As a company dedicated to bioactive natural products target identification, we focus on label-free, unbiased, and proteome-wide approaches. Among these, stability of proteins from rates of oxidation (SPROX) technology has emerged as a powerful and reliable method to identify protein–ligand interactions directly in complex biological systems. SPROX enables the detection of target engagement by measuring ligand-induced changes in protein thermodynamic stability, without the need for compound modification.
What is SPROX?
SPROX is a mass spectrometry-based, label-free chemical proteomics technique that quantifies changes in protein stability upon ligand binding. The method exploits the fact that ligand binding often stabilizes (or destabilizes) a protein's folded structure. These stability changes can be measured by monitoring the oxidation rates of methionine residues under controlled chemical denaturation conditions. In the context of natural product research, SPROX allows direct comparison of protein stability profiles in the presence and absence of a bioactive compound. Proteins that exhibit significant shifts in stability are considered potential direct or indirect targets of the compound.
Principle of SPROX
SPROX technology is founded on the thermodynamic principle that ligand binding alters the conformational stability of proteins. When a bioactive small molecule or natural product interacts with a protein, it can shift the folding–unfolding equilibrium by stabilizing or destabilizing the native structure. This stability change directly affects the solvent accessibility of methionine residues, which are typically buried in the folded state and become exposed upon unfolding. By monitoring changes in methionine oxidation behavior as a function of protein stability, SPROX provides a quantitative and proteome-wide readout of ligand-induced stability shifts. Proteins that display significant stability changes in the presence of a compound are inferred to be directly or indirectly engaged, enabling target identification without the need for chemical labeling or affinity enrichment.
Fig. 1. Schematic diagram of SPROX strategy [1].
Our SPROX-Based Technology Platform
Our SPROX-based platform is specifically optimized for bioactive natural products, including complex mixtures, purified compounds, and extracts. We integrate advanced sample preparation, high-resolution mass spectrometry, and proprietary data analysis pipelines to deliver robust and biologically meaningful results.
Typical Workflow of SPROX Includes:
Step 1
Sample Preparation
Native protein lysates are prepared from relevant biological systems (cell lines, tissues, or organisms). Samples are divided into control and compound-treated groups without chemical labeling.
Step 2
Compound Incubation
The bioactive natural product is incubated with the proteome under physiologically relevant conditions to allow target engagement.
Step 3
Denaturant Titration
Both control and treated samples are distributed across a gradient of denaturant concentrations.
Step 4
Methionine Oxidation Reaction
Hydrogen peroxide is added for a precisely controlled time to oxidize exposed methionine residues.
Step 5
Proteolysis and LC-MS/MS Analysis
Samples are digested (typically with trypsin) and analyzed by high-resolution LC-MS/MS to quantify oxidized and non-oxidized methionine-containing peptides.
Step 6
Data Analysis and Target Prioritization
Stability curves are generated, unfolding midpoint (Cm) shifts are calculated, and statistically significant protein stability changes are identified as candidate targets.
Key Advantages of Our Platform Include:
- Label-free and Native Conditions: No chemical modification of compounds or proteins is required, preserving native binding interactions.
- Proteome-wide Coverage: Thousands of proteins can be monitored simultaneously in a single experiment.
- Quantitative and Thermodynamic Readout: SPROX provides direct information on protein stability changes, offering mechanistic insight beyond simple binding detection.
- Compatibility with Complex Natural Products: Effective for pure compounds, fractions, or complex extracts, where affinity-based approaches often fail.
- Reduced False Positives: Target identification is based on reproducible stability shifts rather than nonspecific enrichment.
- Scalable and Reproducible: Suitable for early discovery screening as well as in-depth mechanistic studies.
Expanded Applications of SPROX Platform
Our SPROX platform not only supports target identification, but also enables a range of downstream studies, including:
- Deconvolution of bioactive natural products
- Mechanism-of-action (MoA) studies
- Target validation and hit prioritization
- Off-target and polypharmacology profiling
- Comparative studies of analogs or derivatives
SPROX technology represents a powerful, label-free solution for uncovering the molecular targets of bioactive natural products. By directly linking ligand binding to changes in protein stability, our SPROX-based technology platform delivers unbiased, proteome-wide, and mechanistically informative insights, enabling confident target identification and accelerated drug discovery.
Online Inquiry
Frequently Asked Questions (FAQ)
Q1: Is SPROX suitable for low-affinity interactions?
Q2: What sample types are compatible with your platform?
A: Cell lysates, tissues, microorganisms, and recombinant systems are all compatible. We tailor experimental design to the biological question.
Reference
- Li G., et al. Currently available strategies for target identification of bioactive natural products[J]. Frontiers in Chemistry, 2021, 9: 761609.