How to Detect Post-Translational Modifications (PTMs) Sites?

Introduction of Post-Translational Modifications (PTMs)

Post-translational modifications (PTMs) are chemical changes to proteins after translation. These modifications—from phosphorylation and acetylation to ubiquitination and glycosylation—play a vital role in modulating protein function, stability, and cellular localization. PTMs are key regulators in signaling networks and are implicated in processes as diverse as cell division, differentiation, and stress response.

Detecting PTM sites is crucial for understanding protein activity in health and disease. Advanced analytical techniques allow researchers to map these modifications with remarkable accuracy, thereby revealing the molecular underpinnings of complex biological systems.

Figure 1. Typical post-translational modifications proteomic analysis workflows. (Virág D, et al., 2020)

Types of PTMs

• Phosphorylation: A phosphate group is added to amino acids. It regulates cell signals and can be quickly reversed.
• Acetylation: An acetyl group attaches to lysine. This change impacts protein stability and gene regulation.
• Ubiquitination: Small proteins are attached to mark proteins for recycling. It controls protein levels in cells.
• SUMOylation: A small modifier binds to change protein location and function.
• Glycosylation: Sugars attach to proteins, helping them fold properly.
• Methylation: A methyl group is added, altering how proteins interact with each other.
• Nitrosylation: A nitric oxide group attaches to protein cysteine residues. This alters protein function and signaling.
• Disulfide Bond Formation: Covalent bonds form between cysteine pairs. They stabilize protein structure and shape.
• Lipidation: Fat molecules attach to proteins. This helps anchor proteins to cell membranes and affects their activity.

Mass Spectrometry-Based Methods for PTMs Identification

Mass spectrometry (MS) is a powerful method used to detect and map PTMs on proteins. Essentially, MS measures the mass of protein fragments, allowing us to determine if a protein has been modified and where that modification is located. Here's a closer look at how it works:

• Breaking Down the Protein: In a typical workflow, proteins are first broken down into smaller pieces called peptides. This is usually done using enzymes like trypsin, which cuts the protein at specific points. Breaking the protein into peptides makes it easier for the mass spectrometer to analyze them.
• Separation Using Liquid Chromatography (LC): Before reaching the mass spectrometer, the peptides are often separated using LC. This step is important because it spreads out the complex mixture of peptides, allowing the instrument to analyze each one more clearly. Think of it like sorting a mixed bag of candies into separate piles by flavor.
• Mass Spectrometry Analysis (MS/MS): Once the peptides are separated, they enter the mass spectrometer. The first stage of analysis (MS) determines the mass of each peptide. Then, selected peptides are further broken apart in tandem mass spectrometry (MS/MS). This second stage provides detailed information about the peptide fragments, helping to pinpoint the exact location of any modifications.
• Identifying PTMs: PTMs, such as adding a phosphate or acetyl group, change the mass of the peptide in a specific way. We can detect these changes by comparing the measured mass with the expected mass of the unmodified peptide. For example, a phosphate group adds about 80 units to the mass, which can be identified in the MS data.
• Data Interpretation: The raw data generated from MS/MS is complex, so specialized software matches the measured masses to known protein sequences. This step helps confirm which peptides have been modified and where the modification is located. The result is a detailed protein map showing its PTM landscape.
• Quantitative Analysis: Modern MS techniques also allow for quantifying the extent of modifications. Methods like Tandem Mass Tag (TMT) and Isobaric Tag for Relative and Absolute Quantitation (iTRAQ) enable researchers to compare modification levels across different samples. This is especially useful when studying how PTMs respond to treatments or conditions.

Figure 1. Mass spectrometry-based workflow for the identification of PTMs.(Brandi J, et al., 2022)

Chromatography Techniques for PTM Characterization

High-Performance Liquid Chromatography (HPLC) for PTM Analysis

HPLC is a powerful separation method commonly used for peptide and protein analysis. In PTM studies, HPLC is often paired with MS to precisely identify and characterize modifications. The technique passes a liquid sample through a column filled with a specialized material that separates the components based on their interactions with the column.

How HPLC Works for PTM Detection

• Reversed-Phase HPLC (RP-HPLC) is the most widely used type for PTMs analysis. It separates peptides based on their hydrophobicity, meaning how well they repel water. Hydrophobic peptides interact more with the column material and take longer to pass through, while hydrophilic peptides move through more quickly.
• Hydrophilic Interaction Liquid Chromatography (HILIC) is particularly useful for detecting PTMs that introduce hydrophilic groups, such as glycosylation or phosphorylation.
• Ion Exchange Chromatography (IEX) separates molecules based on their charge. PTMs like phosphorylation or acetylation often change the charge of a protein, making IEX a useful choice for analyzing these modifications.

