Stability Of Proteins From Rates Of Oxidation Research Articles

Analysis of Brain Protein Stability Changes in a Mouse Model of Alzheimer's Disease.

The stability of proteins from rates of oxidation (SPROX), thermal proteome profiling (TPP), and limited proteolysis (LiP) techniques were used to profile the stability of ∼2500 proteins in hippocampus tissue cell lysates from 2- and 8-months-old wild-type (C57BL/6J; n = 7) and transgenic (5XFAD; n = 7) mice with five Alzheimer's disease (AD)-linked mutations. Approximately 200-500 protein hits with AD-related stability changes were detected by each technique at each age point. The hit overlap from technique to technique was low, and all of the techniques generated protein hits that were more numerous and largely different from those identified in protein expression level analyses, which were also performed here. The hit proteins identified by each technique were enriched in a number of the same pathways and biological processes, many with known connections to AD. The protein stability hits included 25 high-value conformation biomarkers with AD-related stability changes detected using at least 2 techniques at both age points. Also discovered were subunit- and age-specific AD-related stability changes in the proteasome, which had reduced function at both age points. The different folding stability profiles of the proteasome at the two age points are consistent with a different mechanism for proteasome dysfunction at the early and late stages of AD.

Stability-Based Proteomics for Investigation of Structured RNA-Protein Interactions.

RNA-protein interactions are essential to RNA function throughout biology. Identifying the protein interactions associated with a specific RNA, however, is currently hindered by the need for RNA labeling or costly tiling-based approaches. Conventional strategies, which commonly rely on affinity pull-down approaches, are also skewed to the detection of high affinity interactions and frequently miss weaker interactions that may be biologically important. Reported here is the first adaptation of stability-based mass spectrometry methods for the global analysis of RNA-protein interactions. The stability of proteins from rates of oxidation (SPROX) and thermal protein profiling (TPP) methods are used to identify the protein targets of three RNA ligands, the MALAT1 triple helix (TH), a viral stem loop (SL), and an unstructured RNA (PolyU), in LNCaP nuclear lysate. The 315 protein hits with RNA-induced conformational and stability changes detected by TPP and/or SPROX were enriched in previously annotated RNA-binding proteins and included new proteins for hypothesis generation. Also demonstrated are the orthogonality of the SPROX and TPP approaches and the utility of the domain-specific information available with SPROX. This work establishes a novel platform for the global discovery and interrogation of RNA-protein interactions that is generalizable to numerous biological contexts and RNA targets.

Studying Protein-Ligand Interactions by Protein Denaturation and Quantitative Cross-Linking Mass Spectrometry.

Recently, several mass spectrometry methods have utilized protein structural stability for the quantitative study of protein-ligand engagement. These protein-denaturation approaches, which include thermal proteome profiling (TPP) and stability of proteins from rates of oxidation (SPROX), evaluate ligand-induced denaturation susceptibility changes with a MS-based readout. The different techniques of bottom-up protein-denaturation methods each have their own advantages and challenges. Here, we report the combination of protein-denaturation principles with quantitative cross-linking mass spectrometry using isobaric quantitative protein interaction reporter technologies. This method enables the evaluation of ligand-induced protein engagement through analysis of cross-link relative ratios across chemical denaturation. As a proof of concept, we found ligand-stabilized cross-linked lysine pairs in well-studied bovine serum albumin and ligand bilirubin. These links map to the known binding sites Sudlow Site I and subdomain IB. We propose that protein denaturation and qXL-MS can be combined with similar peptide-level quantification approaches, like SPROX, to increase the coverage information profiled for facilitating protein-ligand engagement efforts.

CHalf: Folding Stability Made Simple.

