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 (ΔGf 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 ΔGf values for the two-state folding proteins, lysozyme
and cytochrome c. The protocol also yielded an accurate measure of
the dissociation constant (Kd 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 ΔGf 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 ΔGf value measurements
using the combined HNSB and SPROX protocol is also evaluated.

本描述了一种基于质谱技术的共价标记协议开发过程，该协议利用二甲基-(2-羟基-5-硝基苯甲基)溴化物（HNSB）与色氨酸（Trp）残基的反应来测定蛋白质折叠的自由能（ΔGf值）。在该协议中，通过矩阵辅助激光解吸电离飞行时间分析或使用同位素质量标签的定量、自下而上的蛋白质组学分析，评估了蛋白质中全局保护的色氨酸残基与HNSB反应速率对化学去污剂的依赖性。在本研究的原理验证实验中，该协议为具有二态折叠特性的蛋白质，如溶菌酶和细胞色素c，提供了准确的ΔGf值。此外，该协议还准确测量了N,N′,N″-三乙酰基壳聚糖与溶菌酶结合的解离常数（Kd值），并成功检测了布林佐拉米德与BCA II（一种非二态折叠蛋白质）的结合。HNSB协议可与SPROX（蛋白质从氧化速率中获得的稳定性）技术结合使用，SPROX是一种先前报道的技术，利用蛋白质中蛋氨酸（Met）残基的过氧化氢氧化来测量ΔGf值。将HNSB协议整合到SPROX中，在蛋白质组范围内的SPROX实验中，肽段和蛋白质的覆盖率分别增加了50%和25%。作为这项工作的组成部分，还评估了使用结合HNSB和SPROX的协议进行蛋白质组范围内ΔGf值测量的精确度。