Thermodynamic Analysis of Protein Folding and Stability Using a Tryptophan Modification Protocol

Described here is the development of a mass spectrometry-based covalent labeling protocol that utilizes the reaction of dimethyl­(2-hydroxy-5-nitrobenzyl)­sulfonium bromide (HNSB) with tryptophan (Trp) residues to measure protein folding free energies (ΔG_f values). In the protocol, the chemical denaturant dependence of the rate at which globally protected Trp residues in a protein react with HNSB is evaluated using either a matrix assisted laser desorption ionization time-of-flight analysis of the intact protein or a quantitative, bottom-up proteomics analysis using isobaric mass tags. In the proof-of-principle studies performed here, the protocol yielded accurate ΔG_f values for the two-state folding proteins, lysozyme and cytochrome c. The protocol also yielded an accurate measure of the dissociation constant (K_d value) for the binding of N,N′,N″-triacetylchitotriose to lysozyme, and it successfully detected the binding of brinzolamide to BCA II, a non-two-state folding protein. The HNSB protocol can be used in combination with SPROX (stability of proteins from rates of oxidation), a previously reported technique that exploits the hydrogen peroxide oxidation of methionine (Met) residues in proteins to make ΔG_f value measurements. Incorporating the HNSB protocol into SPROX increased the peptide and protein coverage in proteome-wide SPROX experiments by 50% and 25%, respectively. As part of this work, the precision of proteome-wide ΔG_f value measurements using the combined HNSB and SPROX protocol is also evaluated.