Workflows and Data Analysis in Chemical Proteomics
-
Probes are added to either cell lysates or live cells and incubated under appropriate conditions to allow sufficient labeling.
-
In control samples, either an inactive analog or an excess of a competing small molecule is introduced to distinguish specific from nonspecific binding interactions.
-
Probe-labeled proteins are enriched using streptavidin-coated magnetic beads.
-
Following enrichment, proteins are digested with trypsin to generate peptides suitable for downstream mass spectrometry (MS) analysis.
-
MS data are processed using tools such as MaxQuant or Proteome Discoverer for peptide identification and quantification.
-
Labeling sites introduced by the chemical probes (e.g., alkyne-modified residues) are specifically identified.
-
Background signals are removed by comparative filtering using competition groups or blank controls.
-
Abundance ratio thresholds are set (e.g., comparing DMSO control to the probe-labeled group) to assess binding specificity.
-
Statistical analyses, including t-tests and false discovery rate (FDR) correction, are used to identify high-confidence target proteins.
-
Functional characterization of labeled proteins is performed using tools such as DAVID, Metascape, and STRING to assess molecular functions, subcellular localization, and involvement in signaling pathways.
-
These analyses help elucidate the biological mechanisms of action of the probe or small molecule.
-
A seasoned team of experts in chemical modification-based proteomics
-
High-throughput automated sample processing platforms
-
High-resolution Orbitrap mass spectrometry for precise protein identification
-
In-depth analytical reports along with biological annotation support
Chemical proteomics is a methodology that integrates chemical probes with high-resolution mass spectrometry to systematically investigate protein functional states, molecular interactions, and small-molecule targets under in situ and native states. This approach holds significant promise in fields such as drug discovery, enzymatic function characterization, and post-translational modification analysis. In this article, we provide a comprehensive overview of the experimental workflows and data analysis strategies in chemical proteomics, aiming to facilitate researchers’ in-depth understanding of this emerging technology and to explore how specialized platforms, such as MtoZ Biolabs, can effectively accelerate related studies.
What Is Chemical Proteomics?
Chemical proteomics employs chemical probes that covalently label proteins to enable active-site tagging, affinity enrichment, and mass spectrometry-based identification, thereby offering a comprehensive view of protein functional states.
Its key advantages include:
1. High specificity: Probes can be rationally designed to selectively target specific enzyme classes or structural motifs, such as serine hydrolases or cysteine residues.
2. In situ applicability: The technique can be directly applied at the cellular or even whole-organism level.
3. Function-oriented profiling: It not only identifies proteins but also captures their activity states and small-molecule binding sites.
Dissecting the Experimental Workflows in Chemical Proteomics
A typical chemical proteomics workflow comprises the following essential steps:
1. Design and Synthesis of Chemical Probes
Chemical probes generally consist of three functional components:
(1) Recognition moiety: specifically binds to the active site of the target protein;
(2) Reactive group: covalently attaches to the protein;
(3) Tag moiety: enables downstream enrichment or detection via fluorescence or photoaffinity labeling, commonly using biotin or alkyne functionalities.
Representative types of probes include:
(1) Activity-Based Protein Profiling (ABPP) probes
(2) Photoaffinity labeling probes
(3) Covalent ligand probes
2. Sample Processing and Probe Labeling
3. Click Chemistry
Copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC) is employed to covalently conjugate the alkyne group on the labeled probe with biotin-azide or similar molecules, facilitating subsequent enrichment procedures.
4. Protein Enrichment and Digestion
5. Mass Spectrometry Detection (LC-MS/MS)
Peptides are analyzed using high-resolution mass spectrometry platforms such as Orbitrap or Q-TOF systems to obtain detailed information on peptide sequences, chemical modification sites, and relative abundance levels.
At MtoZ Biolabs, we employ an optimized enrichment and digestion workflow in combination with advanced MS platforms (e.g., Thermo Orbitrap Exploris) to achieve high sensitivity and reproducibility in chemical proteomics experiments.
Data Analysis Strategy
The data analysis workflow in chemical proteomics differs from that of conventional proteomics in that it must account for both chemical modification sites and probe-induced quantitative changes. Typical analysis includes the following steps:
1. Data Preprocessing
2. Specific Binding Protein Screening
3. Functional Annotation and Pathway Enrichment
4. Target Validation (Optional)
Key target proteins may be further validated through orthogonal methods such as Western blotting, co-immunoprecipitation (Co-IP), or functional assays to confirm their roles and binding interactions.
Application Cases: From Mechanistic Studies to Drug Development
Chemical proteomics has demonstrated significant potential across various applications:
1. Discovery of novel drug targets: identifying protein targets that are covalently modified by small molecules (e.g., KRAS G12C, BTK);
2. Enzyme activity profiling: assessing dynamic changes in intracellular enzyme activities using activity-based protein profiling (ABPP) probes;
3. Post-translational modification studies: mapping modification sites such as oxidation, nitration, and alkylation;
4. Investigation of cellular response mechanisms: exploring drug- or stress-induced alterations in protein activity.
MtoZ Biolabs: One-stop Chemical Proteomics Solution
At MtoZ Biolabs, we offer comprehensive end-to-end services, ranging from probe screening recommendations, customized experimental design, enrichment and instrumentation, to data analysis and biological interpretation—empowering your research with efficiency and precision:
As a powerful bridge connecting structure, function, and target discovery, chemical proteomics is increasingly recognized as a key component of functional proteomics. It not only reveals mechanisms of protein regulation but also offers a distinct perspective on drug target identification and mechanistic elucidation. If your research focuses on the protein targets of specific small molecules, the activity dynamics of particular enzyme classes, or function-driven proteomic exploration—we welcome you to contact MtoZ Biolabs to initiate your tailored chemical proteomics research solution.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
Related Services
How to order?