The structure of a protein defines its function and integrity and correlates with the protein folding stability (PFS). Quantifying PFS allows researchers to assess differential stability of proteins in different disease or ligand binding states, providing insight into protein efficacy and potentially serving as a metric of protein quality. There are a number of mass spectrometry (MS)-based methods to assess PFS, such as Thermal Protein Profiling (TPP), Stability of Proteins from Rates of Oxidation (SPROX), and Iodination Protein Stability Assay (IPSA). Despite the critical value that PFS studies add to the understanding of mechanisms of disease and treatment development, proteomics research is still primarily dominated by concentration-based studies. We found that a major reason for the lack of PFS studies is the lack of a user-friendly data processing tool. Here we present the first user-friendly software, CHalf, with a graphical user interface for calculating PFS. Besides calculating site-specific PFS of a given protein from chemical denature folding stability assays, CHalf is also compatible with thermal denature folding stability assays. CHalf also includes a set of data visualization tools to help identify changes in PFS across protein sequences and in between different treatment conditions. We expect the introduction of CHalf to lower the barrier of entry for researchers to investigate PFS, promoting the usage of PFS in studies. In the long run, we expect this increase in PFS research to accelerate our understanding of the pathogenesis and pathophysiology of disease.

Discovery of the Xenon-Protein Interactome Using Large-Scale Measurements of Protein Folding and Stability.

The intermolecular interactions of noble gases in biological systems are associated with numerous biochemical responses, including apoptosis, inflammation, anesthesia, analgesia, and neuroprotection. The molecular modes of action underlying these responses are largely unknown. This is in large part due to the limited experimental techniques to study protein-gas interactions. The few techniques that are amenable to such studies are relatively low-throughput and require large amounts of purified proteins. Thus, they do not enable the large-scale analyses that are useful for protein target discovery. Here, we report the application of stability of proteins from rates of oxidation (SPROX) and limited proteolysis (LiP) methodologies to detect protein-xenon interactions on the proteomic scale using protein folding stability measurements. Over 5000 methionine-containing peptides and over 5000 semi-tryptic peptides, mapping to ∼1500 and ∼950 proteins, respectively, in the yeast proteome, were assayed for Xe-interacting activity using the SPROX and LiP techniques. The SPROX and LiP analyses identified 31 and 60 Xe-interacting proteins, respectively, none of which were previously known to bind Xe. A bioinformatics analysis of the proteomic results revealed that these Xe-interacting proteins were enriched in those involved in ATP-driven processes. A fraction of the protein targets that were identified are tied to previously established modes of action related to xenon's anesthetic and organoprotective properties. These results enrich our knowledge and understanding of biologically relevant xenon interactions. The sample preparation protocols and analytical methodologies developed here for xenon are also generally applicable to the discovery of a wide range of other protein-gas interactions in complex biological mixtures, such as cell lysates.

Analysis of Brain Protein Stability Changes in Mouse Models of Normal Aging and α-Synucleinopathy Reveals Age- and Disease-Related Differences.

Here, we utilize the stability of proteins from rates of oxidation (SPROX) technique, to profile the thermodynamic stabilities of proteins in brain tissue cell lysates from Huα-Syn(A53T) transgenic mice at three time points including at 1 month (n = 9), at 6 months (n = 7), and at the time (between 9 and 16 months) a mouse became symptomatic (n = 8). The thermodynamic stability profiles generated here on 332 proteins were compared to thermodynamic stability profiles generated on the same proteins from similarly aged wild-type mice using a two-way unbalanced analysis of variance (ANOVA) analysis. This analysis identified a group of 22 proteins with age-related protein stability changes and a group of 11 proteins that were differentially stabilized in the Huα-Syn(A53T) transgenic mouse model. A total of 9 of the 11 proteins identified here with disease-related stability changes have been previously detected in human cerebral spinal fluid and thus have potential utility as biomarkers of Parkinson's disease (PD). The differential stability observed for one protein, glutamate decarboxylase 2 (Gad2), with an age-related change in stability, was consistent with the differential presence of a known, age-related truncation product of this protein, which is shown here to have a higher folding stability than full-length Gad2. Mass spectrometry data were deposited at ProteomeXchange (PXD016985).

A Comparison of Two Stability Proteomics Methods for Drug Target Identification in OnePot 2D Format.