HPLC can also be combined with selective enrichment techniques. For example, phosphopeptide enrichment using metal affinity columns enhances the detection of low-abundance phosphorylated peptides. Additionally, size-exclusion chromatography (SEC) can separate intact proteins to study large-scale structural changes caused by PTMs.

Why HPLC is Useful for PTM Analysis

• Provides excellent separation of complex peptide mixtures.
• Can handle a wide range of sample types, including whole cell lysates.
• Facilitates large-scale, high-throughput PTM analysis when integrated with MS.
• Detects even minor changes in protein structure due to PTMs.

Capillary Electrophoresis (CE) for PTM Characterization

Capillary Electrophoresis (CE) is a complementary technique to HPLC, often used when analyzing small amounts of protein or when extremely high-resolution separation is required. CE separates molecules based on their size and charge as they move through a thin capillary tube under an electric field. Because PTMs often alter the charge or mass of a protein, CE can detect these changes with remarkable sensitivity.

How CE Works for PTM Detection

• Capillary Zone Electrophoresis (CZE) is the most common form of CE for PTM analysis. It separates proteins based on their charge-to-size ratio, making it ideal for detecting modifications like phosphorylation or acetylation that change the protein's charge.
• Capillary Isoelectric Focusing (cIEF) separates proteins based on their isoelectric point (pI), which is the pH at which the protein carries no net charge. Since PTMs often shift a protein's pI, cIEF provides detailed insights into the modification state of a protein.
• Capillary Gel Electrophoresis (CGE) is used for size-based separation, useful in cases where PTMs significantly change the size of a protein, such as glycosylation.

Advantages of Using CE for PTM Characterization

• Requires minimal sample amounts, making it suitable for precious or limited samples.
• Delivers exceptionally high-resolution separation, even for subtle PTM-induced changes.
• Provides rapid analysis.
• Can be easily coupled with mass spectrometry for precise identification of PTM sites.

Western Blotting for PTM Detection

Western blotting is a well-established, reliable technique for detecting PTMs and remains a staple in molecular biology laboratories. It is particularly effective for confirming the presence of specific PTMs on proteins and comparing their relative abundance across different samples. By using antibodies that recognize particular modifications, researchers can obtain clear, visual evidence of PTMs and study how they respond to experimental treatments or disease states.

How Western Blotting Works for PTM Detection

The process of western blotting begins with separating proteins based on their size using SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). This step ensures that proteins are spread out in a gel, making them easier to detect individually. Once separated, the proteins are transferred onto a membrane, typically made of nitrocellulose or PVDF (polyvinylidene fluoride). This membrane acts as a stable platform for the subsequent detection steps.

The membrane is exposed to a primary antibody that specifically targets the PTM of interest. These antibodies are often designed to detect phosphorylated, acetylated, ubiquitinated, or otherwise modified protein residues. To enhance the signal, a secondary antibody is applied, which binds to the primary antibody. This secondary antibody is conjugated to a detection enzyme, such as horseradish peroxidase (HRP) or alkaline phosphatase (AP), that produces a measurable signal.

Detection methods

• Chemiluminescence: Produces light in the presence of a substrate, allowing visualization using imaging systems.
• Fluorescence: Uses fluorescently labeled antibodies for detection, offering higher sensitivity and multiplexing capability.
• Colorimetric Detection: Develops a visible color change on the membrane, useful for simple and quick analysis.

Advantages of Western Blotting for PTM Detection

• Specificity and Sensitivity: With the right antibody, Western blotting can detect low-abundance PTMs with remarkable specificity.
• Quantitative Capabilities: While primarily qualitative, Western blotting can provide semi-quantitative data when normalized against loading controls (e.g., actin or GAPDH).
• Versatility: It is applicable for a broad range of PTMs, including phosphorylation, acetylation, and ubiquitination.
• Validation Tool: Western blotting is commonly used to validate findings from mass spectrometry or other high-throughput PTM detection methods.

Choosing the Right Antibody for PTM Detection

The success of a Western blot largely depends on selecting a high-quality primary antibody with strong specificity for the PTM of interest. Antibodies can be monoclonal (produced from a single B cell clone) or polyclonal (derived from multiple B cell clones). Monoclonal antibodies often provide greater specificity, while polyclonal antibodies may offer higher sensitivity.

In some cases, researchers may use phospho-specific antibodies that detect only phosphorylated proteins or pan-specific antibodies that recognize both modified and unmodified forms of a protein. Additionally, antibodies targeting PTMs often require rigorous validation to ensure that they do not produce false-positive or non-specific signals.

Immunoprecipitation (IP) for Enrichment of Modified Proteins

Immunoprecipitation (IP) is a powerful method for isolating specific proteins from complex mixtures, making it a valuable tool for detecting PTMs. By using antibodies that specifically recognize either a target protein or a particular modification, researchers can "fish out" modified proteins from cell lysates, allowing for more detailed downstream analysis.

