What Are Targeted Post-Translational Modifications?
- Phosphorylation: TiO₂, IMAC, PolyMAC
- Acetylation: antibody-based enrichment
- Ubiquitination: K-ε-GG (diGly) antibody-based capture
- A highly sensitive approach implemented on triple quadrupole (QqQ) instruments
- Often used for clinical translation and absolute quantification
- High reproducibility, suitable for large-scale cohort studies
- High-resolution monitoring typically implemented on Orbitrap-based platforms
- Provides richer fragment-ion information, thereby improving specificity
- Particularly suitable for quantifying low-abundance PTMs in complex samples
- Uses stable isotope–labeled peptides as triggers
- Automatically increases monitoring depth
- Highly effective for sites that are difficult to detect
- A robust enrichment system (antibody specificity, enrichment efficiency, background control)
- A high-resolution mass spectrometry platform and a stable LC–MS system
- Accurate target selection and peptide design
- A stringent quality-control framework and cross-batch stability verification
Post-translational modifications (PTMs) are chemical alterations that occur to proteins after translation, including phosphorylation, acetylation, ubiquitination, and others. These modifications not only regulate protein activity, subcellular localization, and stability, but also exert substantial influence on signaling pathways, cellular differentiation, and disease processes. In proteomics, targeted post-translational modifications analysis refers to the selection of specific PTM classes and defined protein/peptide regions (i.e., targeted sites/segments) for precise, quantitative, and highly specific detection by mass spectrometry. Unlike global discovery proteomics, targeted post-translational modifications analysis is typically organized around a clearly defined biological question, for example: whether phosphorylation sites within a particular kinase signaling cascade are genuinely activated; whether key acetylation sites change significantly after drug administration; or whether ubiquitination-based regulation within a pathway is perturbed. This question-driven strategy is highly valuable for drug development, mechanistic investigations, and biomarker discovery.
Why Is Targeted Analysis of Post-Translational Modifications Needed?
1. The Abundance of Post-Translational Modifications Is Extremely Low and Is Often Masked by Overall Protein Signals
For most targeted post-translational modifications, the modified fraction is typically below 5%, and may even be below 1%. When relying only on conventional proteomics acquisition (survey/scanning) modes, many critical sites may not be captured, or quantification can be associated with substantial error. Targeted approaches (e.g., PRM, MRM, SureQuant) can markedly improve analytical sensitivity, enabling accurate characterization of dynamic changes in low-abundance modifications.
2. PTMs Are Highly Site-Specific
A single protein may harbor dozens of modification sites, and individual sites can have distinct functional roles. By focusing on targeted PTM analysis, researchers can perform absolute or relative quantification at specific sites, thereby providing high-confidence evidence for interpreting complex signaling networks.
3. PTM Pathways Have Direct Relevance to Drug Development
(1) The effects of kinase inhibitors are often reflected by changes at phosphorylation sites
(2) Epigenetic drugs regulate acetylation or methylation states
(3) Immunomodulatory drugs frequently involve the ubiquitination–deubiquitination system
Accordingly, targeted PTM measurements can serve as key supporting data for pharmacodynamic assessment, mechanistic validation, and the development of companion diagnostics.
Common Technical Routes for Targeted Post-Translational Modifications
1. Enrichment Strategy (Enrichment)
Because PTMs are typically of low abundance, sample preparation commonly includes an enrichment step:
High-quality enrichment workflows directly determine the reproducibility of targeted PTM datasets.
2. Mass Spectrometry Technical Routes
Targeted PTM analysis commonly employs the following strategies:
(1) MRM (Multiple Reaction Monitoring)
(2) PRM (Parallel Reaction Monitoring)
(3) SureQuant / Internal Standard Triggering
Different strategies match different study objectives: when the goal is highly precise and readily translatable measurements, MRM is often preferable; during exploratory validation requiring high specificity, PRM is a commonly adopted option.
Advantages of Targeted Post-Translational Modifications
1. High Data Credibility, Applicable to Mechanistic Research
Targeted acquisition reduces reliance on database-search-driven variability, increases the likelihood of capturing true signals, and can provide a clear evidentiary chain for mechanistic interpretation.
2. Extremely High Dynamic Range
It enables simultaneous measurement of high-abundance and extremely low-abundance modification sites, making it suitable for models involving drug perturbation, time-course sampling, and dose–response designs.
3. Enables Absolute Quantification
When combined with internal standard peptides, molecular-level absolute abundance can be obtained, an outcome that is typically difficult to achieve using traditional discovery-mode workflows.
Typical Scenarios Suitable for Targeted Post-Translational Modifications
1. Phosphorylation Pathway Studies (e.g., MAPK, PI3K-Akt)
This approach can quantify the true extent of pathway activation rather than relying on indirect antibody-based inference.
2. Drug Target Verification and Pharmacodynamic Evaluation
By measuring the magnitude of change at key modification sites after dosing, targeted PTM analysis can help determine whether a given dose elicits an effective molecular response.
3. Epigenetic Regulation Studies (e.g., Acetylation, Methylation)
It supports investigation of how chromatin-state alterations relate to gene-expression regulation.
4. Immune Signaling and Ubiquitination Studies
These applications are important in contexts such as inflammation, autophagy, and tumor immunity.
How to Carry Out a High-Quality Targeted Post-Translational Modifications Project?
In practice, successful targeted PTM analysis often requires:
None of these elements can be omitted. Because targeted PTM workflows place stringent demands on instrument sensitivity and methodological stability, many research teams choose to collaborate with specialized groups that have established platforms.
Targeted post-translational modifications analysis enables high-sensitivity, site-specific, and highly reproducible assessment of the true status of protein modifications, providing indispensable data support for life science research and drug development. If you are conducting signaling-pathway dissection, investigating drug mechanisms of action, or requiring high-precision validation of key PTMs, targeted PTM analysis represents one of the most reliable experimental strategies. If you need to design an appropriate targeted PTM scheme for your project, MtoZ Biolabs can provide professional experimental workflows and quantitative strategy support.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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