Stability proteomics techniques that do not require drug modifications have emerged as an attractive alternative to affinity purification methods in drug target engagement studies. Two representative techniques include the chemical-denaturation-based SPROX (Stability of Proteins from Rates of Oxidation), which utilizes peptide-level quantification and thermal-denaturation-based TPP (Thermal Proteome Profiling), which utilizes protein-level quantification. Recently, the "OnePot" strategy was adapted for both SPROX and TPP to increase the throughput. When combined with the 2D setup which measures both the denaturation and the drug dose dimensions, the OnePot 2D format offers improved analysis specificity with higher resource efficiency. However, a systematic evaluation of the OnePot 2D format and a comparison between SPROX and TPP are still lacking. Here, we performed SPROX and TPP to identify protein targets of a well-studied pan-kinase inhibitor staurosporine with K562 lysate, in curve-fitting and OnePot 2D formats. We found that the OnePot 2D format provided ∼10× throughput, achieved ∼1.6× protein coverage and involves more straightforward data analysis. We also compared SPROX with the current "gold-standard" stability proteomics technique TPP in the OnePot 2D format. The protein coverage of TPP is ∼1.5 fold of SPROX; however, SPROX offers protein domain-level information, identifies comparable numbers of kinase hits, has higher signal (R value), and requires ∼3× less MS time. Unique SPROX hits encompass higher-molecular-weight proteins, compared to the unique TPP hits, and include atypical kinases. We also discuss hit stratification and prioritization strategies to promote the efficiency of hit followup.

A New Drug Discovery Approach Based on Thermal Proteome Profiling to Develop More Effective Drugs

: The search for disease-related targets and studying drug-protein and protein-protein interactions are central issues that would accelerate the clinical approval of a drug. Also, by developing an accurate method in this regard, time and resource consumption will significantly decrease. The low efficiency of some drugs in humans is a grave issue leading to a low rate of FDA approval after spending billions of dollars and decades of research. Several strategies and methods have been expanded to fill this gap, such as drug affinity responsive target stability (DARTS), stability of proteins from rates of oxidation (SPROX), cellular thermal shift assay (CETSA), and finally, thermal proteome profiling (TPP). The TPP is based on the combination of CETSA and quantitative mass spectrometry. Among recently introduced proteomics technologies, TPP demonstrates the ability to offer detailed proteomic profiles for the large-scale analysis of protein-ligand interactions, including endogenous ligands and proteins like cofactors and metabolites. TPP facilitates the identification of the markers governing drug efficacy and toxicity and provides an unbiased measure for estimating the rate of drug-target engagement. At a glance at TPP steps, after protein extraction, the molecule is exposed to different temperatures and drug concentrations. After discarding solubilized and stabilized proteins, the protein’s identity is investigated by mass spectrometry analysis. As a result of the protein’s structural stabilization after binding to its substrate, TTP helps to accurately identify target proteins with high throughput. In this study, we aimed to introduce the basics of this method and review most recent studies on this technique.

Identification techniques of small molecule drug target proteins without chemical modification and its applications: a review

Identification of the target proteins of small molecule drugs is crucial for understanding the mechanisms of drug actions and its side effects. Conventional methods require chemical modification, which might alter the activities of the drugs. Various label-free techniques have been developed to identify drug target proteins without chemical modifications. This includes drug affinity responsive target stability (DARTS), stability of proteins from rates of oxidation (SPROX), cellular thermal shift assay (CETSA), thermal proteome profiling (TPP) and many others. Here we review the principles and applications of these label-free techniques, their advantages and limitations, as well as the most recent advances.

Plasmodium chaperonin TRiC/CCT identified as a target of the antihistamine clemastine using parallel chemoproteomic strategy

The antihistamine clemastine inhibits multiple stages of the Plasmodium parasite that causes malaria, but the molecular targets responsible for its parasite inhibition were unknown. Here, we applied parallel chemoproteomic platforms to discover the mechanism of action of clemastine and identify that clemastine binds to the Plasmodium falciparum TCP-1 ring complex or chaperonin containing TCP-1 (TRiC/CCT), an essential heterooligomeric complex required for de novo cytoskeletal protein folding. Clemastine destabilized all eight P. falciparum TRiC subunits based on thermal proteome profiling (TPP). Further analysis using stability of proteins from rates of oxidation (SPROX) revealed a clemastine-induced thermodynamic stabilization of the Plasmodium TRiC delta subunit, suggesting an interaction with this protein subunit. We demonstrate that clemastine reduces levels of the major TRiC substrate tubulin in P. falciparum parasites. In addition, clemastine treatment leads to disorientation of Plasmodium mitotic spindles during the asexual reproduction and results in aberrant tubulin morphology suggesting protein aggregation. This clemastine-induced disruption of TRiC function is not observed in human host cells, demonstrating a species selectivity required for targeting an intracellular human pathogen. Our findings encourage larger efforts to apply chemoproteomic methods to assist in target identification of antimalarial drugs and highlight the potential to selectively target Plasmodium TRiC-mediated protein folding for malaria intervention.

