What are the different types of post-translational modifications?

Introduction: Why Study Post-Translational Modifications?

Post-translational modifications, commonly referred to as PTMs, are essential biochemical events that regulate protein structure and functional states. These modifications participate in nearly all aspects of cellular biology, shaping signaling pathways, metabolic activities, gene expression, protein stability, and subcellular distribution. By adding chemical groups such as phosphate, acetyl, methyl, glycan structures, or lipid moieties, PTMs significantly expand the functional diversity of the proteome and allow cells to respond precisely to environmental and physiological cues.

 

Understanding PTMs has become an important theme in proteomics research. With the rapid development of mass spectrometry and enrichment technologies, many types of PTMs can now be detected with high sensitivity and characterized at the site level. MtoZ Biolabs has accumulated practical experience in PTM sample preparation, enrichment, and LC-MS-based workflows, supporting researchers in exploring complex modification networks within biological systems.

 

Overview and Mechanisms of Common PTM Types

1. Phosphorylation

(1) Mechanism and Sites

Phosphorylation occurs when protein kinases transfer a phosphate group to amino acid residues such as serine, threonine, and tyrosine. In certain organisms, histidine and aspartic acid may also be phosphorylated. Phosphatases reverse this process, maintaining a dynamic balance. Due to its rapid and reversible characteristics, phosphorylation functions as a highly adaptable regulatory switch within cellular signaling pathways.

 

(2) Biological Functions

Phosphorylation influences major pathways including MAPK, PI3K-AKT, and JAK-STAT. It modifies protein interactions, affects enzyme activity, and guides critical processes such as cell cycle progression, metabolism, proliferation, and apoptosis.

 

(3) Mass Spectrometry Approaches

Because phosphopeptides are typically low in abundance, enrichment is required. IMAC and TiO₂-based strategies are commonly used. High-resolution LC-MS platforms are applied for identification, and databases such as PhosphoSitePlus support site annotation. DIA-based workflows can further enhance coverage and quantification.

 

2. Acetylation

(1) Mechanism and Sites

Acetylation primarily modifies the epsilon amino group of lysine residues and occasionally protein N-termini. Lysine acetyltransferases such as p300 or CBP catalyze this reaction, while histone deacetylases remove acetyl groups and maintain dynamic regulation.

 

(2) Biological Functions

Histone acetylation regulates chromatin accessibility and transcriptional activity. Non-histone acetylation influences protein stability, subcellular trafficking, signaling recognition, and enzymatic regulation. For example, acetylation of p53 contributes to its activity during DNA damage responses.

 

(3) Mass Spectrometry Approaches

Acetylated peptides are enriched using high-affinity anti-acetyl-lysine antibodies, followed by LC-MS/MS analysis. Since this modification often occurs at low stoichiometry, efficient enrichment and careful sample handling are essential.

 

3. Methylation

(1) Mechanism and Sites

Methylation is catalyzed by protein methyltransferases using S-adenosyl-methionine as a methyl donor. Lysine residues can be mono-, di-, or trimethylated, while arginine residues may undergo symmetric or asymmetric dimethylation.

 

(2) Biological Functions

Methylation regulates chromatin organization and transcription. For example, H3K4me3 correlates with active promoters, while H3K9me3 marks heterochromatin. Non-histone methylation is also involved in DNA repair, RNA processing, and cellular signaling.

 

(3) Mass Spectrometry Approaches

High-resolution MS helps distinguish different methylation states. Antibody enrichment, chemical derivatization, and neutral-loss scanning may be combined for sensitive detection. Databases such as iPTMnet assist in functional interpretation.

 

4. Glycosylation

(1) Mechanism and Sites

Glycosylation attaches glycan structures to proteins, most commonly through N-linked asparagine residues or O-linked serine or threonine residues. These modifications take place in the ER and Golgi where glycan processing is tightly controlled.

 

(2) Biological Functions

Glycosylation is essential for the stability, folding, and trafficking of membrane proteins, receptors, and secreted proteins. It affects ligand binding, cell-cell communication, immune recognition, and disease progression, including roles in cancer and infection.