How Immunoprecipitation Works

The process begins by lysing cells to release their proteins into a solution. An antibody—either one that binds directly to the protein of interest or one that recognizes a specific modification like phosphorylation or ubiquitination—is added to the lysate. This antibody attaches to its target, forming an immune complex. To pull this complex out of the solution, researchers use protein-binding beads (often coated with Protein A, Protein G, or streptavidin, depending on the type of antibody used). When these beads are introduced, they latch onto the antibody-protein complex, which can then be separated from the rest of the sample through centrifugation or magnetic separation.

Once isolated, the bound protein is washed to remove contaminants and then eluted for further analysis. Researchers often follow up with western blotting to confirm the presence of a specific PTM or mass spectrometry to precisely map modification sites.

Types of Immunoprecipitation for PTM Analysis

• Classic IP (Protein-Specific IP): Uses an antibody against the protein of interest to isolate the entire protein population, regardless of modification status. Subsequent analysis (e.g., western blot or MS) can reveal PTMs.
• Modification-Specific IP (PTM-IP): Directly targets a specific PTM using an antibody that recognizes modified amino acids (e.g., anti-phosphotyrosine antibodies for phosphorylated tyrosine residues). This allows for enrichment of only the modified proteins, improving detection sensitivity.
• Co-Immunoprecipitation (Co-IP): Focuses on protein-protein interactions but can also provide indirect evidence of PTMs by identifying interaction partners influenced by specific modifications.

Advantages and Challenges

• Advantages: One of the biggest strengths of IP is its specificity. High-quality antibodies ensure that only the desired protein or modification is captured, reducing background noise and improving signal detection. This makes IP especially useful when working with low-abundance PTMs that might otherwise be undetectable in complex samples.
• Challenges: Not all PTMs have well-characterized or commercially available antibodies, making enrichment difficult. Additionally, certain modifications, like phosphorylation or acetylation, can be unstable, requiring careful sample handling to prevent loss of modifications during preparation. Optimizing lysis conditions, using phosphatase or deacetylase inhibitors, and selecting the right antibody are critical for successful IP-based PTM detection.

PTMs Structural Detection Methods

Understanding how PTMs influence protein structure is essential for uncovering their biological functions. While mass spectrometry and chromatography are excellent for detecting modification sites, structural techniques such as nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography provide a deeper look at how these modifications alter a protein's shape, stability, and interactions.

Nuclear Magnetic Resonance (NMR) Spectroscopy for PTM Analysis

NMR spectroscopy is a powerful tool for studying proteins in solution, offering a dynamic, real-time view of how PTMs affect their structure. Unlike X-ray crystallography, which requires proteins to be crystallized, NMR allows researchers to observe proteins in an environment that closely resembles their natural state in the body.

How NMR works

NMR relies on the magnetic properties of atomic nuclei, primarily hydrogen (protons). When placed in a strong magnetic field, these nuclei resonate at specific frequencies depending on their local chemical environment. By analyzing these signals, scientists can map out the three-dimensional structure of proteins and pinpoint how modifications like phosphorylation or acetylation influence protein folding, flexibility, and interactions.

Why it's useful for PTM analysis

• NMR is particularly valuable for studying small to medium-sized proteins (up to ~50 kDa) and protein domains.
• It provides detailed information on how PTMs affect protein dynamics, including changes in flexibility, stability, and binding to other molecules.
• It helps researchers understand transient interactions—such as how a modified protein temporarily interacts with a signaling partner—which are often difficult to capture with other techniques.

X-ray Crystallography for PTM Structural Studies

X-ray crystallography is the gold standard for determining high-resolution, atomic-level protein structures. By capturing a static snapshot of a protein, this technique reveals how PTMs affect the overall shape, surface properties, and interaction sites.

How it works

The protein of interest must first be crystallized—a process that involves arranging proteins into a well-ordered lattice. Once a high-quality crystal is obtained, it is exposed to an intense X-ray beam. The X-rays scatter upon hitting the atoms in the protein, creating a diffraction pattern that can be analyzed to reconstruct the protein's three-dimensional structure.

Why it's useful for PTM analysis

• It provides high-resolution (down to atomic-level) details of how PTMs modify protein conformation.
• It can pinpoint interactions between PTM-modified proteins and other molecules, such as drugs, DNA, or enzymes.
• It helps in drug discovery by identifying how PTMs influence drug binding sites.

Bioinformatics Tools for PTM Prediction and Analysis

PTM Databases and Computational Models

PTM databases serve as comprehensive resources where researchers can access verified information on modified proteins. These databases consolidate data from peer-reviewed studies, providing detailed information on PTM sites, associated proteins, modification types, and biological contexts.