Protein Footprinting with Radical Probe Mass Spectrometry- Two Decades of Achievement.

Radical Probe Mass Spectrometry (RP-MS) describes a pioneering methodology in structural biology that enables the study of protein structures, their interactions, and dynamics on fast timescales (down to sub-milliseconds). Hydroxyl radicals (•OH) generated directly from water within aqueous solutions induce the oxidation of reactive, solvent accessible amino acid side chains that are then analyzed by mass spectrometry. Introduced in 1998 at the American Society for Mass Spectrometry annual conference, RP-MS was first published on in 1999. This review article describes developments and applications of the RP-MS methodology over the past two decades. The RP-MS method has been variously referred to as synchrotron X-ray radiolysis footprinting, Hydroxyl Radical Protein Footprinting (HRPF), X-ray Footprinting with Mass Spectrometry (XF-MS), Fast Photochemical Oxidation of Proteins (FPOP), oxidative labelling, covalent oxidative labelling, and even the Stability of Proteins from Rates of Oxidation (SPROX). The article describes the utility of hydroxyl radicals as a protein structural probe, the advantages of RP-MS in comparison to other MS-based approaches, its proof of concept using ion mobility mass spectrometry, its application to protein structure, folding, complex and aggregation studies, its extension to study the onset of protein damage, its implementation using a high throughput sample loading approach, and the development of protein docking algorithms to aid with data analysis and visualization. RP-MS represents a powerful new structural approach that can aid in our understanding of the structure and functions of proteins, and the impact of sustained oxidation on proteins in disease pathogenesis.

Proteome-Wide Structural Biology: An Emerging Field for the Structural Analysis of Proteins on the Proteomic Scale.

Over the past decade, a suite of new mass-spectrometry-based proteomics methods has been developed that now enables the conformational properties of proteins and protein-ligand complexes to be studied in complex biological mixtures, from cell lysates to intact cells. Highlighted here are seven of the techniques in this new toolbox. These techniques include chemical cross-linking (XL-MS), hydroxyl radical footprinting (HRF), Drug Affinity Responsive Target Stability (DARTS), Limited Proteolysis (LiP), Pulse Proteolysis (PP), Stability of Proteins from Rates of Oxidation (SPROX), and Thermal Proteome Profiling (TPP). The above techniques all rely on conventional bottom-up proteomics strategies for peptide sequencing and protein identification. However, they have required the development of unconventional proteomic data analysis strategies. Discussed here are the current technical challenges associated with these different data analysis strategies as well as the relative analytical capabilities of the different techniques. The new biophysical capabilities that the above techniques bring to bear on proteomic research are also highlighted in the context of several different application areas in which these techniques have been used, including the study of protein ligand binding interactions (e.g., protein target discovery studies and protein interaction network analyses) and the characterization of biological states.

Chemical Denaturation and Protein Precipitation Approach for Discovery and Quantitation of Protein–Drug Interactions

Described here is a mass spectrometry-based proteomics approach for the large-scale analysis of protein-drug interactions. The approach involves the evaluation of ligand-induced protein folding free energy changes (ΔΔ Gf) using chemical denaturation and protein precipitation (CPP) to identify the protein targets of drugs and to quantify protein-drug binding affinities. This is accomplished in a chemical denaturant-induced unfolding experiment where the folded and unfolded protein fractions in each denaturant containing buffer are quantified by the amount of soluble or precipitated protein (respectively) that forms upon abrupt dilution of the chemical denaturant and subsequent centrifugation of the sample. In the proof-of-principle studies performed here, the CPP technique was able to identify the well-known protein targets of cyclosporin A and geldanamycin in a yeast. The technique was also used to identify protein targets of sinefungin, a broad-based methyltransferase inhibitor, in a human MCF-7 cell lysate. The CPP technique also yielded dissociation constant ( Kd) measurements for these well-studied drugs that were in general agreement with previously reported Kd or IC50 values. In comparison to a similar energetics-based technique, termed stability of proteins from rates of oxidation (SPROX), the CPP technique yielded significantly better (∼50% higher) proteomic coverage and a largely reduced false discovery rate.