 

(3) Mass Spectrometry Approaches

Lectin enrichment, HILIC separation, and enzymatic deglycosylation such as PNGase F are often used. LC-MS/MS provides structural insights into both glycosites and glycan compositions.

 

5. Ubiquitination

(1) Mechanism and Sites

Ubiquitination conjugates an 8.5 kDa protein called ubiquitin to lysine residues of target proteins through E1, E2, and E3 enzymes. Polyubiquitin chains such as K48 or K63 serve distinct regulatory purposes.

 

(2) Biological Functions

Ubiquitination regulates proteasomal degradation, DNA repair, receptor internalization, inflammatory signaling, and cell-cycle control. K48-linked chains often signal protein turnover, while K63-linked chains can modulate signaling pathways.

 

(3) Mass Spectrometry Approaches

Anti-K epsilon GG antibodies are used to enrich diGly remnants produced after trypsin digestion. Both DDA and DIA workflows support global identification of ubiquitination sites.

 

6. Lipidation

(1) Mechanism and Sites

Lipidation introduces lipid groups onto proteins. Examples include N-myristoylation on glycine, palmitoylation on cysteine, and prenylation on cysteine. These lipid groups allow proteins to associate with specific membrane environments.

 

(2) Biological Functions

Lipidation affects membrane anchoring, spatial regulation of signaling pathways, receptor activity, and cytoskeletal dynamics. Myristoylation of Src family kinases, for instance, is essential for their membrane localization and activity.

 

(3) Mass Spectrometry Approaches

Chemical probes, click chemistry, and isotopic labeling facilitate detection. Careful sample processing can help preserve modification integrity.

 

7. Proteolytic Processing

(1) Mechanism and Sites

Proteolytic processing involves specific cleavage by proteases such as caspases or furin to generate mature or functional proteins. These events are central to apoptosis, development, coagulation, and immune responses.

 

(2) Biological Functions

Caspase-mediated cleavage of PARP is widely recognized as a marker of apoptosis. Proteolytic events also regulate biological activation, secretion, and signaling.

 

(3) Mass Spectrometry Approaches

N-terminomics and C-terminomics strategies are commonly used. Quantitative tags such as TMT or iTRAQ support the mapping of cleavage sites and the discovery of regulated proteolysis.

 

MtoZ Biolabs: Empowering Post-Translational Modifications Analysis

1. Modification-Specific Enrichment and Sample-Preparation Strategies

Different PTMs require specific enrichment and sample handling methods. MtoZ Biolabs provides workflows that include:

• Phosphorylation: TiO₂ + IMAC dual enrichment for improved recovery

• Acetylation/Methylation: high-affinity antibody enrichment with de-modification enzyme inhibitors

• Glycosylation: lectin chromatography, HILIC enrichment, and enzymatic deglycosylation

• Ubiquitination: K-ε-GG immunoaffinity enrichment for high site specificity

Buffer systems and protective reagents are adapted to preserve modification integrity throughout processing.

 

2. High-Resolution Platforms and Multiplexed Acquisition Modes

The laboratory supports LC-MS/MS acquisition modes including DDA, DIA, and PRM. These options enable both comprehensive PTM profiling and targeted verification.

 

3. Data-Analysis and Functional Annotation Pipeline

Data processing may include quality control, site localization scoring, database searching, pathway enrichment analysis, and protein network visualization. Tools such as MaxQuant, Spectronaut, PhosphoSitePlus, and STRING support multi-dimensional interpretation.

 

4. Customized Scientific Support for Mechanistic Studies

MtoZ Biolabs assists with experimental design, enrichment strategy selection, parameter optimization, and data interpretation to support mechanistic research and publication goals.

Conclusion

Post-translational modifications are essential mechanisms that connect protein sequence to biological function. Studying PTMs provides insight into cellular regulation, signaling networks, and disease mechanisms. Advances in mass spectrometry and enrichment technologies now allow researchers to explore complex PTM landscapes at unprecedented depth.

 

With optimized enrichment strategies and high-resolution analytical workflows, MtoZ Biolabs supports the scientific community in uncovering the molecular details of PTM regulation and expanding the understanding of protein function within diverse biological systems.