title: Thermodynamic analysis of protein folding and stability using a tryptophan modification protocol.
abstract: 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.
authors.affiliation: Department of Chemistry, Duke University , Durham, North Carolina 27708, United States.
authors.forename: Yingrong
authors.initials: Y
authors.lastname: Xu
authors.forename: Erin C
authors.initials: EC
authors.lastname: Strickland
authors.forename: Michael C
authors.initials: MC
authors.lastname: Fitzgerald
mesh_terms: D000595:Amino Acid Sequence; D045304:Cytochromes c / Q000737:chemistry; D008715:Methionine / Q000737:chemistry; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry; D017510:Protein Folding; D055550:Protein Stability; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013452:Sulfonium Compounds / Q000737:chemistry; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry*
chemical_list: D013452:Sulfonium Compounds; C024586:dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium; D014364:Tryptophan; D045304:Cytochromes c; D008715:Methionine; D009113:Muramidase