Proteome-Wide Characterization of Phosphorylation-Induced Conformational Changes in Breast Cancer.

Because of the close link between protein function and protein folding stability, knowledge about phosphorylation-induced protein folding stability changes can lead to a better understanding of the functional effects of protein phosphorylation. Here, the stability of proteins from rates of oxidation (SPROX) and limited proteolysis (LiP) techniques are used to compare the conformational properties of proteins in two MCF-7 cell lysates including one that was and one that was not dephosphorylated with alkaline phosphatase. A total of 168 and 251 protein hits were identified with dephosphorylation-induced stability changes using the SPROX and LiP techniques, respectively. Many protein hits are previously known to be differentially phosphorylated or differentially stabilized in different human breast cancer subtypes, suggesting that the phosphorylation-induced stability changes detected in this work are disease related. The SPROX hits were enriched in proteins with aminoacyl-tRNA ligase activity. These enriched protein hits included many aminoacyl-tRNA synthetases (aaRSs), which are known from previous studies to have their catalytic activity modulated by phosphorylation. The SPROX results revealed that the magnitudes of the destabilizing effects of dephoshporylation on the different aaRSs were directly correlated with their previously reported aminoacylation activity change upon dephosphorylation. This substantiates the close link between protein folding and function.

Discovery of Tamoxifen and N-Desmethyl Tamoxifen Protein Targets in MCF-7 Cells Using Large-Scale Protein Folding and Stability Measurements.

The proteins in an MCF-7 cell line were probed for tamoxifen (TAM) and n-desmethyl tamoxifen (NDT) induced stability changes using the Stability of Proteins from Rates of Oxidation (SPROX) technique in combination with two different quantitative proteomics strategies, including one based on SILAC and one based on isobaric mass tags. Over 1000 proteins were assayed for TAM- and NDT-induced protein stability changes, and a total of 163 and 200 protein hits were identified in the TAM and NDT studies, respectively. A subset of 27 high-confidence protein hits were reproducibly identified with both proteomics strategies and/or with multiple peptide probes. One-third of the high-confidence hits have previously established experimental links to the estrogen receptor, and nearly all of the high-confidence hits have established links to breast cancer. One high-confidence protein hit that has known estrogen receptor binding properties, Y-box binding protein 1 (YBX1), was further validated as a direct binding target of TAM using both the SPROX and pulse proteolysis techniques. Proteins with TAM- and/or NDT-induced expression level changes were also identified in the SILAC-SPROX experiments. These proteins with expression level changes included only a small fraction of those with TAM- and/or NDT-induced stability changes.

Discovery of Age-Related Protein Folding Stability Differences in the Mouse Brain Proteome.

Described here is the application of thermodynamic stability measurements to study age-related differences in the folding and stability of proteins in a rodent model of aging. Thermodynamic stability profiles were generated for 809 proteins in brain cell lysates from mice, aged 6 (n = 7) and 18 months (n = 9) using the Stability of Proteins from Rates of Oxidation (SPROX) technique. The biological variability of the protein stability measurements was low and within the experimental error of SPROX. A total of 83 protein hits were detected with age-related stability differences in the brain samples. Remarkably, the large majority of the brain protein hits were destabilized in the old mice, and the hits were enriched in proteins that have slow turnover rates (p < 0.07). Furthermore, 70% of the hits have been previously linked to aging or age-related diseases. These results help validate the use of thermodynamic stability measurements to capture relevant age-related proteomic changes and establish a new biophysical link between these proteins and aging.