• PhosphoSitePlus: Specializes in phosphorylation data, while also covering acetylation, ubiquitination, and methylation. It includes curated experimental evidence from human, mouse, and rat studies.
• UniProt: Offers a vast, publicly accessible database that catalogs protein sequences and functional information, including PTMs, across numerous species.
• dbPTM: Provides experimentally validated and computationally predicted PTM data, serving as a valuable resource for researchers exploring novel PTM sites.
• SwissPalm: A specialized database for S-palmitoylation, which is essential for membrane protein localization.

Computational Tools for PTM Site Prediction

Predicting PTM sites computationally is particularly valuable when experimental validation is time-consuming or resource-intensive. Machine learning algorithms and sequence analysis models leverage vast datasets to forecast likely PTM sites. These tools analyze amino acid sequences, structural features, and evolutionary patterns to make accurate predictions.

• NetPhos and NetAcet: Widely used for phosphorylation and acetylation site prediction, these tools apply neural networks trained on experimentally confirmed PTM data.
• GPS (Group-based Prediction System): Predicts phosphorylation sites by grouping similar substrates, offering insights into kinase-substrate relationships.
• ModPred: A versatile tool capable of predicting over 40 different types of PTMs, making it ideal for initial exploratory analyses.
• SignalP: Identifies signal peptides and cleavage sites, which are often linked to PTMs that regulate protein localization and function.

Applications of PTM Analysis

PTM Analysis in Biomedical Research

PTM mapping has unveiled critical insights into cellular signaling pathways, protein interactions, and regulatory networks. By understanding the modification landscape, researchers can elucidate mechanisms underlying processes such as cell growth, differentiation, and apoptosis.

PTMs in Drug Development and Biomarker Discovery

PTM analysis is increasingly used in drug development, aiding in the identification of novel therapeutic targets and biomarkers. The detection of specific PTM patterns can inform treatment strategies and contribute to the development of personalized medicine.

Role of PTM Analysis in Disease Mechanisms

Aberrant PTM patterns are often linked to diseases, including cancer, neurodegenerative disorders, and metabolic syndromes. Mapping these alterations not only enhances our understanding of disease mechanisms but also paves the way for innovative diagnostic and therapeutic approaches.

Case Study

Mass spectrometry investigation of glycosylation on the NXS/T sites in recombinant glycoproteins

Journal: Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

Published: 2013

DOI: 10.1016/j.bbapap.2013.04.022

Background

Glycosylation is a key post-translational modification that influences protein stability, solubility, and function. Therapeutic proteins, especially chimeric ones like IgG-based constructs, rely on correct glycosylation for optimal performance.

Purpose

The study aimed to determine if adding new N-linked glycosylation sites in a chimeric protein (IgG-Fc-ZP3E7) affects the glycosylation of its native IgG-Fc site (96NST98).

Methods

• Protein Constructs: Two proteins were analysed: the control IgG-HC (with one glycosylation site) and the chimeric IgG-Fc-ZP3E7 (with three potential sites: 96NST98, 288NCS290, 291NSS293).
• Glycosylation Analysis: Proteins were treated with PNGaseF to remove N-linked oligosaccharides.
• Analytical Techniques: SDS-PAGE, Western blotting (using ConA, anti-human IgG, and anti-ZP3 antibodies), and LC–MS/MS following a trypsin-AspN double digestion were employed for detailed mapping of glycosylation sites.

Results

• In the control IgG-HC, the 96NST98 glycosylation site was occupied and converted to 96DST98 after PNGaseF treatment.
• In the chimeric IgG-Fc-ZP3E7 protein, only the 291NSS293 site was glycosylated, while the native 96NST98 site remained unoccupied.
• The 288NCS290 site was not detected as glycosylated.
• The presence of additional glycosylation sites interfered with the typical glycosylation of the IgG-Fc region.

Figure 3. Analysis of IgG-HC and IgG-Fc-ZP3E7 by WB.

Figure 4. Analysis of IgG-HC and IgG-Fc-ZP3E7 by SDS-PAGE.

Figure 5. LC-MS/MS analysis of the PNGaseF-treated (deglycosylated) IgG-HC for identification of the potential glycosylation sites for N-linked oligosaccharides.

Conclusion

Introducing extra N-linked glycosylation sites in the IgG-Fc-ZP3E7 protein disrupts the glycosylation of the native Fc glycosylation site. This finding suggests that strategic modification of glycosylation sites can modulate the physicochemical properties of recombinant proteins, offering a novel approach to optimize protein solubility and function for therapeutic applications.

References

• Virág D, et al. Current trends in the analysis of post-translational modifications. Chromatographia, 2020, 83: 1-10. DOI: 10.1007/s10337-019-03796-9
• Brandi J, et al. Advances in enrichment methods for mass spectrometry-based proteomics analysis of post-translational modifications. Journal of Chromatography A, 2022, 1678: 463352. DOI: 10.1016/j.chroma.2022.463352