ms) was adjusted with a custom-built circuitry. The model protein, barstar, was incubated on ice and kept cold denatured until the T jump.
By adjusting the delay, the time allowing protein folding was manipulated. The longer the delay, the more the protein is folded. More folding means less mass is added to the protein owing to the fewer exposed amino acid to solvent.
To measure the outcome, the sample was collected with catalase and methionine to remove the excess peroxide, desalted by C18 Zip Tip and then analyzed using mass spectrometer to determine the extent of oxidation. The representative mass spectra of the barstar post-FPOP as a function of the time between the heating pulse and the FPOP probe are shown in figure 3. The rate constant for equilibration of the folding can be measured by using the centroid of all the oxidized states having the same charge.
This method allows submillisecond protein-folding kinetics to be followed by a subsequent MS measurement at the global protein level. An advantage of this approach is its ability to determine modification and follow kinetics at the amino-acid level, especially for large proteins, by using proteolysis and LC/MS/MS. This MS-based approach has considerable flexibility. We aim to test whether protein-protein and protein-ligand binding dynamics can also be investigated by a two-laser approach.
1. Hambly, D.M. & Gross, M.L. Laser flash photolysis of hydrogen peroxide to oxidize protein solvent-accessible residues on the microsecond timescale. Journal of the American Society for Mass Spectrometry 16, 2057-2063 (2005).
2. Gau, B.C., Sharp, J.S., Rempel, D.L. & Gross, M.L. Fast photochemical oxidation of protein footprints faster than protein unfolding. Analytical chemistry 81, 6563-6571 (2009).
3. Chen, J., Rempel, D.L. & Gross, M.L. Temperature jump and fast photochemical oxidation probe submillisecond protein folding. Journal of the American Chemical Society 132, 15502-15504 (2010).
4. Gruebele, M. Analytical biochemistry: Weighing up protein folding. Nature 468, 640-641 (2010).
Top-down mass spectrometry
The majority of proteins perform biological functions in complexes rather than in isolated states. Determination of the composition and stoichiometry of protein assemblies is very important in structural biology. Our top-down MS approach in this direction is conducted by employing nano-electrospray ionization of the protein complexes in aqueous ammonium acetate buffer and electron capture dissociation (ECD) in an FTICR mass spectrometer. Showcase below is the 147 kDa yeast alcohol dehydrogenase (ADH) tetramer. The spectra show that nano-ESI produced a narrow charge state distribution of the intact ADH complex. ECD of this whole envelop of ions generated two type of product ions – low m/z sequence ions and high m/z charge-reduced precursor ions.
Analysis of the low m/z ions identified c-type sequence ions up to the 55th residue with the N-terminal serine acetylated. The sequenced N-terminal region is away from the complex interface and has the highest B-factor values when referred to the X-ray crystal structure (the lower left subunit is show in B-factor scale, while the N-termini up to the 55th residue of the rest three subunits are highlighted in red). The B-factor is a measure of the flexibility of certain region of the protein sequence when temperature or crystal imperfection occurs. The MS/MS fragments from top-down mass spectrometry and the B-factors from X-ray crystallography build a connection between these two analytical tools. This top-down strategy offers both proteomics and structural-biology information of protein assemblies in one experiment.
1. Native Electropray and Electron-Capture Dissociation in FTICR Mass Spectrometry Provide Top-Down sequencing of a Protein Component in an Intact Protein Assembly. H. Zhang, W. Cui, J. Wen, R. E. Blankenship, and M. L. Gross, J. Am. Soc. Mass Spectrom., 21: 1966-1968, 2010
2. Native Electrospray and Electron-Capture Dissociation FTICR Mass Spectrometry for Top-Down Studies of Protein Assemblies. H. Zhang, W. Cui, J. Wen, R. E. Blankenship, and M. L. Gross, Anal. Chem., 83(14): 5598-5606, 2011
3. Top-Down Mass Spectrometry: Recent Developments, Applications and Perspectives. W. Cui, H. W. Rohrs, and M. L. Gross, Analyst, 136(19): 3854-3864, 2011