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.

Thermodynamic Analysis of the Geldanamycin–Hsp90 Interaction in a Whole Cell Lysate Using a Mass Spectrometry-Based Proteomics Approach

“…The SPROX analyses in this work were performed according to previously described protocols [28,31]. A total of three SPROX experiments were conducted including two in which the standard SPROX protocol [28] was used and one in which the hybrid protocol [31] was used.…”

“…A total of three SPROX experiments were conducted including two in which the standard SPROX protocol [28] was used and one in which the hybrid protocol [31] was used. In each experiment, 180 μL portions of a~5 μg/μL MCF-7 cell lysate, prepared as described in the Supplemental Information, were used to create the (-) control and (+) drug samples that were subjected to SPROX analysis.…”

“…The methionine/tryptophancontaining peptide enriched samples were generated by combining 30 μL aliquots of the eight different iTRAQ labeled samples within a set, (-) or (+). The methionine-containing peptide enrichment was performed using the Pi 3 Methionine Reagent kit according to the manufacturer's protocol, and the tryptophan-containing peptide enrichment was performed as described in reference [31]. The methionine-containing peptide enrichment was performed in the SPROX-Short and SPROXLong experiment; both the methionine-and tryptophancontaining peptide enrichments were performed in the Hybrid-Short experiment.…”

“…The three experiments involved the use of two slightly different SPROX protocols, including one that involved the chemical modification of methionine residues in proteins with H 2 O 2 (referred to hereafter as the SPROX protocol) and one that involved the chemical modification of methionine and tryptophan residues in proteins with H 2 O 2 and HNSB, respectively (referred to hereafter as the Hybrid protocol). By using both methionine-and tryptophan-containing peptide probes to readout the thermodynamic stability of proteins in proteome-wide SPROX experiments, the Hybrid protocol results in a~50% increase in proteomic coverage compared with using only methionine-containing peptide probes [31]. The three experiments also involved the use of different equilibration times.…”

Affinity purification in target identification: the specificity challenge

“…Stability of proteins from rates of oxidation (SPROX) is another approach utilizing the variation in stability of proteins in the presence and absence of a ligand (West et al 2008). Coupled with quantitative mass spectrometry, SPROX measures the rate difference of methionine oxidation of proteins in order to identify specific targets (West et al 2010;Dearmond et al 2011;Strickland et al 2013;Xu et al 2014;Adhikari et al 2015). Finally, with a different concept, Emili and coworkers developed a label-free method based on ligandinduced change of retention time of proteins during chromatographic co-elution (Chan et al 2012).…”