Targeted Mass Spectrometry-Based Approach for Protein-Ligand Binding Analyses in Complex Biological Mixtures Using a Phenacyl Bromide Modification Strategy.

The characterization of protein folding stability changes on the proteomic scale is useful for protein-target discovery and for the characterization of biological states. The Stability of Proteins from Rates of Oxidation (SPROX) technique is one of several mass spectrometry-based techniques recently established for the making proteome-wide measurements of protein folding and stability. A critical part of proteome-wide applications of SPROX is the identification and quantitation of methionine-containing peptides. Demonstrated here is a targeted mass spectrometry-based proteomics strategy for the detection and quantitation of methionine-containing peptides in SPROX experiments. The strategy involves the use of phenacyl bromide (PAB) for the targeted detection and quantitation of methionine-containing peptides in SPROX using selective reaction monitoring (SRM) on a triple quadrupole mass spectrometer (QQQ-MS). As proof-of-principle, the known binding interaction of Cyclosporine A with cyclophilin A protein in a yeast cell lysate is successfully detected and quantified using a targeted SRM workflow. Advantages of the described workflow over other SPROX protocols include a 20-fold reduction in the amount of total protein needed for analysis and the ability to work with the endogenous proteins in a given sample (e.g., stabile isotope labeling with amino acids in cell culture is not necessary).

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

Geldanamycin is a natural product with well-established and potent anti-cancer activities. Heat shock protein 90 (Hsp90) is the known target of geldanamycin, which directly binds to Hsp90's N-terminal ATP binding domain and inhibits Hsp90's ATPase activity. The affinity of geldanamycin for Hsp90 has been measured in multiple studies. However, there have been large discrepancies between the reported dissociation constants (i.e., Kd values), which have ranged from low nanomolar to micromolar. Here the stability of proteins from rates of oxidation (SPROX) technique was used in combination with an isobaric mass tagging strategy to measure the binding affinity of geldanamycin to unpurified Hsp90 in an MCF-7 cell lysate. The Kd values determined here were dependent on how long geldanamycin was equilibrated with the lysate prior to SPROX analysis. The Kd values determined using equilibration times of 0.5 and 24 h were 1 and 0.03 μM, respectively. These Kd values, which are similar to those previously reported in a geldanamycin-Hsp90 binding study that involved the use of a fluorescently labeled geldanamycin analogue, establish that the slow-tight binding behavior previously observed for the fluorescently labeled geldanamycin analogue is not an artifact of the fluorescent label, but rather an inherent property of the geldanamycin-Hsp90 binding interaction. The slow-tight binding property of this complex may be related to time-dependent conformational changes in Hsp90 and/or to time-dependent chemical changes in geldanamycin, both of which have been previously proposed to explain the slow-tight binding behavior of the geldanamycin-Hsp90 complex. Graphical Abstract ᅟ.

Characterization of the Saccharomyces cerevisiae ATP-Interactome using the iTRAQ-SPROX Technique.

The stability of proteins from rates of oxidation (SPROX) technique was used in combination with an isobaric mass tagging strategy to identify adenosine triphosphate (ATP) interacting proteins in the Saccharomyces cerevisiae proteome. The SPROX methodology utilized in this work enabled 373 proteins in a yeast cell lysate to be assayed for ATP interactions (both direct and indirect) using the non-hydrolyzable ATP analog, adenylyl imidodiphosphate (AMP-PNP). A total of 28 proteins were identified with AMP-PNP-induced thermodynamic stability changes. These protein hits included 14 proteins that were previously annotated as ATP-binding proteins in the Saccharomyces Genome Database (SGD). The 14 non-annotated ATP-binding proteins included nine proteins that were previously found to be ATP-sensitive in an earlier SPROX study using a stable isotope labeling with amino acids in cell culture (SILAC)-based approach. A bioinformatics analysis of the protein hits identified here and in the earlier SILAC-SPROX experiments revealed that many of the previously annotated ATP-binding protein hits were kinases, ligases, and chaperones. In contrast, many of the newly discovered ATP-sensitive proteins were not from these protein classes, but rather were hydrolases, oxidoreductases, and nucleic acid-binding proteins. Graphical Abstract ᅟ.

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.